S-phenylglycine T-butyl Ester Research Articles

JAG1 mediates apoptosis in herpes simplex keratitis by suppressing autophagy via ROS/JAG1/NOTCH1/pULK1 signaling pathway

Herpes simplex keratitis (HSK), an ocular disease resulted from herpes simplex virus type 1 (HSV-1) infection, leads to the majority of infectious corneal blindness worldwide. The apoptosis of corneal epithelial cells (CECs) resulted from HSV-1 disrupts the epithelial barrier and exacerbates the infection; however, there is no definitive cure for HSK. Jagged1 (JAG1), one of the primary functional ligands for NOTCH receptors, plays a crucial role in regulating apoptosis and autophagy; however, its role in HSK is unclear. Our transcriptome analysis showed JAG1 was significantly upregulated in HSV-1-infected human CECs. We aimed to explore JAG1’s role in regulating apoptosis in HSV-1-infected human CECs and in HSK mice. HSV-1 infection induced apoptosis and reactive oxygen species (ROS) generation in CECs. HSV-1 also activated the JAG1/NOTCH1 signaling pathway. The ROS scavenger N-acetylcysteine significantly mitigated these effects. Additionally, inhibiting the JAG1/NOTCH1 pathway with short hairpin RNA against JAG1 or a NOTCH1 inhibitor (N-[N-{3,5-difuorophenacetyl}-1-alanyl]-S-phenylglycine t-butyl ester [DAPT]) alleviated HSV-1-induced CEC apoptosis. Transmission electron microscopy and western blotting revealed that HSV-1 infection suppressed ULK1-mediated autophagy in CECs, while DAPT treatment enhanced autophagy by suppressing ULK1 phosphorylation. The activation of autophagy by rapamycin treatment markedly reduced ROS levels and apoptosis in HSV-1-infected CECs, revealing a synergistic effect between the suppressed autophagy and increased ROS levels, ultimately leading to apoptosis. Thus, HSV-1 induces CEC apoptosis by suppressing autophagy through ROS/JAG1/NOTCH1/pULK1 signaling pathway in vitro and in vivo, providing potential therapeutic targets for HSK.

17β-estradiol inhibits Notch1 activation in murine macrophage cell line RAW 264.7.

Macrophages are major effectors in regulating immune response and inflammation. The pro-inflammatory phenotype (M1) is induced by the activation of the Toll-like receptor 4 (TLR4) on the macrophage surface, which recognizes lipopolysaccharide (LPS), a component of Gram-negative bacterial wall, and by the binding of interferon-gamma (IFNγ), a cytokine released by activated T lymphocytes, to its receptor (IFNGR). Among the pathways activated by LPS/IFNγ is the Notch pathway, which promotes the M1 phenotype. Conversely, 17β-estradiol (E2) has been shown to blunt LPS-mediated inflammatory response. While it has been shown that E2 regulates the activity of the Notch1 receptor in human endothelial cells, there is no evidence of estrogen-mediated regulation of Notch1 in macrophages. In this study, RAW 264.7 cells were stimulated with LPS/IFNγ in the presence or absence of E2 and/or N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of γ-secretase, the enzyme involved in Notch activation. The effects of treatment on inducible nitric oxide synthase (iNOS), on components of the Notch pathway, and MAPK (mitogen-activated protein kinase) were assessed by quantitative PCR and Western blotting. We found that E2, through a mechanism involving the inhibition of p38 phosphorylation, reduces the activation of Notch1 induced by LPS/IFNγ. On the contrary, Notch1 exerts a negative control on the estrogen receptor α (ERα) since Notch1 inhibition increases the protein levels of this receptor. In conclusion, we report for the first time a Notch-ERα interaction in macrophages. Our data suggest that E2 may reduce LPS/IFNγ-mediated M1 pro-inflammatory phenotype in macrophages by inhibiting Notch1. This finding encourages further studies on Notch1 inhibitors as novel treatments for inflammation-related diseases.

Chronic exposure to hexavalent chromium induces esophageal tumorigenesis via activating the Notch signaling pathway.

