Journal: The Journal of Cell Biology

Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

doi: 10.1083/jcb.202008101

Figure Lengend Snippet: H3K79me2/3 are increased at the IL1A locus during OIS, corresponding to increased DOT1L methyltransferase expression. IMR90 cells were infected with retrovirus expressing HRas G12V (RAS) or empty vector control. See for time points. (A) Immunoblot analysis of the indicated proteins. β-Actin was used as a loading control. One of three independent experimental replicates is shown. (B) Senescence-associated β-galactosidase (SA-β-Gal) activity, colony formation (CF), and promyelocytic leukemia (PML) body immunofluorescence. Shown are representative images from one of three independent experimental replicates. Scale bar, 10 µm. (C) Quantification of SA-β-Gal activity in B. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >100 cells counted). *, P < 0.0004 by Student’s t test. (D) Quantification of CF in B. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0008 by Student’s t test. (E) Quantification of PML body immunofluorescence in B. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >200 cells counted). *, P < 0.0118 by Student’s t test. (F) Heat map of unmodified, methylated, and acetylated histones determined using LC-MS/MS. Only modifications with FDR <0.1 are shown. H3K79 is marked in green. See for raw data. (G) H3K79un (unmodified), H3K79me1, H3K79me2, and H3K79me3 percent abundance was determined by LC-MS/MS. Data represent mean ± SEM ( n = 2). (H) H3K79me2 and H3K79me3 immunoblot analysis of chromatin fraction. Total histone H3 was used as a loading control. One of three independent experimental replicates is shown. (I) Cross-referencing of H3K79me3 ChIP-seq data and RNA-seq data. A total of 1,952 genes showed increased H3K79me3 occupancy (FC >1.5 RAS versus control; ), and a total of 793 genes were significantly increased by RNA-seq (FC >1.5 RAS versus control; q-value [FDR] <0.25; ). The 95 genes (listed in ) that overlapped were subjected to GSEA. (J) H3K79me3 ChIP-seq track at the IL1A gene locus. Blue indicates H3K79me3 binding in control cells, whereas red indicates H3K79me3 binding in RAS cells. (K) IL6 , IL1B , and CXCL8 mRNA expression. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.003 by Student’s t test. (L) H3K79me3 ChIP-seq track at the IL6, IL1B, and CXCL8 gene loci. Blue indicates binding of H3K79me3 in control cells, whereas red indicates binding of H3K79me3 binding in RAS cells. (M) Schematic of ChIP-qPCR primers for IL1A promoter region (H3K79me2 enrichment, indicated in blue) and gene body (H3K79me3 enrichment, indicated in red). (N and O) H3K79me2 binding to the IL1A promoter region (N) and H3K79me3 binding to the gene body (O) was determined by ChIP-qPCR and normalized to total histone H3 binding at the same site. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by Student’s t test. (P) IL1A mRNA expression. One of five independent experimental replicates is shown ( n = 3). Data represent mean ± SD ( n = 3). *, P < 0.001by Student’s t test. (Q) Cell surface–bound IL1A was determined by flow cytometry. One of four independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by Student’s t test. (R) DOT1L mRNA expression from RNA-seq. Three technical replicates from one experiment are shown. Data represent mean ± SD ( n = 3). *, P < 0.0001 by Student’s t test. (S) Immunoblot analysis of DOT1L. Vinculin was used as a loading control. One of three independent experimental replicates is shown. MFI, median fluorescence intensity.

Article Snippet: Normal diploid IMR90 human fibroblasts (CCL-186; ATCC) and BJ-hTERT immortalized human foreskin fibroblasts (a gift from Dr. Patricia Opresko, University of Pittsburgh, Pittsburgh, PA) were cultured in 2% O 2 in DMEM (10017CV; Corning) with 10% FBS supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, and sodium bicarbonate.

