Cultured Mouse Embryonic Fibroblasts Research Articles

IF1 Promotes Cellular Proliferation and Inhibits Oxidative Phosphorylation in Mouse Embryonic Fibroblasts under Normoxia and Hypoxia.

ATP synthase inhibitory factor subunit 1 (IF1) is an inhibitory subunit of mitochondrial ATP synthase, playing a crucial role in regulating mitochondrial respiration and energetics. It is well-established that IF1 interacts with the F1 sector of ATP synthase to inhibit the reversal rotation and, thus, ATP hydrolysis. Recent evidence supports that IF1 also inhibits forward rotation or the ATP synthesis activity. Adding to the complexity, IF1 may also facilitate mitophagy and cristae formation. The implications of these complex actions of IF1 for cellular function remain obscure. In the present study, we found that IF1 expression was markedly upregulated in hypoxic MEFs relative to normoxic MEFs. We investigate how IF1 affects cellular growth and function in cultured mouse embryonic fibroblasts derived from mouse lines with systemic IF1 overexpression and knockout under normoxia and hypoxia. Cell survival and proliferation analyses revealed that IF1 overexpression exerted limited effects on cellular viability but substantially increased proliferation under normoxia, whereas it facilitated both cellular viability and proliferation under hypoxia. The absence of IF1 may have a pro-survival effect but not a proliferative one in both normoxia and hypoxia. Cellular bioenergetic analyses revealed that IF1 suppressed cellular respiration when subjected to normoxia and was even more pronounced when subjected to hypoxia with increased mitochondrial ATP production. In contrast, IF1 knockout MEFs showed markedly increased cellular respiration under both normoxia and hypoxia with little change in mitochondrial ATP. Glycolytic stress assay revealed that IF1 overexpression modestly increased glycolysis in normoxia and hypoxia. Interestingly, the absence of IF1 in MEFs led to substantial increases in glycolysis. Therefore, we conclude that IF1 mainly inhibits cellular respiration and enhances cellular glycolysis to preserve mitochondrial ATP. On the other hand, IF1 deletion can significantly facilitate cellular respiration and glycolysis without leading to mitochondrial ATP deficit.

The circadian regulator PER1 promotes cell reprogramming by inhibiting inflammatory signaling from macrophages.

Circadian regulation of gene expression is prevalent and plays critical roles in cell differentiation. However, its roles in the reprogramming of differentiated cells remain largely unknown. Here, we found that one of the master circadian regulators PER1 promoted virus-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to induced neurons (iNs) and induced pluripotent stem cells (iPSCs). Unexpectedly, PER1 achieved this by repressing inflammatory activation of contaminating macrophages in the MEF culture, rather than by directly modulating the reprogrammability of MEFs. More specifically, we found that transduced viruses activated inflammatory genes in macrophages, such as Tnf encoding TNFα, one of the central inflammatory regulators and an autocrine activator of macrophages. TNFα inhibited iN reprogramming, whereas a TNFα inhibitor promoted iN reprogramming, connecting the inflammatory responses to iN reprogramming. In addition, macrophages were induced to proliferate and mature by non-macrophage cells serving as feeders, which also supported up-regulation of TNFα in macrophages without virus transduction. Furthermore, the 2 inflammatory responses were repressed by the circadian regulator PER1 in macrophages, making reprogrammability dependent on time-of-day of virus transduction. Similar results were obtained with iPSC reprogramming, suggesting a wide occurrence of macrophage-mediated inhibition of cell reprogramming. This study uncovers mechanistic links between cell reprogramming, bystander inflammatory macrophages, and circadian rhythms, which are particularly relevant to in vivo reprogramming and organoid formation incorporating immune cells.