Hexavalent chromium Cr(VI), as a well-established carcinogen, contributes to tumorigenesis for many human cancers, especially respiratory and digestive tumors. However, the potential function and relevant mechanism of Cr(VI) on the initiation of esophageal carcinogenesis are largely unknown. Here, immortalized human esophageal epithelial cells (HEECs) were induced to be malignantly transformed cells, termed HEEC-Cr(VI) cells, via chronic exposure to Cr(VI), which simulates the progress of esophageal tumorigenesis. In vitro and in vivo experiments demonstrated that HEEC-Cr(VI) cells obtain the ability of anchorage-independent growth, greater proliferative capacity, cancer stem cell properties, and the capacity to form subcutaneous xenografts in BALB/c nude mice when compared to their parental cells, HEECs. Additionally, HEEC-Cr(VI) cells exhibited weakened cell motility and enhanced cell adhesion. Interestingly, HEECs with acute exposure to Cr(VI) failed to display those malignant phenotypes of HEEC-Cr(VI) cells, suggesting that Cr(VI)‍-induced malignant transformation, but not Cr(VI) itself, is the cause for the tumor characteristics of HEEC-Cr(VI) cells. Mechanistically, chronic exposure to Cr(VI) induced abnormal activation of Notch signaling, which is crucial to maintaining the capacity for malignant proliferation and stemness of HEEC-Cr(VI) cells. As expected, N-‍[N-‍(3,5-difluorophenacetyl)‍-L-alanyl]‍-S-phenylglycine t-butyl ester (DAPT), an inhibitor for the Notch pathway, drastically attenuated cancerous phenotypes of HEEC-Cr(VI) cells. In conclusion, our study clarified the molecular mechanism underlying Cr(VI)‍-induced esophageal tumorigenesis, which provides novel insights for further basic research and clinical therapeutic strategies about Cr(VI)‍-associated esophageal cancer.

Interaction of the transforming growth factor-β and Notch signaling pathways in the regulation of granulosa cell proliferation.

The Notch and transforming growth factor (TGF)-β signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-β signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-β and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.

Inhibition of Notch1 signal promotes brain recovery by modulating glial activity after stroke

BackgroundNotch1 signaling inhibiton with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester] (DAPT) treatment could promote brain recovery and the intervention effect is different between striatum (STR) and cortex (CTX), which might be accounted for different changes of glial activities, but the in-depth mechanism is still unknown. The purpose of this study was to identify whether DAPT could modulate microglial subtype shifts and astroglial-endfeet aquaporin-4 (AQP4) mediated waste solute drainage. MethodsSprague-Dawley rats (n=10) were subjected to 90min of middle cerebral artery occlusion (MCAO) and were treated with DAPT (n=5) or act as control with no treatment (n=5). Two groups of rats underwent MRI scans at 24h and 4 week, and sacrificed at 4 week after stroke for immunofluorescence (IF). ResultsCompared with control rats, MRI data showed structural recovery in ipsilateral STR but not CTX. And IF showed decreased pro-inflammatory M1 microglia and increased anti-inflammatory M2 microglia in striatal lesion core and peri-lesions of STR, CTX. Meanwhile, IF showed decreased AQP4 polarity in ischemic brain tissue, however, AQP4 polarity in striatal peri-lesions of DAPT treated rats was higher than that in control rats but shows no difference in cortical peri-lesions between control and treated rats. ConclusionsThe present study indicated that DAPT could promote protective microglia subtype shift and striatal astrocyte mediated waste solute drainage, that the later might be the major contributor of waste solute metabolism and one of the accounts for discrepant recovery of STR and CTX.

Notch Signaling Inhibition Alleviates Allergies Caused by Antarctic Krill Tropomyosin through Improving Th1/Th2 Imbalance and Modulating Gut Microbiota.

