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

Journal: Frontiers in Physiology

Article Title: Nitroalkene inhibition of pro-inflammatory macrophage effector function via modulation of signaling metabolite levels

doi: 10.3389/fphys.2025.1426102

Figure Lengend Snippet: Pharmacological inhibition of NOS2 by NO 2 -OA and 1400W results in the inhibition of NO production and pro-inflammatory cytokine secretion. Wild-type RAW 264.7 macrophages treated with vehicle (0.01% DMSO), NO 2 -OA (5 µM), or 1400W (100 µM) were stimulated with LPS (10 ng/mL). Oleic acid (OA, 5 µM), the non-electrophilic fatty acid precursor of NO 2 -OA, was used as a negative control. Culture media and lysates were collected 12 h post-treatment. (A) Immunoblot analysis was performed for NOS2 on RAW 264.7 macrophage lysates. β-Actin (housekeeping control) is shown. Original blot was probed for both NOS2 and ACOD1/ Irg1 ( ; ) such that the β-actin control is identical in both figures. (B) NOS2 by-products citrulline and nitrite were measured in cell lysates (citrulline) and media (nitrite) using liquid chromatography–high-resolution mass spectrometry and a UV spectrophotometer, respectively. (C) MCP-1 and TNF-α were measured through ELISA. p < 0.05 when compared to respective control (*), LPS (#), LPS + OA (†), and LPS + NO 2 -OA (‡) using ANOVA. Data are from n = 2 independent biological experiments, each comprising three technical replicates.

Article Snippet: To generate Irg1 −/− / ACOD1 −/− cells, wild-type RAW 264.7 cells (ATCC) were transfected with mouse IRG1 CRISPR/Cas9 KO plasmid and mouse IRG1 HDR plasmid (Santa Cruz, #sc-421149, Dallas, TX, United States) using the transfection reagent Viromer RED (Lipocalyx GmbH, Germany) at a 1:1 ratio with selection for clones using puromycin.

Techniques: Inhibition, Negative Control, Western Blot, Control, Liquid Chromatography, Mass Spectrometry, Spectrophotometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Physiology

Article Title: Nitroalkene inhibition of pro-inflammatory macrophage effector function via modulation of signaling metabolite levels

doi: 10.3389/fphys.2025.1426102

Figure Lengend Snippet: Pharmacological inhibition of NOS2 by 1400W does not recapitulate NO 2 -OA-induced changes in metabolism. Wild-type RAW 264.7 macrophages were treated with vehicle (0.01% DMSO, control), oleic acid (OA, 5 μM, control), NO 2 -OA (5 µM), or 1400W (100 µM), with and without LPS activation (10 ng/mL). (A) After 12 h of treatment, cells were harvested, and subsequent measurement of intracellular lactate, succinate, and itaconate was performed through liquid chromatography–high-resolution mass spectrometry. (B) Cells were treated and incubated with universally labeled 13 C glucose ([ 13 C 6 ]H 12 O 6 ) or non-labeled 12 C glucose for carbon tracing in lactate, succinate, and itaconate through liquid chromatography–high-resolution mass spectrometry. APE was calculated for isotopologues. (C) ACOD1 ( Irg1 ) expression in activated and treated macrophages was determined by immunoblotting. Original blot was probed for both iNOS and ACOD1/ Irg1 ( ; ) such that the β-actin control is identical in both figures. p < 0.05 when compared to respective control (*), 1400W (‡), LPS (#), LPS + OA (†), and LPS + NO 2 -OA (‡) using ANOVA; p < 0.05 compared to respective control within M + x (*), LPS (#), LPS + OA (†), and LPS + NO 2 -OA (‡) using ANOVA . Data are from n = 2 independent biological experiments, each comprising three technical replicates.

Article Snippet: To generate Irg1 −/− / ACOD1 −/− cells, wild-type RAW 264.7 cells (ATCC) were transfected with mouse IRG1 CRISPR/Cas9 KO plasmid and mouse IRG1 HDR plasmid (Santa Cruz, #sc-421149, Dallas, TX, United States) using the transfection reagent Viromer RED (Lipocalyx GmbH, Germany) at a 1:1 ratio with selection for clones using puromycin.

