Journal: Cell reports

Article Title: PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics.

doi: 10.1016/j.celrep.2023.112895

Figure Lengend Snippet: Figure 3. PGAM5 interacts with MFN2 and DRP1 (A) Interaction of PGAM5 WT GFP with mitochondrial dynamics regulatory proteins was analyzed by western blotting after coIP. Empty vector was used as the control. 5% of the total lysates were analyzed as input. (B) coIP analysis of PGAM5 and MFN2/DRP1 interaction. 293T cells were transfected with a plasmid encoding PGAM5 WT GFP or empty vector. At 48 h after transfection, cells were treated with either DMSO (vehicle control) or with 20 mM CCCP for 2 h. Cell lysates were immunoprecipitated with anti-GFP beads and analyzed by immunoblot (IB). 5% of the total lysates were analyzed as input. (C) MFN2 was normalized to PGAM5 WT GFP pull-down, and relative binding amounts in DMSO- and CCCP-treated conditions were plotted. The bar graph shows the comparison of the relative MFN2 binding between DMSO- and CCCP-treated conditions. Values are means ± SEM (n = 4). ***p = 0.0004 (paired t test).

Article Snippet: DRP1 K38A (addgene-37243), and MFN2WT (addgene - 28010) were originally purchased from addgene and further sub-cloned into different vectors.

Techniques: Western Blot, Plasmid Preparation, Control, Transfection, Immunoprecipitation, Binding Assay, Comparison

Journal: Cell reports

Article Title: PGAM5 is an MFN2 phosphatase that plays an essential role in the regulation of mitochondrial dynamics.

doi: 10.1016/j.celrep.2023.112895

Figure Lengend Snippet: Figure 4. PGAM5 functions independently of DRP1 but requires MFN2 activity (A) Confocal images of mitochondrial morphology in U2OS cells stably expressing control sh (right) and DRP1 sh (left). Confocal images of mitochondrial morphology in control sh and DRP1 sh stable U2OS cells transiently transfected with either PGAM5 H105A GFP or DMM GFP. Scale bars, 10 mm. (B) Zoomed insets of the images of (A). The region zoomed in on is marked with the white box. (C) Quantification of the mitochondrial aspect ratio of control and DRP1 knockdown cells with or without expressing PGAM5 H105A GFP or WT DMM GFP. Values are presented as mean ± SEM (n = 3) using GraphPad Prism. p values: control (non-transfected): DRP1 sh (non-transfected) **p = 0.0027; DRP1 sh (non- transfected): DRP1 sh/H105A **p = 0.0011; DRP1 sh (non-transfected): DRP1 sh/DMM ****p < 0.0001 (two-way ANOVA with Tukey’s multiple comparison test). (D) Confocal images of mitochondrial morphology in U2OS cells stably expressing control sh (right) and MFN2 sh (left). Confocal images of mitochondrial morphology in control sh and MFN2 sh stable U2OS cells transiently transfected with either PGAM5 WT or DN35 OMM GFP. Scale bars, 10 mm. (E) Zoomed insets of the images of (D). The regions zoomed in on have been marked with white boxes. (F) Quantification of the mitochondrial aspect ratio of control and MFN2 knockdown (KD) cells with or without expressing PGAM5 WT or DN35 OMM GFP. Vales are presented as mean ± SEM (n = 3) using GraphPad Prism. p values: control sh (non-transfected): MFN2 sh ***p = 0.0005; control sh (non-transfected): MFN2 sh/WT OE ***p = 0.0007; control sh (non-transfected): MFN2 sh/DN35 OMM **p = 0.0090 (two-way ANOVA with Tukey’s multiple comparison test).

Article Snippet: DRP1 K38A (addgene-37243), and MFN2WT (addgene - 28010) were originally purchased from addgene and further sub-cloned into different vectors.

