EdU Assay Research Articles

Multiple myeloma (MM) remains incurable, largely owing to the lack of effective therapeutic targets. Wilms’ tumor 1-associated protein (WTAP) has been implicated in MM tumorigenesis, but its underlying molecular mechanisms remain unclear. This study aimed to elucidate the role of WTAP in MM progression. Bone marrow samples from newly diagnosed MM patients were analyzed. WTAP expression was assessed via Quantitative real-time PCR (qRT-PCR) and Western blot. Cell proliferation was evaluated using EdU and flow cytometry assays, while RNA immunoprecipitation (RIP) confirmed protein interactions. WTAP was overexpressed in MM patients and correlated with poor survival. Its knockdown significantly suppressed MM cell proliferation and inflammation. RIP assays identified MAP6D1 as a potential WTAP target. RNA-seq analysis suggested WTAP regulates MM proliferation via the Hippo signaling pathway. WTAP promotes MM proliferation by targeting MAP6D1 and modulating the Hippo pathway, highlighting its potential as a therapeutic target.

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase suppresses glycolysis and proliferation of pulmonary artery smooth muscle cells in hypoxic pulmonary hypertension via inhibition of lactate dehydrogenase A.

Preeclampsia is amultisystem disorder involving in inflammatory responses and metabolic dysfunction of maternal-fetal circulation. Recently, researchers found it threatens renal health of offspring in adulthood. Growing evidence indicated chronic kidney disease is associated with glomeruli deficiencies during intrauterine development. Our previous study showed placenta-derived exosomes from cord plasma with preeclampsia impede fetal glomerular vascularization, during which we postulate microRNAs may function as epigenetic switches for gene silencing of human glomerular endothelial cells. However, the specific miRNAs in placenta-derived exosomes engaged in glomerular vascularization remain unclear. Small RNA sequencing of placental-derived exosomes and bioinformatics analysis were applied to identify differentially expressed miRNAs, followed by real-time polymerase chain reaction for verification. Transient expression and inhibition of candidate miRNA were performed by transfection with chemically synthesized miRNA oligonucleotides. Functional assays of HGECs including cell proliferation assays, EDU assays, migration assays, tube formation assays and monolayer cell barrier permeability assays were performed after transfection. Further, dual luciferase assay was used to explore the target genes of candidate miRNA, followed by RT-qPCR, western blot and rescue assays. Antagomirs transfection of C57BL/6J fetal mice via Amniotic cavity injection in vivo and C57BL/6J fetal mice kidney explants culture in vitro were performed to evaluate number of glomeruli, renal development. Preeclampsia downregulates miR-199a-3p in placenta-derived exosomes from cord plasma. Suppression of endogenous miR-199a-3p in HGECs inhibits angiogenesis, proliferation, migration and permeability. A dual luciferase assay and rescue assays confirmed that miR-199a-3p targets PH domain leucine-rich repeat-containing protein phosphatase 2, regulating the phosphorylation of Akt serine/threonine kinase 1 (S473). C57BL/6J fetal mice with miR-199a-3p downregulation have low glomerulus counts and relative growth rate. miR-199a-3p in placenta-derived exosomes from cord plasma controls VEGF-induced glomerular angiogenesis. Moreover, it provides a distinct perspective for the mechanisms underlying the increased risk for renal disease in the offspring of preeclampsia patients via placenta-derived exosomal miRNAs.

ABSTRACTDespite advances in diagnosis and treatment, the prognosis of non‐small cell lung cancer (NSCLC) remains poor. Therefore, it is urgent to identify potential molecular targets. In this study, we investigated the function and internal mechanism of CPNE3 in the malignant biological behaviour via RACK1/c‐MET signalling in NSCLC, and explored the feasibility of the MET inhibitor in NSCLC treatment. The expression of CPNE3 in normal tissues and lung cancer tissues was compared using a public database. The function of CPNE3 was investigated using CCK‐8 assays, clonogenic assays, EdU assays, Transwell assays and cell cycle analysis. Western blotting was used to detect the protein expression. The interaction between CPNE3 and RACK1 was examined by immunofluorescence staining and co‐immunoprecipitation (co‐IP). The in vitro and in vivo functions of the MET inhibitor JNJ‐38877605 were investigated. CPNE3 is overexpressed in NSCLC and facilitates tumorigenesis and metastasis by interacting with RACK1 through the VWFA domain, which further induces the activation of MET signalling. Accordingly, this process could be suppressed by the MET inhibitor and RACK1 knockdown in vitro and in vivo. CPNE3 is highly expressed in NSCLC and can promote the proliferation and migration of tumour cells. CPNE3 could interact with RACK1 through the VWFA domain and activate MET signalling. These findings may provide new insights into the development of novel therapeutic strategies for NSCLC.