Techniques: Expressing, Infection, Plasmid Preparation, Control, Western Blot, Activity Assay, Immunofluorescence, Methylation, Liquid Chromatography with Mass Spectroscopy, ChIP-sequencing, RNA Sequencing, Binding Assay, ChIP-qPCR, Flow Cytometry, Fluorescence

Journal: The Journal of Cell Biology

Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

doi: 10.1083/jcb.202008101

Figure Lengend Snippet: Oncogenic BRAF induces cellular senescence and increases DOT1L expression; RAS does not affect KDM2B or KDM4D expression. Related to . (A) Timeline of experiments. (B–I, K, and N) BJ-hTERT cells were infected with retrovirus-expressing BRAF V600E (BRAF) or empty vector control. (B) Immunoblot analysis of the indicated proteins. β-Actin was used as a loading control. One of three independent experimental replicates is shown. (C) Senescence-associated β-galactosidase (SA-β-Gal) activity and colony formation (CF). Shown are representative images from one of three independent experimental replicates. Scale bar, 10 µm. (D) Quantification of SA-β-Gal activity in C. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >100 cells counted). *, P < 0.0004 by Student’s t test. (E) Quantification of CF in C. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0008 by Student’s t test. (F) LMNB1 mRNA expression. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by Student’s t test. (G) H3K79me2 and H3K79me3 immunoblot analysis of chromatin fraction. Total histone H3 was used as a loading control. One of three independent experimental replicates is shown. (H) H3K79me2 binding to the IL1A promoter region and H3K79me3 binding to the gene body was determined by ChIP-qPCR and normalized to total histone H3 binding at the same site. One of two independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0140 by Student’s t test. (I) IL1A mRNA expression. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0002 by Student’s t test. (J) IMR90 cells were infected with retrovirus-expressing HRas G12V (RAS) or empty vector control. KDM2B and KDM4D mRNA expression from RNA-seq. Three technical replicates from one experiment are shown. Data represent mean ± SD ( n = 3). Not significant by Student’s t test. (K) DOT1L mRNA expression. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by Student’s t test. (L and M) Primary human melanocytes were infected with retrovirus-expressing BRAF V600E (BRAF) or control. (L) DOT1L and IL1A mRNA expression was determined by RT-qPCR. One independent experiment is shown. Data represent mean ± SD ( n = 3). *, P < 0.005 by Student’s t test. (M) DOT1L and IL1A mRNA expression (GEO accession no. GSE46818 ). Data represent mean ± SD ( n = 3). *, P < 0.005 by Student’s t test. (N) DOT1L immunoblot analysis. Vinculin was used as a loading control. One of three independent experimental replicates is shown. (O) RT-qPCR analysis was performed for DOT1L and IL1A expression on papillomas from mice treated with DMBA/TPA. r 2 is the Pearson’s correlation coefficient ( n = 13). ND, not detected.

Article Snippet: Normal diploid IMR90 human fibroblasts (CCL-186; ATCC) and BJ-hTERT immortalized human foreskin fibroblasts (a gift from Dr. Patricia Opresko, University of Pittsburgh, Pittsburgh, PA) were cultured in 2% O 2 in DMEM (10017CV; Corning) with 10% FBS supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, and sodium bicarbonate.

Techniques: Expressing, Infection, Plasmid Preparation, Control, Western Blot, Activity Assay, Binding Assay, ChIP-qPCR, RNA Sequencing, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