Imaging Subcellular AMPK Activity Using an Excitation‐Ratiometric AMPK Activity Reporter

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a master regulator of cellular metabolism, phosphorylating a variety of downstream targets throughout the cell. Subcellular AMPK activity results in regulation of glycolysis, lipid and protein biosynthesis, mitochondrial function, and gene expression. But how AMPK senses and responds to stimuli in a compartment-specific manner is not well understood, leaving an incomplete picture of compartmentalized AMPK activity. Key tools for studying subcellular AMPK activity are genetically encoded AMPK activity reporters (AMPKARs), which allow for the quantitative visualization of subcelluar AMPK activity. However, many AMPKARs suffer from poor dynamic range and sensitivity, limiting their application. I recently reported the development of a new excitation-ratiometric (ExRai) AMPKAR, a single-fluorophore AMPKAR with enhanced dynamic range for detection of subtle, subcellular AMPK activity. I used ExRai AMPKAR to study subcellular AMPK activity at several locations, including the lysosome and mitochondria, identifying new mechanisms for the regulation of AMPK activity. Here, I describe the use of ExRai AMPKAR to image subcellular AMPK activity in mouse embryonic fibroblasts using both widefield and confocal microscopy. I also describe the culture of mouse embryonic fibroblasts. Through the use of ExRai AMPKAR, subcellular AMPK activity can be illuminated to better understand how this central kinase regulates cellular metabolism. © 2023 The Author. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Imaging subcellular AMPK activity using ExRai AMPKAR Support Protocol 1: Culturing of mouse embryonic fibroblasts for live-cell imaging Support Protocol 2: Live-cell imaging of ExRai AMPKAR using confocal microscopy.

Shared and Distinctive Neighborhoods of Emerin and Lamin B Receptor Revealed by Proximity Labeling and Quantitative Proteomics

Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here, we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts. Our analysis revealed 232 high confidence proximity partners that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

CSN5‐mediated nuclear exclusion of p27 in vascular smooth muscle cells contributes to the exacerbation of neointimal hyperplasia by CSN8 hypomorphism

BackgroundNeointimal hyperplasia is a common pathological response to vascular injury and is caused by the proliferation and migration of vascular smooth muscle cells (VSMCs). The COP9 signalosome (CSN) is a highly conserved protein complex composed of 8 unique protein subunits (CSN1 through CSN8) that functions as the deneddylase for Cullin and thereby regulates ubiquitination. However, CSN subunits may have deneddylation‐independent function. Downregulation of CSN8 was shown to accelerate the growth rate of cultured mouse embryonic fibroblasts but impaired cardiac protein quality control in mice. Despite the strong evidence linking CSN to cell cycle regulation, direct study of CSN in VSMC proliferation is lacking. To fill this critical gap, we performed the present study to test the hypothesis that increased CSN5‐mediated nuclear exclusion of p27 contributes to the promotion of VSMC proliferation in injured vessels by CSN8 hypomorphism (Figure 1).Methods and ResultsTo produce neointimal hyperplasia in vivo, we subjected adult CSN8 hypomorphic (CSN8‐hypo) and the littermate non‐hypomorphic control mice (CTL) to left common carotid artery (LCCA) ligation and collected the LCCA segment proximal to the ligation for analyses. LCCA ligation induced significant increases in CSN8 proteins in wild type mice (p=0.0109), suggesting an important role of the CSN in vascular pathology. Compared to CTL, the CSN8‐hypo mice displayed more severe neointimal thickening at both 1‐ and 4‐week after ligation, a markedly greater increase in PCNA (proliferating cell nuclear antigen) as revealed by western blot analyses (2.05±0.46 vs. 1.37±0.098, p=0.0214), and a significantly greater prevalence of Ki67‐positive VSMCs as detected by double immunostaining for Ki67 and Sm‐22 at 1 week after LCCA ligation (p=0.005). In cell cultures, in response to platelet derived growth factor (PDGF‐BB), CSN8‐hypo VSMCs displayed an accelerated G0/G1 progression (p=0.04) and greater proliferation as assessed by measuring PCNA (p=0.015) and MTT assays (p=0.009), compared to CTL VSMCs. Furthermore, nuclear fractionation revealed that the nuclear to cytoplasmic ratio for p27 and CSN5 proteins were markedly smaller in hypomorphic mice (p=0.0038; p= 0.0246) and hypomorphic VSMCs (p=0.0045; p=0.027); and native gel electrophoresis revealed a significant increase in the cytoplasmic CSN5 mini complex or free CSN5 by CSN8 hypomorphism (in vivo, in vitro; p<0.05). Interestingly, significant increases in PCNA by CSN8 hypomorphism was attenuated by nuclear export inhibitor leptomycin B (LMB) (p=0.04) but not by CSN deneddylase inhibitor (CSN5i‐3). CSN5i‐3, however, suppressed the PDGF‐BB induced PCNA increases in control VSMCs. This suggests that nuclear export (of p27) rather than changes in CSN deneddylation activity is responsible for greater PDGF‐BB induced PCNA increases in CSN8‐hypo VSMCs.ConclusionCSN8 hypomorphism increases VSMC proliferation and promotes neointimal hyperplasia in injured arteries likely through the nuclear exclusion of p27 protein that is mediated by CSN5.