Techniques: Activity Assay, Stable Transfection, Expressing, Control, Transfection, Knockdown, Comparison

Journal: Cancer research

Article Title: STING promotes homeostasis via regulation of cell proliferation and chromosomal stability

doi: 10.1158/0008-5472.CAN-18-1972

Figure Lengend Snippet: (A) Western blot analysis of STING expression in various human and mice cell lines. Bottom panel are quantified bands normalized to b-Actin control. (B) Western blot analyses of lysates from stable D54, HCT116, SCC61, and MC-38 tumor cell lines expressing short hairpin RNAs targeting STING (shSTING) or scrambled control (shScrambled). Bottom panel are quantified bands normalized to b-Actin and their respective non-targeting controls. (C-E) Tumor growth of shSTING knockdown and shScrambled control D54 (C); HCT116 (D); and SCC61 (E) cell lines in athymic nude mice. (F) Tumor growth of MC-38 shSTING knockdown in WT C57BL/6 mice. (G) Tumor growth of A549 with shSTING knockdown in athymic nude mice. Tumor model data are representative of three experiments, each with n = 5 mice per group. (H-N) Kinetic analysis of STING-depleted human tumor cell lines D54 (H), HCT116 (I), and SCC61 (J) as well as murine cell lines MC-38 (K), primary WT and STINGko MEFs (L), and SV40-immortalized WT and STINGko MEFs (M) proliferation in vitro were measured over time by manual cell counting. STING-depleted A549 cells were also tested as negative control (N). In vitro growth curve data are representative of at least three experiments, each with n = 3 per group. P-values were determined using unpaired Student’s t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

Article Snippet: MEFs were immortalized with a retrovirus expressing SV40-large T antigen (Addgene plasmid 13970). p50 −/− primary MEFs were kindly provided by Dr. Giovanna Bernal (University of Chicago).

Techniques: Western Blot, Expressing, In Vitro, Cell Counting, Negative Control

Journal: Cancer research

Article Title: STING promotes homeostasis via regulation of cell proliferation and chromosomal stability

doi: 10.1158/0008-5472.CAN-18-1972

Figure Lengend Snippet: (A) Western analyses of lysates from WT and STING−/− primary MEFs 48 hours post-exposure to increasing doses of IR. The membranes were probed for STING, TBK1, phospho-TBK1 Ser172, IRF3, and phosphor-IRF3 Ser396. Β-actin antibody was used for loading control. (B) IFN-β protein level in WT and STING−/− MEFs supernatant 48 hours following exposure to increasing doses of IR. (C) IFN- β protein secretion in MC-38 with stable shSTING knockdown 48 hours following exposure to increasing doses of IR. (D) Overexpression of STING in HEK293 cells led to a higher IFN-β promoter-driven induction of luciferase activity following exposure to increasing IR dose. (E) Transcriptional profiling of C57BL/6 wild-type (WT) and STINGko primary MEFs 48 hours following 0 or 6 Gy. Venn diagram displays the number of overlapping differentially expressed genes (DEGs) between the basal differences in WT vs. STINGko MEFs and the effects of irradiation in WT MEFs. (F) Top-ranked cellular pathway classification of the 265 DEGs that are activated by IR in WT and STINGko MEFs analyzed using Ingenuity Pathway Analysis (IPA). (G) CDKN1A was predicted as a top upstream regulator that potentially affects most of the 265 DEGs analyzed using IPA Upstream Analysis. (H-K) Western analyses of STING and p21 expression in lysates from primary WT and STINGko MEFs (H), SV40-immortalized WT and STINGko MEFs (I), shSTING HCT116 (J), and shSTING SCC61 (K) harvested 48 hours post-exposure to increasing doses of IR. (L) Kinetic analysis of siCDKN1A HCT116 proliferation in vitro were measured over time in response to 6 Gy IR using the IncuCyte live cell imaging system. In vitro growth curve data are representative of at least two experiments, each with n = 3 per group. P-values were determined using unpaired Student’s t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

Article Snippet: MEFs were immortalized with a retrovirus expressing SV40-large T antigen (Addgene plasmid 13970). p50 −/− primary MEFs were kindly provided by Dr. Giovanna Bernal (University of Chicago).

