Journal: bioRxiv

Article Title: Temporal Coordination of the Transcription Factor Response to H 2 O 2 stress

doi: 10.1101/2023.03.07.531593

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) plot of cells treated with PBS, 50μM and 75μM of H 2 O 2 after unsupervised clustering (n ≥ 10,000). Colors for cells based on the six clusters obtained. (B) UMAP of the same cells in A but colored based on sample. (C) UMAPs of cells from A colored by deviation scores for FOXO1 (left), RelA (middle) and HSF1 motifs (right) (D) UMAPs of cells from A showing deviation scores for p53 (left), Jun(middle) and NRF2 motifs (right) (E) Density colored scatter plots of log nuclear p53 (x-axis) and nuclear fraction of RelA (y-axis) at indicated levels of H 2 O 2 treatment for 5 hours (F) Percentage of cells activating both RelA and p53 (Both), RelA only, p53 only or neither for all concentrations of H 2 O 2 (G) Density colored scatter plots of log nuclear p53 (x-axis) and nuclear fraction of NFAT1 (y-axis) at indicated levels of H 2 O 2 treatment for 5 hours (H) Percentage of cells activating both NFAT1 and p53 (Both), NFAT1 only, p53 only or neither for all concentrations of H 2 O 2 . (I) Density colored scatter plots of log nuclear NRF2 (x-axis) and nuclear fraction of FOXO1(y-axis) at indicated levels of H 2 O 2 treatment for 5 hours (J) Percentage of cells activating both FOXO1 and NRF2 (Both), FOXO1 only, NRF2 only or neither for all concentrations of H 2 O 2 . (K) Density colored scatter plots of log nuclear JUN (x-axis) and nuclear fraction of FOXO1 (y-axis) at indicated levels of H 2 O 2 treatment for 5 hours (L) Percentage of cells activating both FOXO1 and JUN (Both), FOXO1 only, JUN only or neither for all concentrations of H 2 O 2 .

Article Snippet: Primary antibodies used: Anti-FOXO1 (C29H4) from Cell Signaling Cat# 2880S (1:500), Anti-p53 (DO-1) from Santa Cruz Cat# sc-126 (1:500), Anti-Sulfiredoxin from Santa Cruz Cat# sc-166786 (1:100), Recombinant Anti-Peroxiredoxin 1/PAG antibody [EPR5433] (ab109498) (1:1000), NRF2 (D1Z9C) XP Rabbit mAb #12721 (1:500), NFAT1 (D43B1) XP Rabbit mAb #5861 (1:300), NF-kB p65 (D14E12) XP Rabbit mAb #8242 (1:400), Anti-HSF1 antibody 10H8 from Stress Marq Biosciences (1:200), c-Fos (9F6) Rabbit mAb # 2250 (1:1000), and c-Jun (60A8) Rabbit mAb #9165 (1:400) from Cell Signaling.

Techniques:

Journal: bioRxiv

Article Title: Temporal Coordination of the Transcription Factor Response to H 2 O 2 stress

doi: 10.1101/2023.03.07.531593

Figure Lengend Snippet: (A) GSEA of p53 target genes in J14 + H 2 O 2 samples as compared to SRXN1-OE + H 2 O 2 samples (B) GSEA of NRF2 target genes in J14 + H 2 O 2 samples as compared to SRXN1-OE + H 2 O 2 samples. (C) Wald statistic and (D) log 2 fold changes of p53 target genes in H 2 O 2 treated cells vs. PBS controls, H 2 O 2 + J14 treated cells vs J14 treated controls and H 2 O 2 treated SRXN1-OE cells vs. SRXN1-OE PBS controls. (E) Wald statistic and (F) log 2 fold changes of NRF2 target genes in H 2 O 2 treated cells vs. PBS controls, H 2 O 2 + J14 treated cells vs J14 treated controls and H 2 O 2 treated SRXN1-OE cells vs. SRXN1-OE PBS controls. H 2 O 2 concentration is 50μM, J14 20μM.

