Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Identification of the heat shock proteins HSP70 and HSP90 as putative ChAT protein-interactors by proximity-dependent biotin identification (BioID). (A) Optimization of BioID in HEK293 cells expressing wild-type or P17A/P19A-ChAT fused to the HA-tagged promiscuous biotin ligase BirA-R118G (BirA * ). Control cells were transfected with empty vector or plasmids encoding either untagged ChAT or BirA * . Cells were treated for 24 h with either 50 μM biotin to facilitate proximity-dependent biotinylation of ChAT-interacting cellular proteins or with vehicle-control (water). Biotinylated proteins were isolated from cell lysates by streptavidin pull-downs (PD: Strep) and immunoblotted as indicated ( n = 2). (B) Identification of HSP70 and HSP90 as ChAT proximally-interacting proteins. Streptavidin PD samples prepared from biotin-treated HEK293 cells expressing wild-type-ChAT-BirA * or P17A/P19A-ChAT-BirA * fusion proteins were resolved and visualized on a silver-stained SDS-PAGE gels. Two proteins (~70 and ~90 kDa) that were enriched in samples expressing P17A/P19A-ChAT-BirA * were identified by MALDI-TOF-MS or LC-ESI-MS/MS as HSP70 and HSP90, respectively. Control cells were transfected to express untagged wild-type ChAT ( n = 1). (C) Confirmation of endogenous HSP70 and HSP90 as putative ChAT-interacting proteins by immunoblotting of streptavidin PD samples prepared from biotin-treated HEK293 cells expressing HA-tagged wild-type-ChAT-BirA * or P17A/P19A-ChAT-BirA * . Control cells were transfected with empty vector or vector encoding untagged ChAT ( n = 4).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Staining, SDS Page, Tandem Mass Spectroscopy, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Co-immunoprecipitation (co-IP) of ChAT with heat shock proteins HSC70, HSP70, and HSP90 is altered by mutation of N-terminal proline-rich motif in ChAT. (A) Immunoblots showing co-IP of ChAT with endogenous HSC70, HSP70 and HSP90 from HEK293 cells expressing either wild-type or P17A/P19A-ChAT. Control cells were transfected with empty vector. Using HEK293 cells, co-IP of P17A/P19A-ChAT with HSP70 (B) , HSP90 (C) and HSC70 (D) , respectively, is greater than that of wild-type ChAT ( *** p ≤ 0.001, Student's t -test, mean ± SEM, n = 4). (E) Co-IP of ChAT with endogenous HSC70 and HSP90 from mouse cholinergic SN56 cells expressing either wild-type or P17A/P19A-ChAT or CMS-related mutant proteins V18M- or A513T-ChAT. Control cells were transfected with empty vector. (F) Using SN56 cells, Co-IP of P17A/P19A-ChAT ( *** p ≤ 0.001) and V18M-ChAT ( * p ≤ 0.05), but not A531T-ChAT, with HSC70 is greater than that of wild-type ChAT (mean ± SEM, n = 5). (G) While there was a trend toward increased HSP90 interaction with P17A/P19A-ChAT ( p = 0.09), no significant differences were observed for HSP90 interaction with mutant ChAT compared to wild-type ChAT in SN56 cells (mean ± SEM, n = 5). Statistical analysis for (F) and (G) was performed by one-way ANOVA with Dunnett's post-hoc test. (H) Detection of in situ interactions of wild-type ChAT with endogenous HSC70 and HSP90 by proximity ligation assay (PLA) in SN56 cells. Formalin-fixed cells were first co-labeled with goat anti-ChAT together with either mouse anti-HSC70 or mouse anti-HSP90 primary antibodies, then incubated with oligonucleotide-linked secondary antibodies. Following DNA ligation and DNA amplification using the Duolink in Situ Orange Kit (Sigma), in situ ChAT-HSP interactions were imaged by confocal microscopy. Positive in situ ChAT-HSP interactions where visualized as fluorescent red dots while nuclei were stained with DAPI (blue). Control cells were either transfected with empty vector or had primary antibodies omitted from the assay (No 1° antibodies). Images are representative of 3 independent experiments; scale bars are 10 μm.