Techniques: Western Blot, Over Expression, Luciferase, Activity Assay, Irradiation, Expressing, In Vitro, Live Cell Imaging

Journal: PLoS ONE

Article Title: Telomerase Efficiently Elongates Highly Transcribing Telomeres in Human Cancer Cells

doi: 10.1371/journal.pone.0035714

Figure Lengend Snippet: ( A ) Western blot analysis of hTERT-expression in HCT116 parental (par) or DNMT1 and 3b double KO (DKO) infected cells. Numbers at the bottom are the ratios between hTERT and PCNA (loading control) signals, expressed as fold increase over hTERT-infected parental cells. ( B ) qRT-PCR analysis of the steady-state levels of TERRA transcripts originating from 10q and 15q chromosome ends expressed as fold increase over ev-infected par cells. Bars and error bars are averages and standard deviations from three independent experiments. ( C ) Analysis of TRAP amplification products from the indicated cells lines. Three different amounts of total proteins were used for each cell line and to control for specificity one sample was pre-treated with RNase A. Control primers amplifying an internal control (IC) were included in all reactions. ( D ) Quantification of telomerase activity in the indicated samples using qRT-PCR-based TRAP assays. Bars and error bars are averages and standard deviations from three independent experiments, after normalization through ev-infected parental cell samples. Numbers indicate P-values (*: P<0.01). ( E ) qRT-PCR-based quantification of 10q and 15q TERRA transcripts in TRAP extracts (ext) or in cell pellets left after extraction (plt). Relative TERRA amounts are expressed as fractions of total TERRA molecules from pellet plus extract. Bars and error bars are averages and standard deviations from three independent experiments.

Article Snippet: Retroviral plasmids pBABE-puro-hTERT and pBABE-puro-hTERT-HA were from Bob Weinberg's laboratory and were purchased from Addgene (plasmids 1771 and 1772). hTERT expression was monitored by standard western blot analysis of total protein extracts using rat monoclonal anti-hTERT antibodies for total hTERT (kind gift from Elena Giulotto), rabbit polyclonal anti-HA-tag antibodies for hTERT-HA (C29F4, Cell Signaling) and mouse monoclonal anti-PCNA antibodies to control for loading (sc-56, SantaCruz Biotechnology).

Techniques: Western Blot, Expressing, Infection, Control, Quantitative RT-PCR, Amplification, Activity Assay, Extraction

Journal: Nature cell biology

Article Title: The metabolome regulates the epigenetic landscape during naïve to primed human embryonic stem cell transition

doi: 10.1038/ncb3264

Figure Lengend Snippet: A: PCA of RNA-seq data from this study (Elf1, H1 4i–LIF, Lis1, H1) and other studies , , – ,. ComBat was applied on the combined RNA-seq dataset. B: Genes contributing to principal components separating primed vs. naïve hESC. Size of dots are proportional to the square of PC1 value. Top contributing genes are darker. C: Metabolic profile of naïve (Elf1, H1-4iLIF) and primed (H1) hESCs. A trace of OCR changes is shown under a MitoStress protocol (s.e.m, n=6 biological replicates). D: Primed hESCs (H7 and H1) have reduced OCR changes in response to FCCP compared to naïve hESCs (Elf1 and H1-4iLIF), n=18 (H1, H1 4iLIF) or 24 (Elf1, H7) biological replicates; s.e.m.; p=0.122 for H14iLIFvs.Elf1, p=0.0001 for H1vs.Elf1, p=0.0014 for H7vs.Elf1; 2-tailed t-test. E-F: Transition of naïve hESCs Elf1 (E) and WIN1 (F) toward a more primed state by culture in ActivinA-FGF (AF) media reduced OCR changes in response to FCCP after 1 to 3 days (n=29 for Elf AF 1D, n=20 for Elf AF 2D, n=28 for Elf AF 3D, n=33 for Elf1, n=18 for WIN1 and WIN1 AF; s.e.m.; p=0.0013 for ElfAF1Dvs.Elf1, p<0.001 for ElfAF2Dvs.Elf1, ElfAF3Dvs. Elf1 and WIN1AFvs.WIN1; 2-tailed t-test). G: Heatmap log2 fold expression change of mitochondria complexes genes between primed and naïve stages . H–I: Naïve hESCs (Elf1) and primed hESCs (Elf1 AF) have similar mitochondrial DNA copy number (H,n=3) and mitochondrial mutation frequencies (I,n=3). S.e.m.; p=0.7802 (H), p=0.37 and 0.6 (I); 2-tailed t-test. J: HIF1α protein is stabilized in primed hESCs (H7 and Elf1 AF). K: Proteomic workflow used to identify differentially regulated protein expression in primed vs. naive hESCs. L: Volcano plot of differentially expressed proteins in primed hESCs (right, green; Elf1 AF) vs naïve cells (left, blue, Elf1). Significant hits are shown (FDR<0.05). Proteins were quantified by nano-LC-MS/MS on a Fusion Orbitrap. M: JARID2 and LDHA proteins are upregulated in primed hESCs (Elf1 AF and H7) compared to naïve hESCs (Elf1), as revealed by Western blot analysis. Unprocessed original scans of blots are shown in . For raw data, see . n=number of biological replicates.