Lrwd1 impacts cell proliferation and the silencing of repetitive DNA elements.

SummaryLRWD1, also known as ORCA, is a nuclear protein functioning in multiple biological processes. Using its WD40 domain LRWD1 interacts with repressive histone marks and maintains the silencing of heterochromatin regions in mammalian cells. ORCA also associates with the origin recognition complex (ORC) and facilitates prereplication complex formation at late‐replicating origins. However, whether LRWD1 plays a role during development and the functional significance of LRWD1 in vivo remains largely unknown. Using gene‐trap approach we generated Lrwd1 knockout mice and examined the expression of Lrwd1 during embryonic development. We found that Lrwd1 is ubiquitously expressed in the majority of the developing mouse embryo. Depletion of LRWD1 did not affect embryonic development but the postnatal growth of the homozygous mutants is retarded. In vitro cultured mouse embryonic fibroblasts (MEFs) depleted of LRWD1 displayed a reduced proliferation compared to wild type cells. We also showed that the knockout of Lrwd1 in MEFs increased the expression of the epigenetically silenced repetitive elements but with minimal effect on the expression of protein coding genes. Together, these results suggest that LRWD1 plays an important, but not essential, role in postnatal development by regulating cell proliferation likely through modulating DNA replication.

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a mouse model of muscular dystrophy.

SummaryGenetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most commonly inherited muscle disease. Here, we report on reprogramming of fibroblasts from two DMD mouse models into induced myogenic progenitor cells (iMPCs) by MyoD overexpression in concert with small molecule treatment. DMD iMPCs proliferate extensively, while expressing myogenic stem cell markers including Pax7 and Myf5. Additionally, DMD iMPCs readily give rise to multinucleated myofibers that express mature skeletal muscle markers; however, they lack DYSTROPHIN expression. Utilizing an exon skipping-based approach with CRISPR/Cas9, we report on genetic correction of the dystrophin mutation in DMD iMPCs and restoration of protein expression in vitro. Furthermore, engraftment of corrected DMD iMPCs into the muscles of dystrophic mice restored DYSTROPHIN expression and contributed to the muscle stem cell reservoir. Collectively, our findings report on a novel in vitro cellular model for DMD and utilize it in conjunction with gene editing to restore DYSTROPHIN expression in vivo.

Sulfation of glycosaminoglycans depends on the catalytic activity of lithium-inhibited phosphatase BPNT2 in vitro

Golgi-resident bisphosphate nucleotidase 2 (BPNT2) is a member of a family of magnesium-dependent, lithium-inhibited phosphatases that share a three-dimensional structural motif that directly coordinates metal binding to effect phosphate hydrolysis. BPNT2 catalyzes the breakdown of 3′-phosphoadenosine-5′-phosphate, a by-product of glycosaminoglycan (GAG) sulfation. KO of BPNT2 in mice leads to skeletal abnormalities because of impaired GAG sulfation, especially chondroitin-4-sulfation, which is critical for proper extracellular matrix development. Mutations in BPNT2 have also been found to underlie a chondrodysplastic disorder in humans. The precise mechanism by which the loss of BPNT2 impairs sulfation remains unclear. Here, we used mouse embryonic fibroblasts (MEFs) to test the hypothesis that the catalytic activity of BPNT2 is required for GAG sulfation in vitro. We show that a catalytic-dead Bpnt2 construct (D108A) does not rescue impairments in intracellular or secreted sulfated GAGs, including decreased chondroitin-4-sulfate, present in Bpnt2-KO MEFs. We also demonstrate that missense mutations in Bpnt2 adjacent to the catalytic site, which are known to cause chondrodysplasia in humans, recapitulate defects in overall GAG sulfation and chondroitin-4-sulfation in MEF cultures. We further show that treatment of MEFs with lithium (a common psychotropic medication) inhibits GAG sulfation and that this effect depends on the presence of BPNT2. Taken together, this work demonstrates that the catalytic activity of an enzyme potently inhibited by lithium can modulate GAG sulfation and therefore extracellular matrix composition, revealing new insights into lithium pharmacology.