Antarctic krill tropomyosin (AkTM) has been shown in mice to cause IgE-mediated food allergy. The objective of this work was to investigate the role of Notch signaling in AkTM-sensitized mice, as well as to determine the changes in gut microbiota composition and short-chain fatty acids (SCFAs) in the allergic mice. An AkTM-induced food allergy mouse model was built and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was used as an γ-secretase inhibitor to inhibit the activation of Notch signaling. Food allergy indices, some key transcription factors, histologic alterations in the small intestine, and changes in gut microbiota composition were examined. The results showed that DAPT inhibited Notch signaling, which reduced AkTM-specific IgE, suppressed mast cell degranulation, decreased IL-4 but increased IFN-γ production, and alleviated allergic symptoms. Quantitative real-time PCR and Western blotting analyses revealed that expressions of Hes-1, Gata3, and IL-4 were down-regulated after DAPT treatment, accompanied by increases in T-bet and IFN-γ, indicating that Notch signaling was active in AkTM-sensitized mice and blocking it could reverse the Th1/Th2 imbalance. Expressions of key transcription factors revealed that Notch signaling could promote Th2 cell differentiation in sensitized mice. Furthermore, 16S rRNA sequencing results revealed that AkTM could alter the diversity and composition of gut microbiota in mice, leading to increases in inflammation-inducing bacteria such as Enterococcus and Escherichia-Shigella. Correlation analysis indicated that reduced SCFA concentrations in AkTM-allergic mice may be related to decreases in certain SCFA-producing bacteria, such as Clostridia_UCG-014. The changes in gut microbiota and SCFAs could be partially restored by DAPT treatment. Our findings showed that inhibiting Notch signaling could alleviate AkTM-induced food allergy by correcting Th1/Th2 imbalance and modulating the gut microbiota.

Pathogenic variations in MAML2 and MAMLD1 contribute to congenital hypothyroidism due to dyshormonogenesis by regulating the Notch signalling pathway

BackgroundIn several countries, thyroid dyshormonogenesis is more common than thyroid dysgenesis in patients with congenital hypothyroidism (CH). However, known pathogenic genes are limited to those directly involved in hormone biosynthesis....

JAG-1/Notch signaling axis in the spinal cord contributes to bone cancer pain in rats.

Notch signal plays an important role in regulating cell-cell interactions with the adjacent cells. However, it remains unknown whether Jagged1 (JAG-1) mediated Notch signaling regulates bone cancer pain (BCP) via the spinal cell interactions mechanism. Here, we showed that intramedullary injection of Walker 256 breast cancer cells increased the expression of JAG-1 in spinal astrocytes and knockdown of JAG-1 reduced BCP. The supplementation of exogenous JAG-1 to the spinal cord induced BCP-like behavior and promoted expression of c-Fos and hairy and enhancer of split homolog-1 (Hes-1) in the spinal cord of the naïve rats. These effects were reversed when the rats were administered intrathecal injections of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The intrathecal injection of DAPT reduced BCP and inhibited Hes-1 and c-Fos expression in the spinal cord. Furthermore, our results showed that JAG-1 up-regulated Hes-1 expression by inducing the recruitment of Notch intracellular domain (NICD) to the RBP-J/CSL-binding site located within the Hes-1 promoter sequence. Finally, the intrathecal injection of c-Fos-antisense oligonucleotides (c-Fos-ASO) and administration of sh-Hes-1 to the spinal dorsal horn also alleviated BCP. The study indicates that inhibition of the JAG-1/Notch signaling axis may be a potential strategy for the treatment of BCP.

The effects of Wnt, BMP, and Notch signaling pathways on cell proliferation and neural differentiation in a song control nucleus (HVC) of Lonchura striata.