CX-5461 inhibits cell proliferation and induces ferroptosis of colorectal cancer cells by inactivating Nrf2 pathway.

POU2F1 facilitates the malignant phenotypes and aerobic glycolysis of pituitary adenoma by activating LDHA transcription.

The economic value of goose down is attributed to its extensive application in the production of down-based clothing and related products. The primordium formation stage governs the proper morphogenesis of the feather follicle, while the Wnt signaling pathway serves a positive regulatory function during this stage. To identify critical miRNAs and molecular mechanisms regulating the development of goose feather follicle primordium, we performed transcriptomic sequencing of skin tissues collected from six geese at pre- and post-feather follicle primordium developmental stages. Bioinformatics analysis identified 350 differentially expressed miRNAs (DE miRNAs), which were functionally enriched in processes related to system development and multicellular organismal development, etc. As demonstrated by dual-luciferase reporter experiments, miR-200a binds directly to PITX2’s 3′ untranslated region (3′UTR). Furthermore, overexpression of miR-200a decreased the expression levels of genes linked to the Wnt pathway and suppressed the proliferation of GEDFs, as validated by RT-qPCR, CCK8, and EdU assays. Notably, co-transfection experiments demonstrated that miR-200a-mediated regulation of GEDF proliferation through the Wnt pathway is functionally dependent on PITX2. Collectively, this work expands the regulatory network underlying feather follicle development and provides a genetic foundation aimed at breeding geese with enhanced down production quality.

Introduction This study investigated the effects of quercetin on the proliferation and apoptosis of secondary hair follicle stem cells (SHFSCs) isolated from Arbas cashmere goats. Methods SHFSCs were treated with varying quercetin concentrations. CCK-8, EdU assays, and flow cytometry analyses were performed to assess cell proliferation and apoptosis. Transcriptome analyses were used to identify differentially expressed genes and enriched signaling pathways. Results Treatment with 10 μg/mL quercetin for 48 h significantly promoted cell proliferation. The proportion of S-phase cells increased from 15.5% to 21.2%, and the mRNA and protein levels of PCNA and TERT were upregulated. Quercetin inhibited apoptosis by downregulating BAX, TP53, and CASP3, upregulating BCL2, and reducing the number of late apoptotic cells. Mechanistically, quercetin activated the PI3K–Akt, Wnt, and TGF-β signaling pathways, upregulated CCND1 and CDK4 expression, improved mitochondrial membrane potential, reduced ROS levels, and promoted VEGF, FGF, and HGF secretion. Transcriptome analyses revealed that differentially expressed genes were enriched in translational processes, insulin-like growth factor binding, and proliferation-related signaling pathways. Conclusion Quercetin promotes SHFSC proliferation and inhibits apoptosis through multiple pathways, providing a potential regulatory strategy for improving cashmere production in goats.

Exosomes play a crucial role in triple-negative breast cancer (TNBC), influencing various aspects of tumor progression. Given the importance of exosomes in TNBC biology, we proposed a novel exosome-based model that was critically implicated in TNBC. Bulk and single-cell transcriptomics and genetic mutations of TNBC patients were curated for our study. Characteristic exosome genes were selected via LASSO analysis, with subsequent construction of an exosome-based model. The effectiveness in estimating clinical outcomes and treatment responses was then evaluated and validated. MDA-MB-231 and MDA-MB-468 TNBC cells were transiently transfected with FAM129B siRNAs, and cell proliferation and migration were measured via EdU and wound healing assays. The study determined 7 characteristic exosome genes for TNBC: ALCAM, FAM129B, GNB2, KRT6A, PGK1, SERPINE1, and THY1, which were utilized for defining the exosome-based gene signature. It was proven that the signature accurately estimated patient prognosis, and functioned as an independent prognostic predictor. High-risk tumors owned shorter overall survival time, but were suitable for treatment with docetaxel and several small-molecule agents (MK-0752, BRD-K33199242, IC-87114, fumonisin B1, ilomastat, GW-788388, afobazole, and batimastat). High- and low-risk tumors presented the distinct genetic mutation characteristics. The characteristic exosome genes were specifically expressed in the TNBC microenvironment components, indicating their involvement in modulating the microenvironment. High-risk individuals were inferred to better respond to immune checkpoint blockade (CD276, NRP1, TNFRSF4, TNFSF4 or CTLA4). Experimentally, inhibition of FAM129B effectively attenuated proliferative and aggressive phenotypes of TNBC cells. Collectively, our findings proposed the exosome-based gene signature for accurate estimation of clinical outcomes and assisting in individually tailoring therapies in TNBC as well as discovered FAM129B as a potential therapeutic target.