doi: 10.1083/jcb.202008101

Figure Lengend Snippet: DOT1L is necessary for H3K79me2/3 at the IL1A locus and SASP expression but dispensable for other senescence phenotypes. IMR90 cells were infected with retrovirus-expressing HRAS G12V (RAS) or empty vector control with or without shRNA to human DOT1L (shDOT1L) or an shGFP control. Details on time points are in . (A) Immunoblot analysis of total cell lysates (TCL) and chromatin fractions of the indicated proteins. β-Actin was used as a loading control for TCL. Histone H3 was used as a loading control for chromatin fractions. One of three independent experimental replicates is shown. (B) H3K79me2 and DOT1L binding to the IL1A promoter region and H3K79me2 at the promoters of IL6 , CXCL8 , and IL1B was determined by ChIP-qPCR and normalized to total histone H3 binding at the same site. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons. (C) H3K79me3 and DOT1L binding to the IL1A gene body and H3K79me3 at the gene bodies of IL6 , CXCL8 , and IL1B was determined by ChIP-qPCR and normalized to total histone H3 binding at the same site. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons. (D) IL1A mRNA expression was determined by RT-qPCR. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (E) Cell surface–bound IL1A was determined by flow cytometry. One of four independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (F) IL6 , IL1B , and CXCL8 mRNA expression was determined by RT-qPCR. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (G) Secretion of SASP-related factors was detected using an antibody array. Heat map indicates FC. Data are generated from technical replicates of one independent experiment. Raw data can be found in . (H) Senescence-associated β-galactosidase (SA-β-Gal) activity, colony formation (CF), promyelocytic leukemia (PML) body immunofluorescence, γH2AX, and 53BP1 foci. Shown are representative images from one of three independent experimental replicates. Scale bar, 10 µm. (I) Quantification of SA-β-Gal activity in H. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >100 cells counted). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons. (J) Quantification of CF in H. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons. (K) Quantification of PML body immunofluorescence in H. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >200 cells counted). *, P < 0.02 by one-way ANOVA with Tukey’s multiple comparisons. (L) Immunoblot analysis of indicated proteins. β-Actin was used as a loading control. One of three independent experimental replicates is shown. (M) Quantification of γH2AX and 53BP1 foci in H. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >200 cells counted). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons. (N–R) IMR90 cells were infected with retrovirus-expressing HRAS G12V (RAS) or empty vector control. Four days after retroviral infection, cells were treated with 1 µM DOT1L inhibitor EPZ5676. (N) RAS immunoblot analysis. Vinculin was used as loading control. One of three independent experimental replicates is shown. (O) H3K79me2 and H3K79me3 immunoblot analysis was performed on chromatin fractions. Total histone H3 was used as loading control. One of three independent experimental replicates is shown. (P) IL1A, IL6 , IL1B , and CXCL8 mRNA expression was determined by RT-qPCR. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (Q) SA-β-Gal activity. Shown are representative images from one of three independent experimental replicates. Scale bar, 10 µm. (R) Quantification of SA-β-Gal activity in P. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >100 cells counted). *, P < 0.0005 by one-way ANOVA with Tukey’s multiple comparisons. Cont, control; MFI, median fluorescence intensity.

Article Snippet: Normal diploid IMR90 human fibroblasts (CCL-186; ATCC) and BJ-hTERT immortalized human foreskin fibroblasts (a gift from Dr. Patricia Opresko, University of Pittsburgh, Pittsburgh, PA) were cultured in 2% O 2 in DMEM (10017CV; Corning) with 10% FBS supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, and sodium bicarbonate.

Techniques: Expressing, Infection, Plasmid Preparation, Control, shRNA, Western Blot, Binding Assay, ChIP-qPCR, Quantitative RT-PCR, Flow Cytometry, Ab Array, Generated, Activity Assay, Immunofluorescence, Retroviral, Fluorescence

Journal: The Journal of Cell Biology

Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

doi: 10.1083/jcb.202008101

Figure Lengend Snippet: DOT1L knockdown in BRAF-induced senescent cells decreases SASP and maintains cells in a senescence-associated cell cycle arrest. Related to and . (A) IMR90 cells were infected with retrovirus-expressing HRas G12V (RAS) or empty vector control. LMNB1 , CDKN2A , and CDKN1A mRNA expression from RNA-seq. Three technical replicates from one experiment are shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons. (B–F) BJ-hTERT cells were infected with retrovirus-expressing BRAF V600E (BRAF) or empty vector control with or without lentivirus-expressing an shRNA to human DOT1L (shDOT1L) or shGFP control. (B) Immunoblot analysis of total cell lysates (TCL) and chromatin fractions of the indicated proteins. Vinculin was used as a loading control for TCL. Histone H3 was used as a loading control for chromatin fractions. One of three independent experimental replicates is shown. (C) IL1A , IL6 , IL1B , and CXCL8 mRNA expression was determined by RT-qPCR. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons. (D) Senescence-associated β-galactosidase (SA-β-Gal) activity and colony formation (CF). Shown are representative images from one of three experimental replicates. Scale bar, 10 µm. (E) Quantification of SA-β-Gal activity in D. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >100 cells counted). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons. (F) Quantification of CF in D. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by one-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: Normal diploid IMR90 human fibroblasts (CCL-186; ATCC) and BJ-hTERT immortalized human foreskin fibroblasts (a gift from Dr. Patricia Opresko, University of Pittsburgh, Pittsburgh, PA) were cultured in 2% O 2 in DMEM (10017CV; Corning) with 10% FBS supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, and sodium bicarbonate.