Loss of the transcriptional repressor Rev-erb\u03b1 upregulates metabolism and proliferation in cultured mouse embryonic fibroblasts

The transcriptional repressor Rev-erbα is known to down-regulate fatty acid metabolism and gluconeogenesis gene expression. In animal models, disruption of Rev-erbα results in global changes in exercise performance, oxidative capacity, and blood glucose levels. However, the complete extent to which Rev-erbα-mediated transcriptional repression of metabolism impacts cell function remains unknown. We hypothesized that loss of Rev-erbα in a mouse embryonic fibroblast (MEF) model would result in global changes in metabolism. MEFs lacking Rev-erbα exhibited a hypermetabolic phenotype, demonstrating increased levels of glycolysis and oxidative phosphorylation. Rev-erbα deletion increased expression of hexokinase II, transketolase, and ribose-5-phosphate isomerase genes involved in glycolysis and the pentose phosphate pathway (PPP), and these effects were not mediated by the transcriptional activator BMAL1. Upregulation of oxidative phosphorylation was not accompanied by an increase in mitochondrial biogenesis or numbers. Rev-erbα repressed proliferation via glycolysis, but not the PPP. When treated with H2O2, cell viability was reduced in Rev-erbα knockout MEFs, accompanied by increased ratio of oxidized/reduced NADPH, suggesting that perturbation of the PPP reduces capacity to mount an antioxidant defense. These findings uncover novel mechanisms by which glycolysis and the PPP are modulated through Rev-erbα, and provide new insights into how Rev-erbα impacts proliferation.

Arginyltransferase (Ate1) regulates the RGS7 protein level and the sensitivity of light-evoked ON-bipolar responses

Regulator of G-protein signaling 7 (RGS7) is predominately present in the nervous system and is essential for neuronal signaling involving G-proteins. Prior studies in cultured cells showed that RGS7 is regulated via proteasomal degradation, however no protein is known to facilitate proteasomal degradation of RGS7 and it has not been shown whether this regulation affects G-protein signaling in neurons. Here we used a knockout mouse model with conditional deletion of arginyltransferase (Ate1) in the nervous system and found that in retinal ON bipolar cells, where RGS7 modulates a G-protein to signal light increments, deletion of Ate1 raised the level of RGS7. Electroretinographs revealed that lack of Ate1 leads to increased light-evoked response sensitivities of ON-bipolar cells, as well as their downstream neurons. In cultured mouse embryonic fibroblasts (MEF), RGS7 was rapidly degraded via proteasome pathway and this degradation was abolished in Ate1 knockout MEF. Our results indicate that Ate1 regulates RGS7 protein level by facilitating proteasomal degradation of RGS7 and thus affects G-protein signaling in neurons.

FOXO3a acts to suppress DNA double-strand break-induced mutations.

Genomic instability is one of the hallmarks of aging, and both DNA damage and mutations have been found to accumulate with age in different species. Certain gene families, such as sirtuins and the FoxO family of transcription factors, have been shown to play a role in lifespan extension. However, the mechanism(s) underlying the increased longevity associated with these genes remains largely unknown and may involve the regulation of responses to cellular stressors, such as DNA damage. Here, we report that FOXO3a reduces genomic instability in cultured mouse embryonic fibroblasts (MEFs) treated with agents that induce DNA double‐strand breaks (DSBs), that is, clastogens. We show that DSB treatment of both primary human and mouse fibroblasts upregulates FOXO3a expression. FOXO3a ablation in MEFs harboring the mutational reporter gene lacZ resulted in an increase in genome rearrangements after bleomycin treatment; conversely, overexpression of human FOXO3a was found to suppress mutation accumulation in response to bleomycin. We also show that overexpression of FOXO3a in human primary fibroblasts decreases DSB‐induced γH2AX foci. Knocking out FOXO3a in mES cells increased the frequency of homologous recombination and non‐homologous end‐joining events. These results provide the first direct evidence that FOXO3a plays a role in suppressing genome instability, possibly by suppressing genome rearrangements.