MUC3A is upregulated in the serum of patients with extrahepatic cholangiocarcinoma (EHCC). Nevertheless, the specific role of MUC3A in EHCC is elusive. Therefore, this study aimed to analyze the role and mechanism of MUC3A in cell proliferation and epithelial-mesenchymal transition (EMT) in EHCC. UCHL1 and MUC3A expression levels were measured in EHCC cells and tissues. After gain- and loss-of-function assays in EHCC cells, cell proliferation was assessed with CCK-8, EdU, and colony formation assays, while cell invasion, migration, and apoptosis were examined with transwell, scratch, and flow cytometry assays, respectively. Western blot was implemented to detect the levels of migration- and EMT-related markers, as well as proteins related to the downstream pathways of EGFR. A xenograft tumor model was established for in vitro validation. The relationship between UCHL1 and MUC3A was analyzed with Co-immunoprecipitation (Co-IP), Western blotting, ubiquitination analysis, and immunofluorescence. UCHL1 and MUC3A were highly expressed in EHCC. MUC3A knockdown inhibited the viability, EMT, migration, and invasion of ECHH cells while accelerating their apoptosis. MUC3A knockdown repressed tumorigenesis of EHCC in mice. Mechanistically, UCHL1 acted as a deubiquitinase to maintain MUC3A stability and activated the downstream pathways of EGFR, namely Ras/Raf/MEK/ERK and PI3K/AKT. UCHL1 overexpression nullified the repressive impacts of MUC3A knockdown on EHCC cell viability, invasion, and EMT, while EGFR activation abrogated the suppressive impacts of UCHL1 depletion on EHCC cell viability, invasion, and EMT. UCHL1 activates downstream pathways of EGFR by stabilizing MUC3A, thereby fostering EHCC progression.

To investigate the mechanism by which melatonin influences melanin production in goat melanocytes, and to provide a theoretical foundation for the genetic principles underlying animal hair color formation, dorsal skin tissues were collected approximately 5cm behind the shoulder of 4-day-old Jiaohe black goats. Melanocytes were localized using immunohistochemistry and immunofluorescence techniques, isolated from hair follicle tissues through enzyme digestion, and treated with different concentrations of melatonin. Cell viability, proliferation, apoptosis, and melanin synthesis were assessed using CCK8, EdU, flow cytometry, and melanin content assays, respectively. Melanocytes were subjected to transcriptome sequencing, differential gene screening, and functional enrichment analyses. The expression of relevant genes and proteins was verified using real-time quantitative PCR and western blotting, respectively. Melanocytes were determined to be located above the hair follicle papilla and in the outer root sheath, and were successfully isolated and cultured. Melatonin inhibited melanocyte proliferation, promoted apoptosis, and inhibited melanin synthesis. After RNA sequencing, 952 and 1300 differentially expressed genes (DEGs) were screened by comparing the two groups respectively, which were mainly enriched in Melanogenesis and the Wnt signaling pathway. Validation experiments confirmed that melatonin inhibited the expression of genes and proteins related to the Wnt/β-catenin signaling pathway. Notably, treatment with an agonist of the Wnt/β-catenin pathway attenuated the inhibitory effect of melatonin. This study reveals that melatonin can reduce melanocyte viability, inhibit proliferation, promote apoptosis, reduce melanogenesis, and affect melanin synthesis through the Wnt/β-catenin signaling pathway. This study provides a theoretical basis for understanding the regulatory role of melatonin in hair color formation in animals.

Three-dimensional printed PCL/nHA/SA/COL structure-mediated M2 macrophage polarization enhances angiogenesis and osteogenic differentiation of bone marrow mesenchymal stem cells.

Curculigoside mediates the STAT3/PD-L1 pathway to inhibit the proliferation, invasion, and immune escape of breast cancer cells.