Article Snippet: Primary antibodies used: Anti-FOXO1 (C29H4) from Cell Signaling Cat# 2880S (1:500), Anti-p53 (DO-1) from Santa Cruz Cat# sc-126 (1:500), Anti-Sulfiredoxin from Santa Cruz Cat# sc-166786 (1:100), Recombinant Anti-Peroxiredoxin 1/PAG antibody [EPR5433] (ab109498) (1:1000), NRF2 (D1Z9C) XP Rabbit mAb #12721 (1:500), NFAT1 (D43B1) XP Rabbit mAb #5861 (1:300), NF-kB p65 (D14E12) XP Rabbit mAb #8242 (1:400), Anti-HSF1 antibody 10H8 from Stress Marq Biosciences (1:200), c-Fos (9F6) Rabbit mAb # 2250 (1:1000), and c-Jun (60A8) Rabbit mAb #9165 (1:400) from Cell Signaling.

Techniques: Concentration Assay

Journal: Cell

Article Title: Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

doi: 10.1016/j.cell.2013.03.012

Figure Lengend Snippet: Flcn Acts downstream of or in Parallel to mTOR and Interacts with Fnip1/2 (A) Rex1GFPd2 cells were differentiated in N2B27 with and without 20 nM Rapa (left panel) or in N2B27/10%FBS (right panel), and cell lysates probed with indicated antibodies. (B) Rex1GFPd2 cells differentiated in N2B27 with and without 20 nM Rapa were replated at single-cell density in 2i including Rex1-expression selection at the indicated time points. The average percentage of uncommitted cells forming AP-positive colonies relative to the number of cells plated and SD are of two technical replicates. (C) Rex1GFPd2 cells transfected with indicated siRNAs were differentiated for 72 hr in N2B27 with and without 20 nM Rapa and replated in 2i with Rex1-expression selection, and resulting colonies were stained for AP. (D) Proteins were immunoprecipitated with FLAG antibodies from stably transfected Rex1GFPd2 cells cultured in 2i or differentiated for 40 hr and probed with indicated antibodies. (E) mRNA levels were quantified during differentiation and normalized to 2i-cultured cells. Average and SD are of two cell lines. (F) O4GIP ESCs were transfected with indicated siRNAs, and after differentiation for 3 days, exit from pluripotency quantified with a cell-viability assay and normalized to no siRNA transfection controls. Average and SD are of two technical replicates. See also Figure S3 .

Article Snippet: Primary antibodies were Flcn (1:100) , Fnip1 (Novus Biologicals, NBP1-00572, 1:100), GAPDH (Sigma, G8795, 1:1000), Oct4 (Santa Cruz, sc-5279, 1:500), Tfe3 (Sigma, HPA023881, 1:1000), pS6K1 (S371) (Cell Signaling, 9208, 1:100), S6 (Cell Signaling, 2217, 1:100), pS6 (S235/236) (Cell Signaling, 4858, 1:100), and p4EBP1 (T37/46) (Cell Signaling, 2855, 1:100).

Techniques: Expressing, Selection, Transfection, Staining, Immunoprecipitation, Stable Transfection, Cell Culture, Viability Assay