There is obvious sexual dimorphism in the song control system of songbirds. In the higher vocal center (HVC), cell proliferation and neuronal differentiation contribute to the net addition of neurons. However, the mechanism underlying these changes is unclear. Given that Wnt, Bmp, and Notch pathways are involved in cell proliferation and neuronal differentiation, no reports are available to study the role of the three pathways in the song control system. To address the issue, we studied cell proliferation in the ventricle zone overlying the developing HVC and neural differentiation within the HVC of Bengalese finches (Lonchura striata) at posthatching day 15 when HVC progenitor cells are generated on a large scale and differentiate into neurons, after Wnt and Bmp pathways were activated by using a pharmacological agonist (LiCl) or Bmp4, respectively, and the Notch pathway was inhibited by an inhibitor (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester), DAPT). The results indicated that both cell proliferation and neural differentiation toward HVC neurons increased significantly after activation of the Wnt signaling pathway or inhibition of the Notch signaling pathway. Although cell proliferation was increased, neural differentiation was inhibited after treatment with Bmp4. There was obvious synergetic enhancement in the number of proliferating cells after the coregulation of two or three signaling pathways. In addition, synergetic enhancement was also found in the Wnt and Notch pathways in neural differentiation toward neurons within HVC. These results suggest that the three signaling pathways are involved in cell proliferation and neural differentiation of HVC.

CD109 Promotes Drug Resistance in A2780 Ovarian Cancer Cells by Regulating the STAT3-NOTCH1 Signaling Axis.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy owing to relapse caused by resistance to chemotherapy. We previously reported that cluster of differentiation 109 (CD109) expression is positively correlated with poor prognosis and chemoresistance in patients with EOC. To further explore the role of CD109 in EOC, we explored the signaling mechanism of CD109-induced drug resistance. We found that CD109 expression was upregulated in doxorubicin-resistant EOC cells (A2780-R) compared with that in their parental cells. In EOC cells (A2780 and A2780-R), the expression level of CD109 was positively correlated with the expression level of ATP-binding cassette (ABC) transporters, such as ABCB1 and ABCG2, and paclitaxel (PTX) resistance. Using a xenograft mouse model, it was confirmed that PTX administration in xenografts of CD109-silenced A2780-R cells significantly attenuated in vivo tumor growth. The treatment of CD109-overexpressed A2780 cells with cryptotanshinone (CPT), a signal transducer and activator of transcription 3 (STAT3) inhibitor, inhibited the CD109 overexpression-induced activation of STAT3 and neurogenic locus notch homolog protein 1 (NOTCH1), suggesting a STAT3-NOTCH1 signaling axis. The combined treatment of CD109-overexpressed A2780 cells with CPT and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), a NOTCH inhibitor, markedly abrogated PTX resistance. These results suggest that CD109 plays a key role in the acquisition of drug resistance by activating the STAT3-NOTCH1 signaling axis in patients with EOC.

Notoginsenoside R1 Facilitates Cell Angiogenesis by Inactivating the Notch Signaling During Wound Healing.

The development of chronic, nonhealing wounds is a persistent medical problem that drives patient morbidity and increases healthcare costs. Angiogenesis is a critical accompanying activity in the proliferation stage during the wound healing process. Notoginsenoside R1 (NGR1) isolated from Radix notoginseng has been reported to alleviate diabetic ulcers by promoting angiogenesis and decreasing inflammatory responses and apoptosis. In the present study, we investigated the effect of NGR1 on angiogenesis and its therapeutic functions in cutaneous wound healing. For in vitro evaluation, cell counting kit-8 assays, migration assays, Matrigel-based angiogenic assays, and western blotting were conducted. The experimental results showed that NGR1 (10-50 μM) had no cytotoxicity to human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMEC), and NGR1 treatment facilitated the migration of HSFs and enhanced angiogenesis in HMECs. Mechanistically, NGR1 treatment inhibited the activation of Notch signaling in HMECs. For in vivo analysis, hematoxylin-eosin staining, immunostaining, and Masson's trichrome staining were performed, and we found that NGR1 treatment promoted angiogenesis, reduced wound widths, and facilitated wound healing. Furthermore, HMECs were treated with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT; a Notch inhibitor), and DAPT treatment was found to exert pro-angiogenic effects. Simultaneously, DAPT was administrated into experimental cutaneous wound healing model, and we found that DAPT administration prevented the development of cutaneous wounds. Collectively, NGR1 promotes angiogenesis and wound repair via activation of the Notch pathway and exhibits therapeutic effects on cutaneous wound healing.

Effects of Modulation of the Hedgehog and Notch Signaling Pathways on Osteoblast Differentiation Induced by Titanium with Nanotopography.