Novel Insights into the Role of UBE3A in Regulating Apoptosis and Proliferation.

The UBE3A gene codes for a protein with two known functions, a ubiquitin E3-ligase which catalyzes ubiquitin binding to substrate proteins and a steroid hormone receptor coactivator. UBE3A is most famous for its critical role in neuronal functioning. Lack of UBE3A protein expression leads to Angelman syndrome (AS), while its overexpression is associated with autism. In spite of extensive research, our understanding of UBE3A roles is still limited. We investigated the cellular and molecular effects of Ube3a deletion in mouse embryonic fibroblasts (MEFs) and Angelman syndrome (AS) mouse model hippocampi. Cell cultures of MEFs exhibited enhanced proliferation together with reduced apoptosis when Ube3a was deleted. These findings were supported by transcriptome and proteome analyses. Furthermore, transcriptome analyses revealed alterations in mitochondria-related genes. Moreover, an analysis of adult AS model mice hippocampi also found alterations in the expression of apoptosis- and proliferation-associated genes. Our findings emphasize the role UBE3A plays in regulating proliferation and apoptosis and sheds light into the possible effects UBE3A has on mitochondrial involvement in governing this balance.

Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts.

Recent evidence indicates that activation of adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and modulator of cellular energy and redox, regulates cell mitosis. However, the underlying molecular mechanisms for AMPKα subunit regulation of chromosome segregation remain poorly understood. This study aimed to ascertain if AMPKα1 deletion contributes to chromosome missegregation by elevating Polo-like kinase 4 (PLK4) expression. Centrosome proteins and aneuploidy were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J) or AMPKα1 homozygous deficient (AMPKα1−/−) mice by Western blotting and metaphase chromosome spread. Deletion of AMPKα1, the predominant AMPKα isoform in immortalized MEFs, led to centrosome amplification and chromosome missegregation, as well as the consequent aneuploidy (34–66%) and micronucleus. Furthermore, AMPKα1 null cells exhibited a significant induction of PLK4. Knockdown of nuclear factor kappa B2/p52 ameliorated the PLK4 elevation in AMPKα1-deleted MEFs. Finally, PLK4 inhibition by Centrinone reversed centrosome amplification of AMPKα1-deleted MEFs. Taken together, our results suggest that AMPKα1 plays a fundamental role in the maintenance of chromosomal integrity through the control of p52-mediated transcription of PLK4, a trigger of centriole biogenesis.

PKN1 controls the aggregation, spheroid formation, and viability of mouse embryonic fibroblasts in suspension culture

The role of protein kinase N1 (PKN1) in cell aggregation and spheroid formation was investigated using mouse embryonic fibroblasts (MEFs) deficient in kinase activity caused by a point mutation (T778A) in the activation loop. Wild type (WT) MEFs formed cell aggregates within a few hours in suspension cultures placed in poly-2-hydroxyethylmethacrylate (poly-HEMA) coated flat-bottom dishes. By contrast, PKN1[T778A] (PKN1 T778A/T778A homozygous knock-in) MEFs showed significantly delayed aggregate formation and higher susceptibility to cell death. Video analysis of suspension cultures revealed decreased cell motility and lesser frequency of cell-cell contact in PKN1[T778A] MEFs compared to that in WT MEFs. Aggregate formation of PKN1[T778A] MEFs was compensated by shaking the cell suspension. When cultured in U-shaped ultra-low attachment well plates, initially larger-sized and loosely packed aggregates of WT MEFs underwent compaction resulting in a single round spheroid. On the other hand, image-based quantitative analysis of PKN1[T778A] MEFs revealed irregular compaction with decreased roundness, solidity, and sphericity within 24 h. Flow cytometry of PKN1[T778A] MEFs revealed decreased surface-expression of N-cadherin and integrins α5 and αV. These results suggest that kinase activity of PKN1 controls cell aggregation and spheroid compaction in MEF suspension culture, possibly by regulating the cell migration and cell-surface expression of N-cadherin and integrins.

Effects of substrate patterning on cellular spheroid growth and dynamics measured by gradient light interference microscopy (GLIM).