Journal: Cell

Article Title: Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

doi: 10.1016/j.cell.2013.03.012

Figure Lengend Snippet: Related to Figure 3 (A) ESCs were transfected with siRNAs overnight, and after 36 hr in indicated culture conditions, cell lysates were probed with specified antibodies. Equal exposures indicate that the increase in S6 phosphorylation during differentiation exceeds the effect of Tsc2 depletion in undifferentiated ESCs. (B) Silver-stained gel of FLAG-IP eluates from ESCs expressing indicated constructs. Bands corresponding to the IP’ed protein are marked with an asterisk. FLAG-Flcn co-IPs Fnip1 (arrowhead) and FLAG-Fnip1 co-IPs a band at the size of Flcn (open arrowhead). No other specific bands are visible compared to empty vector controls. (C) Fold changes of indicated transcript levels in ESCs overexpressing the Flcn-Fnip1 complex (empty, 3xFLAG-Flcn, 3xFLAG-Fnip1), stably knocked down for Tfe3 (empty shRNA [ctrl.2], Tfe3 shRNA [Tfe3.3]) or depleted of mTOR activity by rapamycin (EtOH, Rapa) relative to empty shRNA (ctrl.2). For comparison, two O4GIP EpiSC lines are included. Average and SD are of two independent experiments. (D) ESCs overexpressing the Flcn-Fnip1 complex (empty, 3×FLAG-Flcn, 3×FLAG-Fnip1) were exposed for 24 hr to the indicated culture conditions, and ESCs quantified by replating single cells in 2i with Rex1-expression selection. Average clonogenicity relative to empty vector control and SD are of four independent experiments. (E) Deconvolution of Fnip1/2 siRNA pools. O4GIP ESCs were transfected with indicated siRNAs, and after 3 days of differentiation, exit from pluripotency quantified with a cell-viability assay and normalized to no siRNA transfection controls. Average and SD are of two technical replicates. (F) O4GIP-7 EpiSCs were transfected with indicated siRNAs and differentiated by removal of FGF2 and Activin A for 3 days. After reapplication of EpiSC culture conditions and Oct4-expression selection using puromycin, EpiSCs were visualized by AP activity.

Article Snippet: Primary antibodies were Flcn (1:100) , Fnip1 (Novus Biologicals, NBP1-00572, 1:100), GAPDH (Sigma, G8795, 1:1000), Oct4 (Santa Cruz, sc-5279, 1:500), Tfe3 (Sigma, HPA023881, 1:1000), pS6K1 (S371) (Cell Signaling, 9208, 1:100), S6 (Cell Signaling, 2217, 1:100), pS6 (S235/236) (Cell Signaling, 4858, 1:100), and p4EBP1 (T37/46) (Cell Signaling, 2855, 1:100).

Techniques: Transfection, Phospho-proteomics, Staining, Expressing, Construct, Plasmid Preparation, Stable Transfection, shRNA, Activity Assay, Comparison, Selection, Control, Viability Assay

Journal: Cell

Article Title: Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

doi: 10.1016/j.cell.2013.03.012

Figure Lengend Snippet: Flcn Regulates Subcellular Localization of Tfe3 (A) Control (ctrl.1), Flcn shRNA (Flcn.4), and Tfe3 shRNA (Tfe3.3) cell lines were stained for Tfe3 and DNA in 2i conditions (left panel) and 24 hr after inhibitor withdrawal (right panel). Tfe3 was detected in the nucleus (arrowhead) and cytoplasm (open arrowhead). (B) Cytoplasmic (C) and nuclear (N) fractions of control and Flcn shRNA cells probed with indicated antibodies (open arrowheads indicate Fnip1 and Flcn bands; both bands recognized by Tfe3 antibodies are specific and likely represent phosphorylation variants; Hong et al., 2010 ). (C) Box and whisker plots of nuclear/cytoplasmic Tfe3 ratios in ESCs transfected with indicated siRNAs in 2i conditions and 24 hr after inhibitor withdrawal. Indicated cell numbers (white) from three experiments with two ESC lines were quantified. ( ∗∗ ) and ( ∗ ) indicate Student’s t test values < 1 × 10 −100 and 1 × 10 −50 , respectively. (D–F) Tfe3 is localized to the nucleus (arrowhead) at E3.5 (D). At E4.5, Tfe3 is found in the nucleus and cytoplasm of Nanog-positive epiblast cells (arrowhead) but stays nuclear in GATA4-positive (open arrowhead) primitive endodermal cells (E). At E5.5, Tfe3 is enriched in the cytoplasm (arrowhead) of Oct4-positive epiblast cells and remains nuclear (open arrowhead) in extraembryonic endoderm cells (F). See also Figure S4 .

Article Snippet: Primary antibodies were Flcn (1:100) , Fnip1 (Novus Biologicals, NBP1-00572, 1:100), GAPDH (Sigma, G8795, 1:1000), Oct4 (Santa Cruz, sc-5279, 1:500), Tfe3 (Sigma, HPA023881, 1:1000), pS6K1 (S371) (Cell Signaling, 9208, 1:100), S6 (Cell Signaling, 2217, 1:100), pS6 (S235/236) (Cell Signaling, 4858, 1:100), and p4EBP1 (T37/46) (Cell Signaling, 2855, 1:100).