The events of bone formation and osteoblast/titanium (Ti) interactions may be affected by Hedgehog and Notch signalling pathways. Herein, we investigated the effects of modulation of these signalling pathways on osteoblast differentiation caused by the nanostructured Ti (Ti-Nano) generated by H2SO4/H2O2. Osteoblasts from newborn rat calvariae were cultured on Ti-Control and Ti-Nano in the presence of the Hedgehog agonist purmorphamine or antagonist cyclopamine and of the Notch antagonist N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) or agonist bexarotene. Osteoblast differentiation was evaluated by alkaline phosphatase activity and mineralization, and the expression of Hedgehog and Notch receptors was also evaluated. In general, purmorphamine and DAPT increased while cyclopamine and bexarotene decreased osteoblast differentiation and regulated the receptor expression on both Ti surfaces, with more prominent effects on Ti-Nano. The purmorphamine and DAPT combination exhibited synergistic effects on osteoblast differentiation that was more intense on Ti-Nano. Our results indicated that the Hedgehog and Notch signalling pathways drive osteoblast/Ti interactions more intensely on nanotopography. We also demonstrated that combining Hedgehog activation with Notch inhibition exhibits synergistic effects on osteoblast differentiation, especially on Ti-Nano. The uncovering of these cellular mechanisms contributes to create strategies to control the process of osseointegration based on the development of nanostructured surfaces.

Astrocyte-neuron communication mediated by the Notch signaling pathway: focusing on glutamate transport and synaptic plasticity.

Maintaining glutamate homeostasis after hypoxic ischemia is important for synaptic function and neural cell activity, and regulation of glutamate transport between astrocyte and neuron is one of the important modalities for reducing glutamate accumulation. However, further research is needed to investigate the dynamic changes in and molecular mechanisms of glutamate transport and the effects of glutamate transport on synapses. The aim of this study was to investigate the regulatory mechanisms underlying Notch pathway mediation of glutamate transport and synaptic plasticity. In this study, Yorkshire neonatal pigs (male, age 3 days, weight 1.0-1.5 kg, n = 48) were randomly divided into control (sham surgery group) and five hypoxic ischemia subgroups, according to different recovery time, which were then further subdivided into subgroups treated with dimethyl sulfoxide or a Notch pathway inhibitor (N-[N-(3, 5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester). Once the model was established, immunohistochemistry, immunofluorescence staining, and western blot analyses of Notch pathway-related proteins, synaptophysin, and glutamate transporter were performed. Moreover, synapse microstructure was observed by transmission electron microscopy. At the early stage (6-12 hours after hypoxic ischemia) of hypoxic ischemic injury, expression of glutamate transporter excitatory amino acid transporter-2 and synaptophysin was downregulated, the number of synaptic vesicles was reduced, and synaptic swelling was observed; at 12-24 hours after hypoxic ischemia, the Notch pathway was activated, excitatory amino acid transporter-2 and synaptophysin expression was increased, and the number of synaptic vesicles was slightly increased. Excitatory amino acid transporter-2 and synaptophysin expression decreased after treatment with the Notch pathway inhibitor. This suggests that glutamate transport in astrocytes-neurons after hypoxic ischemic injury is regulated by the Notch pathway and affects vesicle release and synaptic plasticity through the expression of synaptophysin.

Inhibition of Notch 1 signaling in the subacute stage after stroke promotes striatal astrocyte-derived neurogenesis.

Inhibition of Notch1 signaling has been shown to promote astrocyte-derived neurogenesis after stroke. To investigate the regulatory role of Notch1 signaling in this process, in this study, we used a rat model of stroke based on middle cerebral artery occlusion and assessed the behavior of reactive astrocytes post-stroke. We used the γ-secretase inhibitor N-[N-(3,5-diuorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester (DAPT) to block Notch1 signaling at 1, 4, and 7 days after injury. Our results showed that only administration of DAPT at 4 days after stroke promoted astrocyte-derived neurogenesis, as manifested by recovery of white matter fiber bundle integrity on magnetic resonance imaging, which is consistent with recovery of neurologic function. These findings suggest that inhibition of Notch1 signaling at the subacute stage post-stroke mediates neural repair by promoting astrocyte-derived neurogenesis.