Techniques: Inhibition, Control, Activation Assay, Liquid Chromatography, Mass Spectrometry, Incubation, Labeling, Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: Nitroalkene inhibition of pro-inflammatory macrophage effector function via modulation of signaling metabolite levels

doi: 10.3389/fphys.2025.1426102

Figure Lengend Snippet: Deletion of Irg1 reduces levels of succinate and itaconate, but not IL-1β production. Wild-type RAW 264.7 macrophages (WT) and ACOD1 −/− / Irg1 −/− RAW 264.7 macrophages ( Irg1 −/− ) were treated with vehicle (0.01% DMSO, control), or NO 2 -OA (5 µM) was stimulated with LPS (10 ng/mL) for 12 h prior to harvest. (A) Lactate, succinate, and itaconate were measured through liquid chromatography–high-resolution mass spectrometry. (B) MCP-1, TNF-α, and IL-1β were measured in treated cells using ELISA. p < 0.05 when compared to respective control (*), WT LPS (#), WT LPS + NO 2 -OA (‡), Irg −/− vehicle (Ω), Irg −/− NO 2 -OA (◊), and Irg −/− LPS (⌂) using ANOVA. Data are from n = 2 independent biological experiments, each comprising three technical replicates.

Article Snippet: To generate Irg1 −/− / ACOD1 −/− cells, wild-type RAW 264.7 cells (ATCC) were transfected with mouse IRG1 CRISPR/Cas9 KO plasmid and mouse IRG1 HDR plasmid (Santa Cruz, #sc-421149, Dallas, TX, United States) using the transfection reagent Viromer RED (Lipocalyx GmbH, Germany) at a 1:1 ratio with selection for clones using puromycin.

Techniques: Control, Liquid Chromatography, Mass Spectrometry, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Soluble E-cadherin Drives Brain Metastasis in Inflammatory Breast Cancer

doi: 10.1101/2025.06.18.660428

Figure Lengend Snippet: (A) Human apoptosis array showed upregulation of XIAP in IBC cells that overexpress sEcad compared with controls. (B) Mass spectrometry and (C) BioID assays identified XIAP as a novel interaction partner of sEcad. (D) Exogenous immunoprecipitation assay confirmed the interaction between sEcad and XIAP. (E) Endogenous immunoprecipitation assay confirmed the interaction between sEcad and XIAP. (F) Domain mapping demonstrates that sEcad interacted with the BIR2 domain of XIAP. (G) Immunoblotting shows that sEcad enhanced XIAP expression, activated NFκB signaling, and inhibited the cleavage of caspase-3 in IBC cells.

Article Snippet: Human E-Cadherin Antibody (MAB1838), Recombinant Human E-Cadherin Protein-CF (sEcad protein) (8505-EC) , Human E-Cadherin Quantikine ELISA Kit (#DCADE0B), Human CXCL1/GRO alpha Quantikine ELISA Kit (#DGR00B), Human IL-8/CXCL8 Quantikine ELISA Kit (#D8000C), Human Dkk-1 Quantikine ELISA Kit (#DKK100B), Proteome Profiler Human Cytokine Array Kit (#ARY005B), and Proteome Profiler Human Apoptosis Array Kit (ARY009) were purchased from R&D system (USA).

Techniques: Mass Spectrometry, Immunoprecipitation, Western Blot, Expressing

Journal: Cancer Discovery

Article Title: Enhancing PD-L1 Degradation by ITCH during MAPK Inhibitor Therapy Suppresses Acquired Resistance