Article Snippet: Naïve hESCs (Elf1) were infected with a non-degradable form of HIF1α over-expressing construct (Addgene plasmid 19005, Yan et al.) or a pBABE empty vector construct in presence of 4 ng/ml Polybrene (Invitrogen).

Techniques: RNA Sequencing, Expressing, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Nature cell biology

Article Title: The metabolome regulates the epigenetic landscape during naïve to primed human embryonic stem cell transition

doi: 10.1038/ncb3264

Figure Lengend Snippet: A: screen shot of RNA expression and H3K27me3 marks of EGLN1 (PHD2) in naïve hESCs [Elf1 , WIRB3 naïve and BGO1 naïve )], primed hESCs [WIRB3 primed , H1 and H9 and Elf1 treated with STAT3 inhbitor (100 µM) for 6h. B: HIFα is hydroxylated on prolyl residues by EGLN1 (PHD2), leading to VHL-mediated proteolysis. C-D: Sequencing trace files, DNA sequences and protein sequences of HIF1α CRISPR-Cas9 knock-out (KO) mutant clones (gHIF1 6.2.1, C; gHIF1 6.3.1, D). E: schematic representation of wild type HIF1α protein and proteins resulting from CRISPR-Cas9 knock-out (KO) mutants gHIF1 6.2.1 and gHIF1 6.3.1. bHLH= basic helix-loop-helix domain, PAS= Per-Arnt-Sim domain, NTAD= N-terminus transcriptional activation domain, CTAD= C-terminus transcriptional activation domain. F: HIF1α is not expressed in CRISPR-Cas9 KO mutants. Western blot analysis of HIF1α expression in cells pushed toward the primed stage by culture in TeSR1 for 5 days in wild type Elf1 cells (iCas9 Elf1), and two CRISPR-Cas9 KO mutants of HIF1α (gHIF1 6.2.1 and gHIF1 6.3.1). G: qPCR analysis of hESCs transitioning to primed reveals that naïve markers (DNMT3L and NNMT) are still expressed higher in Elf1 HIF1α CRISPR-Cas9 KO cells compared to wild type Elf1, while primed marker IDO1 and HIF target genes (PDK1 and VEGFA) are downregulated (n=3; s.e.m.; p=0.024 for DNMT3L, p=0.0005 for NNMT, p=0.001 for IDO1, p=0.12 for PDK1, p=0.004 for VEGFA; 2-tailed t-test). H: KO of HIF1α prevents the metabolic switch occurring during the transition of hESCs from naïve to primed state as shown by measuring OCR after FCCP addition using SeaHorse. n=3 for gHIF1 6.3.1 2iLIF and AF and n=4 for Elf iCas9 and gHIF1 6.2.1 2iLIF and AF; s.e.m.; p=0.0117 for gHIF1 6.2.1 vs. Elf iCas9, p=0.0032 for gHIF1 6.3.1 vs. Elf iCas9; 2-tailed t-test. Unprocessed original scans of blots are shown in . For raw data, see . n= number of biological replicates.