The interaction between autophagy and the epithelial-mesenchymal transition mediated by NICD/ULK1 is involved in the formation of diabetic cataracts

BackgroundCataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation.MethodsA rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest.ResultsIn LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy.ConclusionsWe describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.

TGF-β3 induces lactate production in Sertoli cell through inhibiting the Notch pathway.

In the testis, spermatocytes and spermatids rely on lactate produced by Sertoli cells (SCs) as energy source. Transforming growth factor-beta 3 (TGF-β3) is one of the generally accepted paracrine regulatory factors of SC-created blood-testis barrier (BTB), yet its role in SC glycolysis and lactate production still remains unclear. To investigate the effect of TGF-β3 on glycolysis and lactate production in SCs and determine the role of lethal giant larvae 2 (Lgl2) and Notch signaling activity during this process. Primary cultured rat SCs and TM4 cells were treated with different concentrations of TGF-β3. In some experiments, cells were transfected with siRNA specifically targeting Lgl2 and then treated with TGF-β3 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Lactate concentration, glucose and glutamine (Gln) consumption in the culture medium, activity of phosphofructokinase (PFK), lactate dehydrogenase (LDH), and glutaminase (Gls), ATP level, oxygen consumption, extracellular acidification, and mitochondrial respiration complex activity were detected using commercial kits. The protein level of Lgl2, LDH, monocarboxylate transporter 4 (MCT4), and activity of Akt, ERK, p38 MAPK, and Notch pathway were detected by Western blot. The stage-specific expression of Jagged1 was examined by immunohistochemistry (IHC) and qPCR after laser capture microdissection. Spermatogenesis in rat testis injected with recombinant Jagged1 (re-Jagged1) was observed by HE staining, and lactate concentration in testis lysate was measured at a different day point after re-Jagged1 treatment. Significant enhancement of lactate concentration was detected in a culture medium of both primary SCs and TM4 cells treated with TGF-β3 at 3 or 5ng/ml. Besides, other parameters of glycolysis, that is, glucose and Gln consumption, enzyme activity of PFK, LDH, and Gls displayed different levels of increment in primary SCs and TM4 cells after TGF-β3 treatment. Mitochondria respiration of SCs was shown to decrease in response to TGF-β3. Lgl2, MCT4, activity of ERK, and p38 MAPK were up-regulated, whereas Akt and Notch pathway activity were inhibited by TGF-β3. Silencing of Lgl2 in SCs affected lactate production and attenuated the previous effects of TGF-β3 on SC glycolysis except for Gln consumption, Gls activity, and activity of Akt, ERK, and p38. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment in SCs antagonized glycolysis suppression caused by Lgl2-silencing. In vivo analysis revealed a stage-specific expression of Jagged1 in contrary with TGF-β3. Activating Notch signaling by re-Jagged1 resulted in restorable hypospermatogenesis and lowered lactate level in rat testis. TGF-β3 induces lactate production in SC through up-regulating Lgl2, which weakened the Notch signaling activity and intensified glycolysis in SCs. Thus, besides the known function of TGF-β3 as the BTB regulator, TGF-β3-Lgl2-Notch may be considered an important pathway controlling SC glycolysis and spermatogenesis.

Analysis and pharmacological modulation of senescence in human epithelial stem cells.

Human epithelial stem cells (ESCs) are characterized by long‐term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly‐ectodermal dysplasia‐clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ‐secretase inhibitor; N‐[N‐(3, 5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine T‐butyl ester). All cells were able to differentiate in vitro but exhibited variable self‐renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up‐ or down‐regulated during their lifetime, thus allowing to identify druggable gene networks and off‐the‐shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.

Phenotype transition of fibroblasts incorporated into patient-derived oral carcinoma organoids.