doi: 10.1158/2159-8290.CD-21-1463

Figure Lengend Snippet: The E3 ligase ITCH interacts with PD-L1 and PD-L2. A, Spectral count of indicated proteins from shotgun mass spectrometry–based analysis of anti-FLAG immunoprecipitates from the M229 R5 cell line engineered to express PD-L2-FLAG ( n = 2) or empty vector (control, n = 4). Average total spectral count in samples: 5,156 (control), 4,229 (anti-FLAG), mean ± SEMs. Proteins are shown with confidence score for interaction [Significance Analysis of INTeractome (SAINT)] higher than 0.95. B, Proteins identified in proximity to PD-L1 in the M229 R5 cell line stably expressing PD-L1–APEX2 (H 2 O 2 treated vs. not treated, n = 3). Black dots, proteins significantly enriched in H 2 O 2 -treated cell. C, Immunoprecipitation (IP) of HEK 293T cells (transfected with PD-L1 and ITCH-FLAG, alone or together) using anti-FLAG M2 beads, followed by Western blots (WB) of indicated proteins. TUBULIN, loading control. D, IP of HEK 293T cells (transfected with PD-L2-FLAG and/or ITCH-HA) using anti-HA beads, followed by WBs of indicated proteins. TUBULIN, loading control. E, IP of the human melanoma cell line M238 R1 stably expressing ITCH using anti–PD-L1 antibody (rabbit-IgG as control), followed by WBs of indicated proteins. TUBULIN, loading control. F, IP of the human non–small cell lung carcinoma cell line H358, with or without ITCH overexpression (OE), using anti–PD-L1 (rabbit-IgG as control) followed by WBs of indicated proteins. TUBULIN, loading control. Vec, empty vector. G, PD-L1 protein levels compared by splitting 7,194 patient-derived tumors with matched RNA-seq data and PD-L1 protein levels (measured by reverse-phase protein array from 32 TCGA cancer types) into top versus bottom 20% of ITCH RNA expression levels. P value, Student t test. H, Kaplan–Meier survival curve of 471 cutaneous melanoma patients from TCGA data set with high (top 20%) versus low (bottom 20%) ITCH RNA expression levels. Time of diagnosis is set as the starting time for follow-up. P value, log-rank test.

Article Snippet: The human cell lines HEK 293T (ATCC, CRL-3216, RRID:CVCL_0063), M229 R5 (RRID:CVCL_IM73), M238 R1 (RRID:CVCL_IM74), H358 (ATCC, CRL-5807, RRID:CVCL_1559), and MDA-MB-231 (ATCC, CRM-HTB-26, RRID:CVCL_0062) and mouse cell lines mSK-Mel254, KPC, and CT26 (ATCC, CRL-2639, RRID:CVCL_7254) were maintained in high-glucose DMEM (Omega Scientific, DM-22) with 10% heat-inactivated FBS (Omega Scientific, FB-02) and 2 mmol/L glutamine.

Techniques: Mass Spectrometry, Plasmid Preparation, Control, Stable Transfection, Expressing, Immunoprecipitation, Transfection, Western Blot, Over Expression, Derivative Assay, RNA Sequencing, Protein Array, RNA Expression, Biomarker Discovery

Journal: Cancer Discovery

Article Title: Enhancing PD-L1 Degradation by ITCH during MAPK Inhibitor Therapy Suppresses Acquired Resistance

doi: 10.1158/2159-8290.CD-21-1463

Figure Lengend Snippet: ITCH polyubiquitinates PD-L1 and promotes T-cell activation by downregulating tumor cell–surface levels of PD-L1. A–C, Left, Western blots (WB) of M238 R1 ( A ), H358 ( B ), or MDA-MB-231 ( C ) cells stably expressing control shRNA (shCtr) or ITCH-targeting shRNAs (shITCH-1 and shITCH-2). GAPDH or TUBULIN, loading control. Right, cell-surface levels of PD-L1 were measured by cell-surface staining and flow cytometry analysis (right). MFI, mean fluorescence intensity. Mean ± SEMs ( n = 3). D, Left, WBs of M238 R1 cells stably expressing empty vector (Vec) or overexpressing (OE) ITCH. TUBULIN, loading control. Right, cell-surface levels of PD-L1 were measured by cell-surface staining and FACS analysis. Mean ± SEMs ( n = 3). E, HEK 293T cells transiently expressing PD-L1-FLAG, with or without ITCH cotransfection, were pretreated with or without MG-132 (20 μmol/L) for 4 hours, followed by anti-FLAG immunoprecipitation (IP) and detection of ubiquitin (UB) by WBs. TUBULIN, loading control. F and G, M238 R1 ( F ) or H358 ( G ) cells expressing shCtr or ITCH shRNAs were pretreated with MG-132 (20 μmol/L) for 4 hours, followed by anti–PD-L1 IP and UB detection by WBs. TUBULIN, loading control. H, M238 R1 cells expressing Vec or OE ITCH were pretreated with MG-132 (20 μmol/L) for 4 hours followed by anti–PD-L1 IP and then WB detection of UB. GAPDH, loading control. I, Schematic of the coculture assay used in J – L . J and K, Left, WBs of indicated proteins in NYESO-HLA-A2–expressing M238 R1 ( J ) or H358 ( K ) cells with shCtr or ITCH shRNA stable expression. TUBULIN, loading control. Right, IL2 production was measured by ELISA after coculture experiments with indicated cell lines. Mean ± SEMs ( n = 3). L, IL2 production was measured by ELISA after coculture experiment with indicated cell lines and treatment. Anti–PD-1 and anti–PD-L1 antibodies were added to the culture media at the final concentration of 10 μg/mL (each antibody) and preincubated with Jk T cells for 30 minutes before coculture with target cells. Mean ± SEMs ( n = 4). M and N, IL2 production measured by ELISA after coculture of human PBMCs (hPBMC) with M238 R1 ( M ) or H358 ( N ) cell lines. Anti-CD3 and anti-CD28 antibodies were added at the final concentration of 1 μg/mL (each antibody) to activate hPBMCs. Anti–PD-1 and anti–PD-L1 antibodies were added at the final concentration of 10 μg/mL (each antibody). Mean ± SEMs ( n = 4). P values, Student t test. **, P < 0.01; ***, P < 0.001.