Techniques: Control, shRNA, Staining, Phospho-proteomics, Whisker Assay, Transfection

Journal: Cell

Article Title: Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

doi: 10.1016/j.cell.2013.03.012

Figure Lengend Snippet: Genome-wide Tfe3-Target Determination Identifies Esrrb as a Downstream Effector of Flcn-Fnip1/2-Tfe3 (A) Gene tracks of loci identified by Tfe3 ChIP-Seq with control (ctrl.2), Flcn shRNA (Flcn.4), and Tfe3 shRNA (Tfe3.3) cell lines, overlaid with Nanog-, Oct4- and Tcf3-bound regions. (B) ESCs expressing indicated constructs were treated for 3 hr with 0.1 μM Tam in 2i. Average mRNA fold changes relative to EtOH treatment and SD are from two independent experiments with three different cell lines per genotype. ( ∗ ) indicates Student’s t test values < 0.005. (C) Average mRNA changes in ESCs transfected with the indicated siRNA combinations. Average relative expression normalized to no and negative siRNA treatments and SD are of three independent experiments. ( ∗ ) indicates Student’s t test values < 0.05. (D) O4GIP ESCs expressing Tfe3-ERT2 were transfected with indicated siRNAs, differentiated for 3 days in the presence of 0.1 μM Tam or EtOH, and switched back to 2i with puromycin selection, and remaining ESC colonies were quantified with a cell-viability assay. Average fold changes over negative siRNA-transfected, EtOH-treated cells and SD are from two independent experiments. ( ∗∗ ) and ( ∗ ) indicate Student’s t test values < 0.001 and 0.03, respectively. (E) Commitment of O4GIP cells transfected with indicated siRNA combinations, including two independent Esrrb siRNAs. Exit from pluripotency was quantified with a cell-viability assay and normalized to negative siRNA treatment. Average and SD are of two technical replicates. See also Figure S6 .

Article Snippet: Primary antibodies were Flcn (1:100) , Fnip1 (Novus Biologicals, NBP1-00572, 1:100), GAPDH (Sigma, G8795, 1:1000), Oct4 (Santa Cruz, sc-5279, 1:500), Tfe3 (Sigma, HPA023881, 1:1000), pS6K1 (S371) (Cell Signaling, 9208, 1:100), S6 (Cell Signaling, 2217, 1:100), pS6 (S235/236) (Cell Signaling, 4858, 1:100), and p4EBP1 (T37/46) (Cell Signaling, 2855, 1:100).

Techniques: Genome Wide, ChIP-sequencing, Control, shRNA, Expressing, Construct, Transfection, Selection, Viability Assay

Journal: Cell

Article Title: Exit from Pluripotency Is Gated by Intracellular Redistribution of the bHLH Transcription Factor Tfe3

doi: 10.1016/j.cell.2013.03.012

Figure Lengend Snippet: Nuclear Tfe3 Maintains an ESC State (A) Tfe3-ERT2-expressing cells differentiated into neurons (arrowhead) in the absence (left panel) but retained undifferentiated morphology and could be serially passaged in the presence of 0.1 μM Tam (right panel). (B) mRNA expression in Rex1GFPd2 and O4GIP TET cells relative to ESCs in 2i conditions. Average and SD are of three independent experiments. (C) mRNA expression in Rex1GFPd2 TET cells after Tam withdrawal relative to presence of Tam. Average and SD are of two technical replicates. (D) Immunohistochemistry in Rex1GFPd2 TET cells treated for 6 days with and without 0.1 μM Tam. (E) TET cells were plated at single-cell density in indicated culture conditions. Resulting colonies were stained for AP (lower panel) and quantified. Average numbers relative to Tam and SD are from two independent experiments (left bar: Rex1GFPd2, right bar: O4GIP). (F) Rex1GFPd2 TET cells were transfected with indicated siRNAs and replated after 2 days at clonal density in the presence of Tam. Average clonogenicity relative to negative siRNA and SD are of two independent experiments. (G) Rex1GFPd2 TET cells stably transfected with a GFP-expressing plasmid were microinjected into blastocysts, and resulting embryos analyzed at E11.5. Widespread contribution was detected in 4/7 embryos. For comparison, a non-GFP-expressing embryo (arrowhead) of the same litter is shown. (H) Passage 8 Rex1GFPd2 TET cells were cultured for 2 days in 2i and injected into C57BL/6 blastocysts. Contribution of the TET cell agouti gene to coat color is visible against black host fur. (I) Schematic representation of the Flcn-Fnip1/2 pathway and combinatorial inputs into Esrrb transcription. (….) denotes additional Tfe3 targets that contribute to self-renewal. See also Figure S7 .