The development of three-dimensional (3D) cellular architectures during development and pathological processes involves intricate migratory patterns that are modulated by genetics and the surrounding microenvironment. The substrate composition of cell cultures has been demonstrated to influence growth, proliferation and migration in 2D. Here, we study the growth and dynamics of mouse embryonic fibroblast cultures patterned in a tissue sheet which then exhibits 3D growth. Using gradient light interference microscopy (GLIM), a label-free quantitative phase imaging approach, we explored the influence of geometry on cell growth patterns and rotational dynamics. We apply, for the first time to our knowledge, dispersion-relation phase spectroscopy (DPS) in polar coordinates to generate the radial and rotational cell mass-transport. Our data show that cells cultured on engineered substrates undergo rotational transport in a radially independent manner and exhibit faster vertical growth than the control, unpatterned cells. The use of GLIM and polar DPS provides a novel quantitative approach to studying the effects of spatially patterned substrates on cell motility and growth.

Immobilization of boron-rich compound on Fe3O4 nanoparticles: Stability and cytotoxicity

Magnetic nanoparticles based on Fe3O4 and their modifications of surface with therapeutic substances are of great interest, especially drug delivery for cancer therapy includes boron-neutron capture therapy. The results of boron-rich compound (carborane borate) attachment to previously aminated by (3-aminopropyl)-trimethoxysilane iron oxide nanoparticles are presented. Energy-dispersive X-ray analysis and Fourier transform infrared spectroscopy with attenuated total reflection (ATR) accessory confirmed change of nanoparticles elemental content after modification and formation of new bond between Fe3O4 and attached molecules. Scanning and transmission electron microscopy showed that Fe3O4 nanoparticles average size is 18.9 nm. Phase parameters were investigated by powder X-ray diffraction, Fe3O4 nanoparticles magnetic behavior was evaluated by Mössbauer spectroscopy. Chemical and colloidal stability was studied using simulated body fluid (phosphate buffer – PBS). Modified nanoparticles have excellent stability in PBS (pH = 7.4), characterized by X-ray diffraction, Mössbauer spectroscopy and dynamic light scattering. Fe3O4 biocompatibility was elucidated in-vitro using cultured mouse embryonic fibroblasts. The obtained results show the increasing of IC50 from 0.110 mg/ml for Fe3O4 to 0.405 mg/ml for Fe3O4-Carborane nanoparticles. Obtained data confirm biocompatibility and stability of synthesized nanoparticles and potential to use them in boron-neutron capture therapy.

Fe₃O₄ Nanoparticles for Complex Targeted Delivery and Boron Neutron Capture Therapy.

Magnetic Fe3O4 nanoparticles (NPs) and their surface modification with therapeutic substances are of great interest, especially drug delivery for cancer therapy, including boron-neutron capture therapy (BNCT). In this paper, we present the results of boron-rich compound (carborane borate) attachment to previously aminated by (3-aminopropyl)-trimethoxysilane (APTMS) iron oxide NPs. Fourier transform infrared spectroscopy with Attenuated total reflectance accessory (ATR-FTIR) and energy-dispersive X-ray analysis confirmed the change of the element content of NPs after modification and formation of new bonds between Fe3O4 NPs and the attached molecules. Transmission (TEM) and scanning electron microscopy (SEM) showed Fe3O4 NPs’ average size of 18.9 nm. Phase parameters were studied by powder X-ray diffraction (XRD), and the magnetic behavior of Fe3O4 NPs was elucidated by Mössbauer spectroscopy. The colloidal and chemical stability of NPs was studied using simulated body fluid (phosphate buffer—PBS). Modified NPs have shown excellent stability in PBS (pH = 7.4), characterized by XRD, Mössbauer spectroscopy, and dynamic light scattering (DLS). Biocompatibility was evaluated in-vitro using cultured mouse embryonic fibroblasts (MEFs). The results show us an increasing of IC50 from 0.110 mg/mL for Fe3O4 NPs to 0.405 mg/mL for Fe3O4-Carborane NPs. The obtained data confirm the biocompatibility and stability of synthesized NPs and the potential to use them in BNCT.

Generation and Culture of Mouse Embryonic Fibroblasts.