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Expressing, Transfection, Plasmid Preparation, In Situ, Proximity Ligation Assay, Labeling, Incubation, DNA Ligation, Amplification, Confocal Microscopy, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Inhibition of HSP90 ATPase activity specifically reduces steady-state levels of mutant ChAT protein. Immunoblots from SN56 cells expressing wild-type (A) , P17A/P19A- (B) , V18M- (C) , or A513T-ChAT (D) that were treated for 24 h with 0.5–2 μM with 17-AAG, an inhibitor of HSP90 activity, or with DMSO-vehicle. (E) Treatment of cells with 17-AAG at concentrations up to 2 μM has no effect on the steady-state levels of wild-type ChAT protein. Conversely, steady-state protein levels of P17A/P19A- (F) , V18M- (G) , and A513T-ChAT (H) are reduced following treatment of cells with 17-AAG compared to vehicle-control ( *** p ≤ 0.001; one-way ANOVA with Dunnett's post-hoc test, mean ± SEM, n = 4). (I) Proteasome inhibition by co-treatment with 5 μM MG132 for 18 h attenuates the effects of inhibition of HSP90 (1 μM 17-AAG, 24 h) on ChAT steady-state protein levels in ChAT-expressing SN56 cells. Control cells were treated with DMSO-vehicle. Proteasome inhibition was validated by immunoblotting for the accumulation of ubiquitinated cellular proteins ( n = 4). (J) Lysosomal inhibition by co-treatment with 50 μM chloroquine (CQ) for 18 h did not prevent the effects of 17-AAG treatment (1 μM, 24 h) on steady-state ChAT protein levels in SN56 cells. Lysosomal inhibition was validated by immunoblotting for the accumulation of the lysosome-associated protein LC3B-II ( n = 3).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Inhibition, Activity Assay, Mutagenesis, Western Blot, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Chaperone-Mediated Regulation of Choline Acetyltransferase Protein Stability and Activity by HSC/HSP70, HSP90, and p97/VCP

doi: 10.3389/fnmol.2017.00415

Figure Lengend Snippet: Inhibition of HSC/HSP70 and HSP90 activity enhances ChAT ubiquitination and reduces cellular ChAT enzymatic activity. (A) Immunoblots following anti-ChAT IP from ChAT-expressing SN56 cells that were co-treated for 24 h with 40 μM VER-155008 and for the final 6 h with 20 μM MG132 to inhibit degradation of ubiquitinated ChAT. Control cells were transfected with empty vector and/or treated with DMSO-vehicle. Inhibition of HSC/HSP70 by VER-155008 treatment enhanced ChAT ubiquitination, where levels of ubiquitinated mutant ChAT, particularly P17A/P19A-ChAT, are greater than that of wild-type ChAT ( n = 3). (B) Immunoblots following anti-ChAT IP from ChAT-expressing SN56 cells that were co-treated for either 8 or 24 h with 1 μM 17-AAG and for the final 6 h with 20 μM MG132. Inhibition of HSP90 by treatment with 1 μM 17-AAG for 8 h, but not for 24 h, enhanced ChAT ubiquitination where levels of ubiquitinated mutant ChAT, particularly P17A/P19A-ChAT, are greater than that of wild-type ChAT ( n = 3). (C) Cellular activity of wild-type ChAT is reduced following treatment of SN56 cells for 24 h with either 40 μM VER-155008 or 1 μM 17-AAG compared to vehicle-treated (DMSO) cells ( ** p ≤ 0.01). Treatment with VER-155008 trended toward a greater reduction in ChAT activity compared to 17-AAG ( p = 0.074; one-way ANOVA with Tukey's post-hoc test, mean ± SEM, n = 3).