Techniques: Knockdown, Infection, Expressing, Plasmid Preparation, Control, RNA Sequencing, shRNA, Western Blot, Quantitative RT-PCR, Activity Assay

Journal: The Journal of Cell Biology

Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

doi: 10.1083/jcb.202008101

Figure Lengend Snippet: Overexpression (OE) of IL1A in DOT1L knockdown cells rescues SASP gene expression and corresponds to C/EBPβ expression. IMR90 cells were infected with retrovirus-expressing HRAS G12V (RAS) or empty vector control followed by a simultaneous infection with or without shRNA to human DOT1L (shDOT1L) or shGFP control and lentivirus-expressing V5-tagged IL1A or empty vector control. (A) Immunoblot analysis of the indicated proteins. Vinculin was used as a loading control. One of three independent experimental replicates is shown. (B) IL1A , IL6 , IL1B , and CXCL8 mRNA expression was determined by RT-qPCR. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (C) Immunoblot analysis of indicated proteins. β-Actin was used as a loading control. One of three independent experimental replicates is shown. (D) CEBPB mRNA expression was determined by RT-qPCR. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (E) H3K79me3 ChIP-seq track at the CEBPB gene locus. Blue indicates binding of H3K79me3 in control cells, whereas red indicates binding of H3K79me3 binding in RAS cells.

Article Snippet: Normal diploid IMR90 human fibroblasts (CCL-186; ATCC) and BJ-hTERT immortalized human foreskin fibroblasts (a gift from Dr. Patricia Opresko, University of Pittsburgh, Pittsburgh, PA) were cultured in 2% O 2 in DMEM (10017CV; Corning) with 10% FBS supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, and sodium bicarbonate.

Techniques: Over Expression, Knockdown, Gene Expression, Expressing, Infection, Plasmid Preparation, Control, shRNA, Western Blot, Quantitative RT-PCR, ChIP-sequencing, Binding Assay

Journal: The Journal of Cell Biology

Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

doi: 10.1083/jcb.202008101

Figure Lengend Snippet: DOT1L overexpression (OE) increases H3K79me2/3 at the IL1A locus and is sufficient for SASP gene expression but does not affect other senescence phenotypes. IMR90 cells were infected with retrovirus-expressing human DOT1L or empty vector control. In some experiments, as a positive control, IMR90 cells were infected with retrovirus-expressing HRAS G12V (RAS) or empty vector control. (A) DOT1L mRNA expression. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.001 by Student’s t test. (B) DOT1L immunoblot analysis. Vinculin was used as a loading control. One of five independent experimental replicates is shown. (C) H3K79me2 and H3K79me3 immunoblot analysis on chromatin fractions. Total histone H3 was used as loading control. One of three independent experimental replicates is shown. (D) H3K79me2 and DOT1L binding to the IL1A promoter region and H3K79me2 at the promoters of IL6 , CXCL8 , and IL1B was determined by ChIP-qPCR and normalized to total histone H3 binding at the same site. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by Student’s t test. (E) H3K79me3 and DOT1L binding to the IL1A gene body and H3K79me3 at the gene bodies of IL6 , CXCL8 , and IL1B was determined by ChIP-qPCR and normalized to total histone H3 binding at the same site. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.05 by Student’s t test. (F) IL1A mRNA expression was determined by RT-qPCR. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.006 by Student’s t test. (G) Cell surface–bound IL1A was determined by flow cytometry. One of four independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.001 by Student’s t test. (H) Secretion of SASP-related factors were detected by antibody array. Heat map indicates FC of DOT1L OE versus control. Data are generated from technical replicates of one independent experiment. Raw data can be found in . (I) IL6 , IL1B , and CXCL8 mRNA expression was determined by RT-qPCR. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0001 by Student’s t test. (J) Immunoblot analysis of the indicated proteins. β-Actin was used as a loading control. One of three independent experimental replicates is shown. (K) Senescence-associated β-galactosidase (SA-β-Gal) activity, promyelocytic leukemia (PML) body immunofluorescence, and γH2AX and 53BP1 foci. Shown are representative images from one of three independent experimental replicates. Scale bar, 10 µm. (L) Quantification of SA-β-Gal activity in K. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >100 cells counted). *, P < 0.05 by Student’s t test. (M) Quantification of PML body immunofluorescence in K. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >200 cells counted). *, P < 0.01 by Student’s t test. (N) Quantification of 53BP1 and γH2AX foci in K. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >200 cells counted). *, P < 0.005 by Student’s t test. (O) C/EBPβ protein expression was determined by immunoblot. Vinculin was used as a loading control. One of six independent experimental replicates is shown. Cont, control; MFI, median fluorescence intensity.