Article Snippet: The human cell lines HEK 293T (ATCC, CRL-3216, RRID:CVCL_0063), M229 R5 (RRID:CVCL_IM73), M238 R1 (RRID:CVCL_IM74), H358 (ATCC, CRL-5807, RRID:CVCL_1559), and MDA-MB-231 (ATCC, CRM-HTB-26, RRID:CVCL_0062) and mouse cell lines mSK-Mel254, KPC, and CT26 (ATCC, CRL-2639, RRID:CVCL_7254) were maintained in high-glucose DMEM (Omega Scientific, DM-22) with 10% heat-inactivated FBS (Omega Scientific, FB-02) and 2 mmol/L glutamine.

Techniques: Activation Assay, Western Blot, Stable Transfection, Expressing, Control, shRNA, Staining, Flow Cytometry, Fluorescence, Plasmid Preparation, Cotransfection, Immunoprecipitation, Ubiquitin Proteomics, Co-culture Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cancer Discovery

Article Title: Enhancing PD-L1 Degradation by ITCH during MAPK Inhibitor Therapy Suppresses Acquired Resistance

doi: 10.1158/2159-8290.CD-21-1463

Figure Lengend Snippet: AK087 is an ITCH activator that downregulates tumor cell–surface PD-L1/L2 and suppresses MAPKi resistance in vivo . A, Structure and chemical name of AK087. B, M238 R1 cells with ITCH-FLAG overexpression were treated with 0, 40, or 80 μmol/L of AK087 for 6 days and then treated with MG-132 (20 μmol/L) for 4 hours, followed by anti-FLAG or anti–PD-L1 immunoprecipitation (IP) and Western blot (WB) detection of indicated proteins [ubiquitin (UB)]. GAPDH, loading control. C, HEK 293T cells cotransfected with PD-L1-FLAG and ITCH-HA were treated with 0, 40, or 80 μmol/L of AK087 for 6 days and then treated with MG-132 (20 μmol/L) for 4 hours, followed by anti-HA or anti-FLAG IP and WB detection of indicated proteins. GAPDH, loading control. D, M238 R1 cell-surface levels of PD-L1 (left) and PD-L2 (right) as measured by cell-surface staining and FACS analysis after 0, 20, 40, or 80 μmol/L AK087 treatment for 6 days. Mean ± SEMs ( n = 3). E, As in D , except for H358 (PD-L2 is undetectable in H358). F and G, WBs of total PD-L1 protein levels in M238 R1 ( F ) and H358 ( G ) cells after 6 days of treatment with vehicle (V or DMSO) or 20, 40, or 80 μmol/L of AK087 (AK). GAPDH, loading control. H, Growth curves of YUMM1.7ER tumors on trametinib (Tram; 0.45 mg/kg/day) in combination with daily vehicle (4% Tween80 + 8% DMSO in double-distilled water) or AK087 (10 mg/kg/day, from D0–D17) treatment in C57BL/6 mice. Mean ± SEMs ( n = 9). CR, complete response. I, Weekly body weights of mice in H . J, Growth curves of NILER1-4 tumors on daily vehicle (4% Tween80 + 8% DMSO in double-distilled water), AK087 (10 mg/kg/day, from D0–D9), Tram (2 mg/kg/day) in combination with daily vehicle or AK087 (10 mg/kg/day, from D0–D18) treatment in C57BL/6 mice. Mean ± SEMs ( n = 9–10). K, Weekly body weights of mice in J . P values, Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The human cell lines HEK 293T (ATCC, CRL-3216, RRID:CVCL_0063), M229 R5 (RRID:CVCL_IM73), M238 R1 (RRID:CVCL_IM74), H358 (ATCC, CRL-5807, RRID:CVCL_1559), and MDA-MB-231 (ATCC, CRM-HTB-26, RRID:CVCL_0062) and mouse cell lines mSK-Mel254, KPC, and CT26 (ATCC, CRL-2639, RRID:CVCL_7254) were maintained in high-glucose DMEM (Omega Scientific, DM-22) with 10% heat-inactivated FBS (Omega Scientific, FB-02) and 2 mmol/L glutamine.