Cancer-associated fibroblasts (CAFs) are abundantly infiltrated in oral squamous cell carcinoma (OSCC), but the contact-dependent mechanisms that regulate CAFs phenotype in precursor cells, such as paracancerous fibroblasts (PFs), remain unclear. Here, a fibroblast-attached organoid (FAO) model was initiated to determine phenotype transition of fibroblasts triggered by contact with OSCC. Organoids and fibroblasts were generated using OSCC and adjacent tissues. Cell-clusters containing fibroblasts and tumour cells were aggregated to allow for FAOs expansion. Immunoblotting assay was performed to compare expression of Notch intracellular domain (NICD) in CAFs and PFs. Colony formation assay was employed to evaluate morphological activation of fibroblasts. Compared to traditional 3D co-culture, FAOs better modulated the spatial distribution of fibroblasts with tumour nests. The presence of CAFs with multiple branches was stably observed in FAOs during serial passage. Incorporation with organoids promoted the ability of PFs to form multiple branches. Immunoblotting assay confirmed higher NICD level in CAFs than PFs. Treatment with Notch inhibitor, N-[N-(3, 5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (i.e. DAPT) blocked morphological activation of fibroblasts incorporated into FAO. We developed a robust strategy to study contact-dependent mechanisms underlying tumour-stromal interaction, and suggested that Notch activity contributes to biogenesis of OSCC-associated fibroblasts.

Notch pathway inhibitor DAPT accelerates in vitro proliferation and adipogenesis in infantile hemangioma stem cells.

The Notch signaling pathway is crucial in both adipogenesis and tumor development. It serves a vital role in the development and stability of blood vessels and may be involved in the proliferative phase of infantile hemangiomas, which express various related receptors. Therefore, it was hypothesized that the Notch signaling pathway inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a γ-secretase inhibitor, might help accelerate the regression of infantile hemangiomas. The present in vitro study evaluated whether inhibition of the Notch signaling pathway using DAPT could alter adipogenesis in hemangioma stem cells (HemSCs) derived from infantile hemangioma (IH) specimens. A total of 20 infants (age, ≤6 months) with hemangiomas who had not yet received any treatment were selected, and their discarded hemangioma tissues were obtained. HemSCs were isolated from the fresh, sterile IH specimens and treated with DAPT. Reverse transcription-quantitative PCR and western blotting were used to demonstrate the inhibition of the Notch signaling pathway by DAPT. A proliferation assay (Cell Counting Kit-8), oil red O staining, flow cytometry and a transwell assay were used to detect proliferation, adipogenesis, apoptosis and migration of HemSCs. Treatment with DAPT upregulated the expression levels of CCAAT/enhancer-binding protein (C/EBP) α, C/EBPβ, peroxisome proliferator-activated receptor-γ, adiponectin and insulin-like growth factor 1, and promoted the proliferation, apoptosis, migration and lipid accumulation in HemSCs in vitro. Targeting the Notch signaling pathway using DAPT may potentially accelerate the regression of infantile hemangiomas.

Validating Multivariate Classification Algorithms in Raman Spectroscopy-Based Osteosarcoma Cellular Analysis

Raman microspectroscopy has been widely demonstrated as an ideal analytical tool for preclinical drug development and clinical applications. However, it is still not easy to accurately identify the subtle spectral variations in different biological samples, which requires a feasible combination between novel spectra collection instrumentation and effective data mining algorithms. In this study, three distinct multivariate classification approaches, which were principal component analysis-linear discriminant analysis (PCA-LDA), support vector machine (SVM), and principal component analysis-support vector machine (PCA-SVM), were validated and compared for obtaining reliable and chemically significant results from the analysis of bio-spectral data. Their performances were evaluated by classifying the spectral characteristics of osteosarcoma cells treated with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) from untreated cells. Based on the discriminated spectral variations, the results indicate that PCA combined with the radial basis function (RBF) kernel SVM model achieved the highest classification accuracy. In general, this study confirms that PCA-SVM algorithm improves the automatic processing accuracy and efficiency of micro-Raman spectroscopy, which may be adopted in further cell screening and analysis applications.