Article Snippet: Primary antibodies were Flcn (1:100) , Fnip1 (Novus Biologicals, NBP1-00572, 1:100), GAPDH (Sigma, G8795, 1:1000), Oct4 (Santa Cruz, sc-5279, 1:500), Tfe3 (Sigma, HPA023881, 1:1000), pS6K1 (S371) (Cell Signaling, 9208, 1:100), S6 (Cell Signaling, 2217, 1:100), pS6 (S235/236) (Cell Signaling, 4858, 1:100), and p4EBP1 (T37/46) (Cell Signaling, 2855, 1:100).

Techniques: Expressing, Immunohistochemistry, Staining, Transfection, Stable Transfection, Plasmid Preparation, Comparison, Cell Culture, Injection

Journal: Frontiers in Physiology

Article Title: Use of a 3D Floating Sphere Culture System to Maintain the Neural Crest-Related Properties of Human Dental Pulp Stem Cells

doi: 10.3389/fphys.2018.00547

Figure Lengend Snippet: (A) Intracellular patch-clamp recordings performed on neuronal differentiated hDPSCs. Cells exhibit large outward ( I out ) and tiny inward transient currents ( I in ). Inward transient currents showing a small amount of currents at the peak of activation and kinetics compatible with transient sodium currents. Outward currents displaying larger values at the peak and kinetics compatible with currents produced by voltage-dependent potassium channels, responsible of action potential repolarization. (B) Western blot analysis (left) of Kir4.1 in undifferentiated 3D-derived hDPSCs and in hDPSCs following neuronal commitment. Immunofluorescence analysis against Kir4.1 was performed on undifferentiated STRO-1 + /c-Kit + /CD34 + hDPSCs following 3D culture (right). High magnification is referred to yellow insert. (C) Western blot analysis of Fas and FasL, and (D) immunofluorescence analysis of FasL expression were carried out on undifferentiated hDPSCs, cultured either as floating spheres or under adherent conditions. Densitometry of Fas and FasL bands is shown at the bottom ( ∗∗∗ P < 0.001, Fas expression in hDPSCs 3D spheres vs. adherent hDPSCs). No statistically significant differences were reported for FasL expression between the two culture conditions. (E) Western blot analysis of FasL performed on hDPSCs induced toward neuronal, osteogenic and myogenic lineages, respectively. Actin bands were presented as control of protein loading. Densitometric analysis revealed that FasL expression was higher following induction of neuronal commitment in hDPSCs, when compared to osteogenic and myogenic commitments ( ∗ P < 0.05 vs. osteogenic diff hDPSCs, # P < 0.05 vs. myogenic diff hDPSCs). Immunofluorescence analysis on differentiated hDPSCs showed FasL expression following induction to neuronal commitment, as demonstrated by positive immunolabeling against MAP-2 and FasL. Bar: 10 μm.

Article Snippet: Immunofluorescence analysis was carried out as described above by using the following primary antibodies: rabbit anti-Kir4.1 (1:100; StressMarq Biosciences), rabbit anti-FasL (1:100; Cell Signaling).

Techniques: Patch Clamp, Activation Assay, Produced, Western Blot, Derivative Assay, Immunofluorescence, Expressing, Cell Culture, Immunolabeling