Techniques: In Vivo, Over Expression, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Control, Staining

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: SW900 (squamous cell lung carcinoma) , ATCC , ATCC Cat# HTB-59, RRID:CVCL_1731.

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software

Journal: Molecular Oncology

Article Title: Melphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma

doi: 10.1016/j.molonc.2015.12.013

Figure Lengend Snippet: Mel‐flufen induces cytotoxicity in UC cells. J82, RT4, 5637 and TCC‐SUP UC cells were treated with mel‐flufen or melphalan for 72 h. (A) Photos showing cell morphology upon treatment. (B) Cell viability after 72 h of continuous treatments with either mel‐flufen (upper panel) or melphalan (lower panel). Data shown is the mean ± SD from 3 experiments. The ratio between mel‐flufen and melphalan for the cell lines is presented in Supplementary Figure. S1.

Article Snippet: Cell lines, cell culture, and chemicals The UC cell lines J82 (ATCC ® HTB‐1TM), TCC‐SUP (ATCC ® HTB‐5TM), 5637 (ATCC ® HTB‐9TM), and RT4 (ATCC ® HTB‐2TM) were obtained from American Type Culture Collection (ATCC, Manassas, VA) ( Fogh et al., 1977 ; Nayak et al., 1977 ; O'Toole et al., 1978 ; Rigby and Franks, 1970 ).

Techniques:

Journal: Molecular Oncology

Article Title: Melphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma

doi: 10.1016/j.molonc.2015.12.013

Figure Lengend Snippet: Mel‐flufen treatment of UC cells results in increased loading of melphalan. J82 cells were treated with mel‐flufen (1 μM) or melphalan (10 μM). The intracellular concentration of mel‐flufen, de‐esterified mel‐flufen or melphalan was measured using HPLC‐MS/MS. (A) Solid line: intracellular concentration of mel‐flufen, de‐esterified mel‐flufen or melphalan after mel‐flufen treatment. Dotted line: intracellular concentration of melphalan after melphalan treatment. (B) AUC 0–60 min after mel‐flufen (1 μM) or melphalan (10 μM) treatment. Please note the 10‐fold difference in concentration and the 1.7 fold higher loading of melphalan after mel‐flufen treatment. (A–B) is based on 3 biological replicates. (C) γH2AX was examined in J82 cells after 1 h treatment of mel‐flufen or melphalan and 24 h post incubation in which GAPDH served as loading control.

Article Snippet: Cell lines, cell culture, and chemicals The UC cell lines J82 (ATCC ® HTB‐1TM), TCC‐SUP (ATCC ® HTB‐5TM), 5637 (ATCC ® HTB‐9TM), and RT4 (ATCC ® HTB‐2TM) were obtained from American Type Culture Collection (ATCC, Manassas, VA) ( Fogh et al., 1977 ; Nayak et al., 1977 ; O'Toole et al., 1978 ; Rigby and Franks, 1970 ).