Article Snippet: Normal diploid IMR90 human fibroblasts (CCL-186; ATCC) and BJ-hTERT immortalized human foreskin fibroblasts (a gift from Dr. Patricia Opresko, University of Pittsburgh, Pittsburgh, PA) were cultured in 2% O 2 in DMEM (10017CV; Corning) with 10% FBS supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, and sodium bicarbonate.

Techniques: Over Expression, Gene Expression, Infection, Expressing, Plasmid Preparation, Control, Positive Control, Western Blot, Binding Assay, ChIP-qPCR, Quantitative RT-PCR, Flow Cytometry, Ab Array, Generated, Activity Assay, Immunofluorescence, Fluorescence

Journal: The Journal of Cell Biology

Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

doi: 10.1083/jcb.202008101

Figure Lengend Snippet: DOT1L overexpression (OE) in BJ-hTERT cells induces the SASP but not a senescence-associated cell cycle arrest or DNA damage accumulation; p-p65 is not affected by DOT1L OE. Related to . (A–F and I) BJ-hTERT cells were infected with retrovirus-expressing human DOT1L or empty vector control. (A) Immunoblot analysis of DOT1L. Vinculin was used as a loading control. One of three independent experimental replicates is shown. (B) H3K79me2 and H3K79me3 immunoblot analysis on chromatin fractions. Histone H3 was used as a loading control. One of three independent experimental replicates is shown. (C) IL1A mRNA expression was determined by RT-qPCR. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0001 by Student’s t test. (D) IL6 , IL1B , and CXCL8 mRNA expression was determined by RT-qPCR. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0001 by Student’s t test. (E) Senescence-associated β-galactosidase (SA-β-Gal) activity. Shown are representative images from one of three independent experimental replicates. Scale bar, 10 µm. (F) Quantification of SA-β-Gal in E. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >100 cells counted). Not significant by Student’s t test. (G–J) IMR90 cells were infected with retrovirus-expressing human DOT1L or empty vector control. (G) CDKN2A and CDKN1A mRNA expression from RNA-seq. Three technical replicates from one experiment are shown. Data represent mean ± SD ( n = 3). *, P < 0.005 by Student’s t test. (H) LMNB1 expression from RNA-seq. Three technical replicates from one experiment are shown. Data represent mean ± SD ( n = 3). *, P < 0.0001 by Student’s t test. (I) Immunoblot analysis of p-p65 and total p65 in IMR90 and BJ-hTERT cells as indicated. β-Actin was used as a loading control. One of three independent experimental replicates is shown. (J) CEBPB mRNA expression was determined by RT-qPCR. One of six independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.005 by Student’s t test.