Myocardial fibrosis has been found to accelerate heart dysfunction after myocardial infarction (MI). Butein is a chalcone compound possessing multiple biological properties. However, its effect on MI-induced myocardial fibrosis remains unclear. A mouse MI model was established by left anterior descending ligation. Human cardiac fibroblasts (HCFs) were stimulated with TGF-β1 in vitro. Mouse cardiac function was evaluated by assessing EF and FS. Masson's trichrome staining showed the fibrosis area in murine hearts. Western blotting evaluated protein levels of fibrosis markers and signaling-related markers. CCK-8, EdU, and Transwell assays were used to evaluate HCF proliferation and migration. Butein improved MI-induced cardiac dysfunction and reduced the fibrosis area in mice. Butein inactivated TGF-β1/Smad signaling in MI mice and TGF-β1-stimulated HCFs. Butein inhibited TGF-β1-induced proliferation, migration, and collagen synthesis in HCFs, which were similar to the effects of LY2109761, a pharmacological inhibitor of TGF-β signaling. Butein mitigated MI development by inhibiting cardiac fibrosis and ECM deposition by inactivating the TGF-β1/Smad signaling pathway.

PurposeThe unlimited proliferation of breast cancer (BC) cells is the basis for recurrence and metastasis. Ambra1 is involved in the regulation of cell proliferation, but its role may be cancer type-dependent, and the underlying mechanisms need further exploration. In addition, it remains unclear whether Ambra1 is involved in regulating the proliferation of BC cells. This study aims to explore the regulatory effect of Ambra1 on the proliferation of BC cells, as well as the underlying mechanisms.MethodsThe effects of Ambra1 on cell proliferation were detected in MCF-7 and MDA-MB-231 cells using CCK-8, EdU, and colony formation assays. The role of Ambra1 in regulating p27 via the Akt-FoxO1 pathway was determined in MCF-7, MDA-MB-231, and 293T cells through Western blotting, qRT-PCR, and co-immunoprecipitation. Subsequently, the role of p27 in Ambra1-mediated regulation of cell proliferation was validated in cell models and xenograft mouse models.ResultsAmbra1 deficiency significantly inhibited the proliferation of BC cells. p27 played a crucial role in this process. Furthermore, Ambra1 regulates the phosphorylation of the Ser256 residue of FoxO1 through Akt, thereby altering the nuclear distribution of FoxO1 and the transcription of p27.ConclusionAmbra1 can control the proliferation of BC cells by regulating the Akt-FoxO1-p27 signaling pathway. Therefore, this protein is a potential therapeutic target for BC.

In poultry production, the laying rate is a critical economic trait, as high egg production significantly enhances profitability. Mitogen-activated protein kinase-activated protein kinase 3 (MK3) is a member of the mitogen-activated protein kinase (MAPK) family, which plays an important role in follicular development. Our previous RNA-seq analysis revealed that MK3 expression was significantly altered in the ovaries of laying hens exposed to normal versus light-deprivation conditions. Based on previous RNA-seq analysis of chicken ovaries, this study focused on the MK3 gene to explore its role in regulating apoptosis of follicular granulosa cells in laying hens. The results demonstrated that MK3 overexpression induced granulosa cell apoptosis by modulating the expression of key proliferation- and apoptosis-related genes, including FAS, Caspase3, BCL2, and C-myc. These findings were further validated using specific siRNA-mediated knockdown of MK3. Flow cytometry, CCK-8, and EdU assays consistently showed that MK3 facilitated apoptosis and inhibited granulosa cell proliferation. Additionally, dual-luciferase reporter assays revealed that the transcription factor WT1 bound to the MK3 promoter and enhanced its transcriptional activity. Mechanistically, MK3 regulated granulosa cell apoptosis through the TNF/P38 MAPK pathway. This conclusion was corroborated by treatment with the P38 inhibitor GS-444217 and specific siRNA targeting components of the pathway. In summary, MK3 promotes granulosa cell apoptosis in the follicles of laying hens, is transcriptionally regulated by WT1, and exerts its pro-apoptotic effects via the TNF/P38 MAPK pathway.

KIAA0319 plays a critical role in cortical neuronal maturation and synaptic development through a dyslexia-associated gene network.