Techniques: Concentration Assay, Tandem Mass Spectroscopy, Incubation, Control

Journal: Molecular Oncology

Article Title: Melphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma

doi: 10.1016/j.molonc.2015.12.013

Figure Lengend Snippet: Mel‐flufen induces apoptosis in UC cell lines. (A) J82 cells were treated with mel‐flufen (red bars) or melphalan (grey bars) for 1 h and post incubated for 48 h. The nuclear morphology of the cells was assessed by DAPI‐staining and subsequent visualization in the FL‐1 channel of a Fluorescence microscope. The percentage of cells showing apoptotic nuclear morphology of 200 cells examined is given. Results shown are the mean ± SD from three biological replicates. Statistical difference between mel‐flufen and melphalan were reached at doses 0.5 μM, 1 μM and 5 μM (P = 0.0009, P = 0.0087 and P = 0.002 respectively). (B) Apoptosis was confirmed in J82 cells by analysing PARP‐1 and caspase‐9 cleavage by western blot treated as in (A) β‐tubulin and GAPDH was used as loading control for PARP‐1 and caspase‐9 analyses respectively. For PARP, the fold ratio CL/FL was determined by densitometry and is presented. (C) Bak or Bax activation in J82 cells was analysed by flow cytometry after 1 h treatment with mel‐flufen or melphalan, or after 24 h with cisplatin. Left: histogram showing Bak or Bax activation as a shift to the right in the diagram. Filled: untreated; unfilled: mel‐flufen treated. Right: Fold Bax or Bak mean IFL is presented relative to untreated cells. Data shown are the mean ± SD from three biological replicates. Upper panel: Bax, t‐test mel‐flufen 1 μM vs. melphalan 1 μM P = 0.012. Lower panel: Bak, t‐test mel‐flufen 1 μM vs. melphalan 1 μM P = 0.07.

Article Snippet: Cell lines, cell culture, and chemicals The UC cell lines J82 (ATCC ® HTB‐1TM), TCC‐SUP (ATCC ® HTB‐5TM), 5637 (ATCC ® HTB‐9TM), and RT4 (ATCC ® HTB‐2TM) were obtained from American Type Culture Collection (ATCC, Manassas, VA) ( Fogh et al., 1977 ; Nayak et al., 1977 ; O'Toole et al., 1978 ; Rigby and Franks, 1970 ).

Techniques: Incubation, Staining, Fluorescence, Microscopy, Western Blot, Control, Activation Assay, Flow Cytometry

Journal: Molecular Oncology

Article Title: Melphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma

doi: 10.1016/j.molonc.2015.12.013

Figure Lengend Snippet: Combined mel‐flufen and gemcitabine or cisplatin treatment decreases cell survival in UC cells. (A) Left: J82 cells were treated with either mel‐flufen (0.1 or 0.5 μM), gemcitabine (0.025 μM), or a combination, during 1 h and post incubated for 72 h. The number of cells were counted using Trypan blue and is presented as % of untreated control. Data shown is the mean ± SD of three experiments. Student's t‐test was used to achieve the indicated P‐values. Right: PARP‐1 cleavage was examined in J82 cells after mel‐flufen, gemcitabine, and combined treatment. GAPDH was used as loading control. Fold ratio CL/FL PARP was determined by densitometry. (B) Left: J82 cells were treated with either mel‐flufen (0.1 μM) or cisplatin (1 μM) for 1 h and post incubated for another 48 h and counted as in (A). Right: PARP‐1 cleavage was examined in J82 cells after mel‐flufen, cisplatin, and their combination. GAPDH was used as loading control.

Article Snippet: Cell lines, cell culture, and chemicals The UC cell lines J82 (ATCC ® HTB‐1TM), TCC‐SUP (ATCC ® HTB‐5TM), 5637 (ATCC ® HTB‐9TM), and RT4 (ATCC ® HTB‐2TM) were obtained from American Type Culture Collection (ATCC, Manassas, VA) ( Fogh et al., 1977 ; Nayak et al., 1977 ; O'Toole et al., 1978 ; Rigby and Franks, 1970 ).