Article Snippet: Normal diploid IMR90 human fibroblasts (CCL-186; ATCC) and BJ-hTERT immortalized human foreskin fibroblasts (a gift from Dr. Patricia Opresko, University of Pittsburgh, Pittsburgh, PA) were cultured in 2% O 2 in DMEM (10017CV; Corning) with 10% FBS supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, and sodium bicarbonate.

Techniques: Over Expression, Infection, Expressing, Plasmid Preparation, Control, Western Blot, Quantitative RT-PCR, Activity Assay, RNA Sequencing

Journal: The Journal of Cell Biology

Article Title: DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A

doi: 10.1083/jcb.202008101

Figure Lengend Snippet: STING is necessary for DOT1L expression to promote the SASP. IMR90 cells were infected with retrovirus-expressing HRas G12V (RAS) or empty vector control with or without lentivirus expressing an shRNA to human STING (shSTING) or shGFP control with or without overexpression of DOT1L (DOT1L OE) or empty vector control. (A) STING mRNA expression was determined by RT-qPCR. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons. (B) RAS and STING immunoblot analysis. β-Actin was used as a loading control. One of three independent experimental replicates is shown. (C) DOT1L mRNA expression was determined by RT-qPCR. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons. (D) H3K79me2 and H3K79me3 immunoblot analysis on the chromatin fraction. Total histone H3 was used as loading control. One of three independent experimental replicates is shown. (E) IL1A , IL6 , IL1B , and CXCL8 mRNA expression was determined by RT-qPCR. One of five independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons. (F) Immunoblot analysis for the indicated proteins. Vinculin was used as a loading control. One of three independent experimental replicates is shown. (G) Senescence-associated β-galactosidase (SA-β-Gal) activity, colony formation (CF), promyelocytic leukemia (PML) body immunofluorescence, and γH2AX and 53BP1 foci. Shown are representative images from one of three independent experimental replicates. Scale bar, 10 µm. (H) Quantification of SA-β-Gal in G. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >100 cells counted). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (I) Quantification of CF in G. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (J) Quantification of PML body foci in G. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >200 cells counted). *, P < 0.02 by one-way ANOVA with Tukey’s multiple comparisons. (K) Quantification of 53BP1 foci in G. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >200 cells counted). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (L) Quantification of γH2AX foci in G. One of three independent experimental replicates is shown. Data represent mean ± SD ( n = 3, where each dot represents >200 cells counted). *, P < 0.01 by one-way ANOVA with Tukey’s multiple comparisons. (M) Proposed model of DOT1L-mediated SASP induction. Upon OIS, STING induces DOT1L expression, which methylates H3K79 at the IL1A locus. IL1A is then transported to the cell surface, where it contributes to the feed-forward mechanism of SASP induction.

Article Snippet: Normal diploid IMR90 human fibroblasts (CCL-186; ATCC) and BJ-hTERT immortalized human foreskin fibroblasts (a gift from Dr. Patricia Opresko, University of Pittsburgh, Pittsburgh, PA) were cultured in 2% O 2 in DMEM (10017CV; Corning) with 10% FBS supplemented with L-glutamine, nonessential amino acids, sodium pyruvate, and sodium bicarbonate.

Techniques: Expressing, Infection, Plasmid Preparation, Control, shRNA, Over Expression, Quantitative RT-PCR, Western Blot, Activity Assay, Immunofluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: MAMs formation boosted in mice fed with HFHC diets and PA-stimulation hepatocytes. A Transmission electron microscopy (TEM) images of hepatic tissue from C57BL/6 J mice fed a regular chow diet or a high-fat, high-carbohydrate (HFHC) diet for 16 weeks, showing the morphology of MAMs (× 60,000, scale bar 600 nm; n = 4 mice per group). B Confocal microscopy images displaying the colocalization of the ER and mitochondria in AML12 and HepG2 cells treated with BSA or palmitic acid (PA). Colocalization is indicated by the merged fluorescence of DsRed2-ER-5 (red) and Mito-Tracker Green. The Pearson’s coefficient graph quantifies the degree of colocalization (× 1200, scale bar 5 μm; n = 4 per group). C - D Western blot analysis of MAMs-related Ca 2 ⁺ channel proteins, including IP3R1, GRP75, and VDAC1, in homogenates and MAMs fractions from the liver of NAFLD mice and PA-stimulated AML12 cells. β-tubulin or calreticulin was used as a loading control. Data are presented as means ± SD. * P < 0.05, ** P < 0.01 indicate significant differences between the indicated treatment groups; n.s. indicates no significant difference