In addition to leukocytes, a variety of cells also participate in the innate immune response, including endothelial cells, epithelial cells, and fibroblasts. Thus, the study of these cells is highly relevant in broadening our understanding of mechanisms that modulate innate immunity. With the rise of genetically engineered animals, it is now common to confirm in vitro data acquired using immortalized cell lines with more physiologically relevant primary cells from these animals ex vivo. Indeed, many studies exploring innate immune system function employ mouse embryonic fibroblasts (MEFs). These cells are relatively simple to generate and are a powerful tool to explore regulatory networks, examine biochemical profiling of protein complexes, and investigate novel signaling pathways associated with innate immune system signaling. Here, we provide a robust protocol to isolate, maintain, and store primary MEFs. This protocol is designed for users with minimal experience using mouse models. We have also added precautions and common pitfalls associated with these procedures.

Use of Primary Mouse Embryonic Fibroblasts in Developmental Toxicity Assessments.

Mouse embryonic fibroblasts (MEFs) are commonly collected as a means to maintain the culture and growth of embryonic stem cells (ESCs). However, their utility can extend well beyond their use exclusively in ESC culture. With collection from various transgenic mouse models, use of MEFs may serve as a more simplistic means to reconstitute in vivo/in utero toxicological assessments in an in vitro format for evaluation of function of specific proteins during toxic insults. The ease of collection, rapid growth kinetics, and large-scale expansion to perform multiple, high-throughput experiments are just some of the advantages of MEF use. Here, we describe procedures for successful MEF isolation and culture. As an example of MEF utility, we use MEFs collected form wild-type (WT) and Nrf2 knockout mice. After collection, MEFs were pretreated with the Nrf2 activator, dithiol-3-thione (D3T; 10μM) for 12h, and then treated with either hydrogen peroxide (0-2000μM) or mercury (0-100μM) for another 24h. Viability was measured via MTT assay after 24h of treatment.

PO-173 Critical requirement of the Sos1 and Sos2 RasGEFs for maintenance of mitochondrial homeostasis

IntroductionThe homologous, ubiquitously expressed, members (Sos1 and Sos2) of the Sos family of RasGEFs participate in multiple signalling pathways but their specific cellular functions are not clearly defined yet.Material and methodsUsing a tamoxifen (4OHT)-inducible, conditional Sos1 null mutation, here we generated and analysed wild type (WT), single Sos1-KO, constitutive Sos2-KO and double Sos1/2-DKO primary mouse embryonic fibroblasts (MEFs) in an effort to ascertain the functional specificity or redundancy of Sos1 and Sos2 at the cellular level.Results and discussionsSos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organisation that were not observed in WT and Sos2 KO counterparts. Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation of cytoplasmic bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Consistent with a mitophagic phenotype, in vivolabelling using specific fluorophores revealed increased levels of mitochondrial oxidative stress in the Sos1-KO and the Sos1/2-DKO cells as compared to Sos2-KO or WT MEFs. Treatment of the MEF cultures with antioxidants such as GSH and NAC corrected the altered perimeter size and proliferative rate of Sos1-KO and Sos1/2-DKO MEFs to levels similar to those of WT and Sos2-KO, but not recover oxidative stress. Furthermore, treatment with the specific mitochondrial superoxide scavenger mitoTEMPO recovered endogenous redox-homeostasis in Sos1-KO and Sos1/2-DKO to normal levels. Analysis of MEFs concomitantly loaded with MitoTracker Green (Δψ-independent) and MitoTracker Red (Δψ-dependent) showed a significantly increased of dysfunctional mitochondria in the Sos1/2-DKO MEFs in comparison to all other genotypes. Finally, Seahorse-based measurements of mitochondrial parameters showed that basal and maximal mitochondrial respiration, spare respiratory capacity, and ATP production, were also significantly decreased in Sos1/2-DKO cells as compared to MEFs of all the other genotypes.ConclusionOur data uncover a direct mechanistic link between Sos RasGEF proteins and the control of mitochondrial oxidative stress and respiration. Dysfunctional mitochondria are a key element in a variety of serious diseases, including cancer, and thus a promising therapeutic target.This work was supported by grants PI16/02137 (FIS) and CIBERONC (ONCG19//2017) from ISCIII; SA043U16 from FEDER-JCyL; FS/35–2017 from FSB and AECC, Spain.