Techniques: Incubation, Control

Journal: Molecular Oncology

Article Title: Melphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma

doi: 10.1016/j.molonc.2015.12.013

Figure Lengend Snippet: Kinome profiling of UC cells after mel‐flufen treatment reveal Src as a driver of mel‐flufen refractoriness and identifies possible combination regimen. (A) The phosphorylation status of kinases and growth factor receptors were profiled in J82 cell lysates after mel‐flufen (1 μM, 24 h), melphalan (1 μM, 24 h), or cisplatin (1 μM, 24 h) exposure using PathScan RTK signalling antibody array. The phosphorylation levels of the kinases in untreated J82 cells were arbitrary set to 100 and the values in the treated samples calculated accordingly. Red bars: kinases/growth factor receptors showing at least 1.3 fold increased phosphorylation level as compared to untreated cells; Green bars: kinases/growth factor receptors showing at least 1.3 fold decreased phosphorylation level as compared to untreated cells; Grey bars: non‐altered kinases/growth factor receptors. (B) Validation of Src phosphorylation at Y416 in J82 cells using western blot with GAPDH as loading control (upper panel). Quantification of Src phosphorylation in J82 cells after indicated treatments. The western blot values were obtained by densitometry of the bands and the PathScan RTK signalling antibody array from values obtained as in (A). (C) Left: J82 cells were treated with dasatinib for 48 h and effect on Src phosphorylation at Y416 was examined by western blot with GAPDH as loading control. Right: J82 cells were treated with 20 nM dasatinib for 16 h and thereafter, with mel‐flufen for 1 h and post incubated for 48 h. The effect on Src phosphorylation at Y416 was examined by western blot in which GAPDH was used as loading control. (D) J82 cells were treated as in (C, right) and effect on cell morphology (left) and cytotoxicity (right) examined. Left: Photos showing cell morphology after indicated treatments. Right: Cells were counted using Trypan blue and is depicted as % of untreated control. Data presented is the mean ± SD in three experiments. Student's t‐test was applied.

Article Snippet: Cell lines, cell culture, and chemicals The UC cell lines J82 (ATCC ® HTB‐1TM), TCC‐SUP (ATCC ® HTB‐5TM), 5637 (ATCC ® HTB‐9TM), and RT4 (ATCC ® HTB‐2TM) were obtained from American Type Culture Collection (ATCC, Manassas, VA) ( Fogh et al., 1977 ; Nayak et al., 1977 ; O'Toole et al., 1978 ; Rigby and Franks, 1970 ).

Techniques: Phospho-proteomics, Ab Array, Biomarker Discovery, Western Blot, Control, Incubation

Journal: Molecular Oncology

Article Title: Melphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma

doi: 10.1016/j.molonc.2015.12.013

Figure Lengend Snippet: Mel‐flufen intracellular loading and cell death is controlled by aminopeptidases and Aminopeptidase N display increased expression in clinical UC specimens. (A) Left: J82 cells were treated with 1 μM mel‐flufen alone or in combination with 5 μM bestatin. The concentrations of melphalan were measured using HPLC‐MS/MS. Data shown is the mean of three biological replicates. Right: Mel‐flufen‐induced cytotoxicity ± bestatin (5 μM) was analysed in J82 cells at 72 h after 1 h pulse treatment with indicated doses of mel‐flufen using MTT‐assay. Data shown are the mean ± SD of three biological replicates. (B) Immunohistochemical staining of ANPEP in primary urothelial carcinoma. Micrographs of UC specimens determined as negative or positive for ANPEP staining. Scale bar = 200 and 100 μm, respectively. (C) Distribution of immunohistochemistry ANPEP staining intensity (upper) and semi‐quantitative scoring of number of positive cells (lower) in UC specimen from 83 patients. (D) ANPEP expression in relation to overall survival (top) and disease‐free survival (bottom) by Kaplan–Meier method. Days from cystectomy is stated. ANPEP expression was stratified as high (Histoscore > 5, red solid line) or low (Histoscore < 5, blue dotted line). High tumour expression was significantly associated with better OS (P = 0.02, upper panel), but not significant for DFS (P = 0.094, lower panel).

Article Snippet: Cell lines, cell culture, and chemicals The UC cell lines J82 (ATCC ® HTB‐1TM), TCC‐SUP (ATCC ® HTB‐5TM), 5637 (ATCC ® HTB‐9TM), and RT4 (ATCC ® HTB‐2TM) were obtained from American Type Culture Collection (ATCC, Manassas, VA) ( Fogh et al., 1977 ; Nayak et al., 1977 ; O'Toole et al., 1978 ; Rigby and Franks, 1970 ).

Techniques: Expressing, Tandem Mass Spectroscopy, MTT Assay, Immunohistochemical staining, Staining, Immunohistochemistry