Article Snippet: Cells were washed with HBSS, formalin-fixed (4% paraformaldehyde in HBSS) for 15 min, permeabilized with 0.1% Triton X-100, blocked for 1 h in HBSS supplemented with 3% donkey serum, then finally incubated for 1 h with primary antibodies targeting ChAT (1:100; Chemicon, goat primary) together with either endogenous HSC70 (1:100; StressMarq, mouse primary), HSP90 (1:200; StressMarq, mouse primary) or CHIP (1:200; Santa Cruz, rabbit primary); all steps were performed at room temperature.

Techniques: Inhibition, Activity Assay, Western Blot, Expressing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

doi: 10.1074/jbc.m110.141580

Figure Lengend Snippet: FIGURE 1. Schematic representation of the Hsp90 occupancy assay. A drug-treated cancer cell lysate (sample) was passed over a gel filtration spin column at 4 °C, and the sample was split into two aliquots. In one sample, total Hsp90 was determined by quantitative immunoblotting using sepa- rate antibodies to detect both Hsp90 and Hsp90 isoforms. In the second sample, open Hsp90 binding sites were titrated with [3H]17-AAG at 4 °C. Percent of Hsp90 occupancy was calculated from a ratio of Hsp90 open binding sites to total Hsp90.

Article Snippet: Anti-Hsp90 antibody (clone 68) was from BD Biosciences; anti-Hsp90 antibody (clone H-114) and anti-HER2 (C-18) from Santa Cruz Biotechnology; and anti-EGFR, Akt (protein kinase B), and cRaf antibodies were from Cell Signaling (Beverly, MA).

Techniques: Filtration, Western Blot, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

doi: 10.1074/jbc.m110.141580

Figure Lengend Snippet: FIGURE 2. Dissociation of [3H]17-AAG from purified Hsp90 and SK-BR-3 lysates is highly temperature-dependent. Purified Hela Hsp90 (100 nM) or lysate Hsp90[3H]17-AAG complexes were formed as described under “Ex- perimental Procedures” and passed over size exclusion spin columns. Col- umn eluates were incubated with 10 M cold 17-AAG, and samples were removed at different time points. A loss of bound radioactive 17-AAG counts from Hsp90 was measured at both 4 and 37 °C (A) and Hsp90 in SK-BR-3 cancer cell lysate at 4 °C (B). The data were fit to a monoexponential decay equation.

Article Snippet: Anti-Hsp90 antibody (clone 68) was from BD Biosciences; anti-Hsp90 antibody (clone H-114) and anti-HER2 (C-18) from Santa Cruz Biotechnology; and anti-EGFR, Akt (protein kinase B), and cRaf antibodies were from Cell Signaling (Beverly, MA).

Techniques: Purification, Incubation

Journal: Journal of Biological Chemistry

Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

doi: 10.1074/jbc.m110.141580

Figure Lengend Snippet: FIGURE 3. Binding of [3H]17-AAG to purified Hela Hsp90 and SK-BR-3 lysates at 4 °C. The binding reaction was initiated by adding 10 M [3H]17- AAG to 100 nM purified Hsp90 or to SK-BR-3 lysate containing 100 nM Hsp90. Drug association was measured at 4 °C by a time-dependent in- crease in protein bound counts for purified Hsp90 (A) or Hsp90 in SK-BR-3 cell lysate (B). Data were fitted by nonlinear regression to a single exponen- tial equation to obtain a (kobs) value. Half-life was calculated using the equa- tion t1⁄2 0.693/kobs.

Article Snippet: Anti-Hsp90 antibody (clone 68) was from BD Biosciences; anti-Hsp90 antibody (clone H-114) and anti-HER2 (C-18) from Santa Cruz Biotechnology; and anti-EGFR, Akt (protein kinase B), and cRaf antibodies were from Cell Signaling (Beverly, MA).