Article Snippet: Naïve hESCs (Elf1) were infected with a non-degradable form of HIF1α over-expressing construct (Addgene plasmid 19005, Yan et al.) or a pBABE empty vector construct in presence of 4 ng/ml Polybrene (Invitrogen).

Techniques: RNA Expression, Sequencing, CRISPR, Knock-Out, Mutagenesis, Clone Assay, Activation Assay, Western Blot, Expressing, Marker

Journal: Nature cell biology

Article Title: The metabolome regulates the epigenetic landscape during naïve to primed human embryonic stem cell transition

doi: 10.1038/ncb3264

Figure Lengend Snippet: A–B: Sequencing trace files, DNA sequences, protein sequences and 3D protein structures predicted from sequence (Pymol) of various NNMT CRISPR-Cas9 KO mutant clones (gNNMT 7.2.1, A; gNNMT 6.2.4, B). Green color represents the truncated NNMT protein in the CRISPR-Cas9 mutant. C: Schematic representation of wild type NNMT protein and proteins resulting from the CRISPR-Cas9 KO mutants gNNMT 7.2.1 and gNNMT 6.2.4. D: Elf1 NNMT CRISPR-Cas9 KO cells have higher amounts of SAM than wild type Elf1 cells (n=6; s.e.m.; p=1.23E-05 for gNNMT7.2.1, p=5.47E-06 for gNNMT6.2.4; 2-tailed t-test). E: Western blot analysis reveals higher HIF1α expression and H3K27me3 marks in Elf1 CRISPR-Cas9 KO mutants gNNMT 7.2.1 and gNNMT 6.2.4 compared to control Elf1 (iCas9) cells. F: qPCR analysis of the naïve marker DNMT3L in wild type Elf1 cells (n=6) and Elf1 CRISPR-Cas9 KO mutants gNNMT 7.2.1 (n=5) and gNNMT 6.2.4 (n=3). s.e.m.; p=0.0009 for gNNMT 6.2.4 vs. Elf1, p=0.027 for gNNMT 7.2.1 vs. Elf1; 2-tailed t-test. G: log2 fold expression change of NNMT, WNT ligands and HIF target genes in Elf1 CRISPR-Cas9 KO gNNMT 7.2.1 compared to wild type Elf1 cells (RNAseq). H: PCA plot of CRISPR NNMT knockout line and different naïve and primed lines sequenced in this study. gNNMT 6.2.2 and gNNM 7.3.5 are heterozogous controls. PC1 (x-axis) explains majority of the variation in the data (61%), and the gNNMT 7.2.1 knockdown line moved along x-axis substiantially away from other naïve lines and toward the primed state. I: Hypergeometric test p-values for the overlap between genes expressed higher (lower) in gNNMT 7.2.1 compared to Elf1 and genes expressed higher (lower) in primed lines compared to naïve lines from multiple studies. Color shade is proportional to negative log10 of p-values. gNNMT 7.2.1 transcriptomic signature has significant overlap with all published primed transcriptomic datasets, supporting its transition toward a primed stage. J: Model of the intricate relationship between metabolism and epigenetic in hESCs. Unprocessed original scans of blots are shown in . For raw data, see . n= number of biological replicates.

Article Snippet: Naïve hESCs (Elf1) were infected with a non-degradable form of HIF1α over-expressing construct (Addgene plasmid 19005, Yan et al.) or a pBABE empty vector construct in presence of 4 ng/ml Polybrene (Invitrogen).

Techniques: Sequencing, CRISPR, Mutagenesis, Clone Assay, Western Blot, Expressing, Control, Marker, Knock-Out, Knockdown