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Transmission Assay, Electron Microscopy, Micro-arrays for Mass Spectrometry, Confocal Microscopy, Fluorescence, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: SIRT1 overexpression inhibited the Mito-ER contact, Ca 2+ dyshomeostasis, and mitochondria dysfunction induced by PA stimulation in hepatocytes. A Confocal microscopy images displayed the colocalization of ER and mitochondria in AML12 cells with stable overexpression of SIRT1 under BSA or PA exposure for 24 h. The colocalization is indicated by the merge of DsRed2-ER-5 and Mito-Tracker Green; and the Pearson’s coefficient graph quantifies the colocalization (× 1200, Scar bar, 5 μm; n = 4 per group). B Western blot analysis of expression of MAMs-related Ca 2 ⁺ channels (IP3R1, GRP75, and VDAC1), β-tubulin served as a loading control. C Measurement of Ca 2 ⁺ dynamics. The left panels show the time course of the D1ER ratio and pCAG-Mito-Gcamp5G. The fluorescence was recorded every 10 s. 100 µM ATP was injected at 100 s. The quantification of baseline and ATP-induced delta-peak or delta troughs are shown in the right panels ( n = 3 per group). D DCFH-DA/DAPI staining for analyzing the production of ROS (× 1200, Scar bar, 5 μm, n = 4 per group). E Flow cytometry analysis of mitochondrial superoxide levels ( n = 3 per group). F: BODIPY 493/503 staining for analyzing the mitochondrial membrane potential (× 200, Scar bar, 50 μm; n = 4 per group). Data were presented as the means ± SD. * P < 0.05 and ** P < 0.01 are significantly different between the indicated treatment groups; n.s., no significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Over Expression, Confocal Microscopy, Western Blot, Expressing, Control, Fluorescence, Injection, Staining, Flow Cytometry, Membrane