Although gemcitabine (GEM) is the standard of care for most patients with pancreatic cancer (PC), its efficacy is limited by resistance development. Furthermore, p21-activated kinase-1 (PAK1) has been demonstrated to be involved in regulating the development of PC with GEM resistance. This study is designed to explore the role and mechanism of PAK1 in the GEM resistance of PC cells. PAK1, ubiquitin-specific peptidase 33 (USP33), methyltransferase-like 3 (METTL3), and insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) mRNA levels were detected using RT-qPCR. PAK1, MDR1, MRP1, USP33, METTL3, and IGF2BP3 protein levels were examined by western blot. GEM resistance, cell viability, proliferation, apoptosis, invasion, and migration were assessed using MTT, EdU, flow cytometry, transwell, and wound healing assays. After ubibrowser database analysis, the interaction between USP33 and PAK1 was verified using co-immunoprecipitation (Co-IP) assay. Meanwhile, the interaction between METTL3 and USP33 m6A was analyzed using methylated RNA immunoprecipitation (MeRIP)-qPCR and RNA immunoprecipitation (RIP) assay. A xenograft model analyzed the effects of PAK1 on GEM resistance of PC in vivo. PAK1 was upregulated in GEM-resistant PC tissues and cells. PAK1 knockdown enhanced cell sensitivity to GEM; repressed cell proliferation, invasion, and migration; and induced cell apoptosis in vitro. Mechanistically, USP33 triggered the deubiquitination of PAK1 and prevented its degradation. METTL3 stabilized USP33 mRNA through the m6A-IGF2BP3-dependent mechanism and naturally increased USP33 expression. USP33 silencing increased the drug sensitivity of PC in vivo. METTL3 supports GEM resistance of PC cells partly by regulating USP33-mediated PAK1 deubiquitination, providing a promising therapeutic target for GEM-resistant PC cells.

BackgroundEmerging evidence showed that ubiquitin-specific proteases (USPs) are promising therapeutic targets for hematopoietic malignancies. The roles of USP34 on acute myeloid leukemia (AML) pathogenesis remain completely unknown.MethodsGene expression and prognostic relevance were analyzed utilizing public AML datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA), and validated with the Gene Expression Profiling Interactive Analysis (GEPIA), BloodSpot and the human protein atlas (HPA) databases. Systematical analyses are performed to characterize the immune infiltration and biological function of USP34 in AML. MTS, colony formation and EdU assays were also employed to confirm the effect of USP34 on cell proliferation in AML cells.ResultsUSP34 expression was significantly higher in AML compared to its matched normal samples. Higher USP34 expression predicted worse prognosis in AML, and USP34 acted as an independent prognostic predictor for AML patients (HR:1.365, 95%CI:1.032–1.805, p = 0.029). Moreover, the expression of USP34 was associated with several immune cells, including Treg cells, indicating its potential role in modulating immune responses. Finally, functional experiments revealed that USP34 knockdown reduced cell proliferation in Molm-13 and Kasumi cells.ConclusionsUSP34 is an independent prognostic predictor in AML and may contribute to leukemogenesis via intervening immune processes and promoting proliferation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12672-025-03662-1.

The current study aimed to explore the effect of Cadmium (Cd) on nucleus pulposus derived mesenchymal stem cells (NPMSCs) and the possible mechanism of IVDD caused by Cd. In this study, cell viability assay, EdU assay, TUNEL staining, flow cytometry assay, mRNA transcriptome sequencing, quantitative real-time polymerase chain reaction (PCR) assay, immunofluorescence assay and western blot assay were used to prove that Cadmium induces apoptosis of NPMSCs. Cd impaired the proliferation of NPMSCs and promoted cell apoptosis, and this effect was time and concentration dependent. Further study also found that the expression levels of senescence-related molecules (P16, P21 and P53) in the Cd group were up-regulated and the expression levels of pro-apoptotic molecules Bax and Caspase-3 in the Cd group were significantly up-regulated, while the expression level of anti-apoptotic molecule Bcl-2 was significantly down-regulated compared with those of the Control group. The MAPK signaling pathway-related proteins were detected, and the results found that the ratios of p-P38/P38 and p-JNK/JNK in the Cd group were significantly increased, while the ratios of p-ERK/ERK was significantly less compared with the control group, and it was in a concentration-dependent relationship. Cd can inhibit the activity and proliferation of NPMSCs in a dose and time-dependent manner, and promote cell aging and apoptosis. Cd may promote the apoptosis of NPMSCs by activating MAPK signaling pathway.