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: SIRT1 overexpression relieved ROS overgeneration and mitochondria dysfunction through the reduction of MAMs-mediated mitochondria Ca 2+ overload. A Confocal microscopy images displayed the colocalization of ER and mitochondria in AML12 cells with stable overexpression of SIRT1 transfected with Mock or Mito-ER linker vectors. The merge of ER Tracker Blue-White and Mito-Tracker Green indicates the colocalization. The Pearson’s coefficient graph quantifies the colocalization (× 1200, Scar bar, 5 μm; n = 4 per group). B Western blot analysis of expression of MAMs-related Ca 2 ⁺ channels (IP3R1, GRP75, and VDAC1), with β-Tubulin serving as a loading control. C Mitochondria Ca 2 ⁺ levels. The left panels show the time course of the pCAG-Mito-Gcamp5G. The fluorescence was recorded every 10 s. 100 µM ATP was injected at 100 s. The quantification of baseline and ATP-induced delta-peak are shown in the right panels ( n = 3 per group). D Flow cytometry analysis of ROS levels ( n = 3 per group). Data were presented as the means ± SD. * P < 0.05 and ** P < 0.01 are significantly different between the indicated treatment groups; n.s., no significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Over Expression, Confocal Microscopy, Transfection, Western Blot, Expressing, Control, Fluorescence, Injection, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: Activated SIRT1 blocked the aberrant formation of MAMs through the distribution of the interaction of MDM2 with MAMs-related Ca 2+ channels protein. A Differential gene expression of MAMs-related genes was analyzed by RNA-Seq from SIRT1-NC + PA vs. SIRT1-NC and SIRT1-NC + PA vs. SIRT1-OE + PA. Gray dots: Nonsignificant; Red dots: Significantly upregulated; Blue dots: Significantly downregulated; Black dots: MAMs-related genes. B Western blot analysis of MDM2 expression in MAMs fractions from the liver tissue of NAFLD mice treated with SRT1720 and PA-stimulated AML12 cells overexpressing SIRT1. Calreticulin served as a loading control. C Western blot analysis of MDM2 in whole-cell homogenates from the liver tissue of NAFLD mice treated with SRT1720 and PA-stimulated AML12 cells overexpressing SIRT1. β-Tubulin served as a loading control. D Interaction of MDM2 and MAMs-related Ca 2 ⁺ channels was analyzed by Co-IP in PA-stimulated AML12 cells overexpressing SIRT1 and in NAFLD mice treated with SRT1720. Data were presented as means ± SD. * P < 0.05 and ** P < 0.01 indicate significant differences between the indicated treatment groups; n.s., no significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Gene Expression, RNA Sequencing, Western Blot, Expressing, Control, Co-Immunoprecipitation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: Inhibition of MDM2 expression reduced the MAMs formation enrichment caused by PA-stimulation and Mito-ER linker transfected in hepatocytes. A Confocal microscopy images showing the colocalization of ER and mitochondria in AML12 cells transfected with MDM2 constructs under BSA or PA exposure for 24 h. Colocalization is indicated by the merge of DsRed2-ER-5 and Mito-Tracker Green, and Pearson’s coefficient graph quantifies the colocalization (× 1200; scale bar, 5 μm; n = 4 per group). B Western blot analysis of expression of MAMs-related Ca 2 ⁺ channels (IP3R1, GRP75, and VDAC1) in homogenates and MAMs fractions from AML12 cells. β-tubulin served as a loading control. C Co-IP analysis for IP3R1-GRP75-VDAC1 complex in AML12 cells transfected with MDM2 vector. D Confocal microscopy images showing the colocalization of ER and mitochondria. Colocalization is indicated by the merge of ER Tracker Blue-White and Mito-Tracker Green, and Pearson’s coefficient graph quantifies the colocalization (× 1200; scale bar, 5 μm; n = 4 per group). E Western blot analysis of expression of MAMs-related Ca 2 ⁺ channels (IP3R1, GRP75, and VDAC1) in homogenates and MAMs fractions from AML12 cells. β-tubulin served as a loading control. Data were presented as means ± SD. * P < 0.05 and ** P < 0.01 indicate significant differences between the indicated treatment groups; n.s., not significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Inhibition, Expressing, Transfection, Confocal Microscopy, Construct, Western Blot, Control, Co-Immunoprecipitation Assay, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Regulatory role of sirtuin-1 by targeting MDM2 to mitochondria-associated membranes formation in the treatment of NAFLD

doi: 10.1186/s12964-025-02251-7

Figure Lengend Snippet: Knockdown MDM2 and IP3R1 inhibited Ca 2+ dyshomeostasis and mitochondria dysfunction caused by PA stimulation in hepatocytes. A , B Measurements of mitochondrial Ca 2 ⁺ levels in AML12 cells transfected with MDM2 or IP3R1 vectors under BSA or PA exposure for 24 h. The upper panel shows the time course of pCAG-Mito-Gcamp5G fluorescence, recorded every 10 s. 100 µM ATP was injected at 100 s. The quantification of baseline and ATP-induced delta-peak values are shown in the right panels ( n = 3 per group). C Western blot analysis of mitochondrial respiratory chain proteins (ATP5A, UQCRC2, NDUFB8), with β-tubulin as a loading control. D Rhodamine 123 staining and BODIPY 493/503 staining for analyzing mitochondrial membrane potential and lipid droplets. Data were presented as means ± SD. * P < 0.05 and ** P < 0.01 are significantly different between the indicated treatment groups; n.s., not significant

Article Snippet: The human hepatocellular carcinoma cell line HepG2 and mouse hepatocyte cell line AML12 were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle medium (DMEM) and DMEM/F12 medium, respectively.

Techniques: Knockdown, Transfection, Fluorescence, Injection, Western Blot, Control, Staining, Membrane