Techniques: Binding Assay, Purification

Journal: Journal of Biological Chemistry

Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

doi: 10.1074/jbc.m110.141580

Figure Lengend Snippet: FIGURE 4. Hsp90 open binding sites can be titrated with [3H]17-AAG. Recombinant human Hsp90 protein (100 nM) was incubated with increas- ing concentrations of unlabeled 17-AAG overnight at 4 °C followed by re- moval of unbound 17-AAG with prechilled size exclusion columns. Free Hsp90 sites were titrated with [3H]17-AAG at 4 °C as described under “Ex- perimental Procedures.” The data from triplicate binding experiments were fit to a four parameter logistic equation.

Article Snippet: Anti-Hsp90 antibody (clone 68) was from BD Biosciences; anti-Hsp90 antibody (clone H-114) and anti-HER2 (C-18) from Santa Cruz Biotechnology; and anti-EGFR, Akt (protein kinase B), and cRaf antibodies were from Cell Signaling (Beverly, MA).

Techniques: Binding Assay, Recombinant, Incubation

Journal: Journal of Biological Chemistry

Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

doi: 10.1074/jbc.m110.141580

Figure Lengend Snippet: FIGURE 5. Determination of Hsp90 occupancy in living cells. H1650 cells were incubated with increasing concentrations of IPI-504 for 6 h at 37 °C. Total Hsp90 protein levels were determined by quantitative immunoblot- ting using separate anti Hsp90 and Hsp90 antibodies and recombinant proteins as internal standards (A). Percent Hsp90 occupancy was deter- mined by titration of open binding sites at 4 °C and total Hsp90 (B and C). Data are from a representative experiment with n 2.

Article Snippet: Anti-Hsp90 antibody (clone 68) was from BD Biosciences; anti-Hsp90 antibody (clone H-114) and anti-HER2 (C-18) from Santa Cruz Biotechnology; and anti-EGFR, Akt (protein kinase B), and cRaf antibodies were from Cell Signaling (Beverly, MA).

Techniques: Incubation, Western Blot, Recombinant, Titration, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

doi: 10.1074/jbc.m110.141580

Figure Lengend Snippet: FIGURE 6. Effect of Hsp90 occupancy on client protein abundance. H1650 cells were incubated for 6 or 24 h with increasing concentrations of IPI-504. HER2, mEGFR, Akt, and cRaf protein levels were assessed by immunoblotting (A and C). The fraction of degraded proteins was assessed by densitometry compared with untreated samples and plotted together with the occupancy curves (B and D).

Article Snippet: Anti-Hsp90 antibody (clone 68) was from BD Biosciences; anti-Hsp90 antibody (clone H-114) and anti-HER2 (C-18) from Santa Cruz Biotechnology; and anti-EGFR, Akt (protein kinase B), and cRaf antibodies were from Cell Signaling (Beverly, MA).

Techniques: Quantitative Proteomics, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

doi: 10.1074/jbc.m110.141580

Figure Lengend Snippet: FIGURE 7. Hsp90 occupancy is a better predictor of in vivo pharmacody- namic effects by IPI-504 than tumor or plasma PK. H1650 tumor-bearing mice were treated with a single dose of 100 mg/kg intravenous IPI-504. Tu- mors and blood plasma were harvested at designated time points post dose. Drug levels of Hsp90 active species (IPI-504, 17-AAG, and 17-AG) were quantified by LC-MS/MS in plasma and in tumor (A). EGFR protein levels (B) and Hsp90 occupancy (C) were measured in tumor tissue. Data are ex- pressed as averages S.D. (vehicle (veh), n 2; and 1–48 h, n 3).

Article Snippet: Anti-Hsp90 antibody (clone 68) was from BD Biosciences; anti-Hsp90 antibody (clone H-114) and anti-HER2 (C-18) from Santa Cruz Biotechnology; and anti-EGFR, Akt (protein kinase B), and cRaf antibodies were from Cell Signaling (Beverly, MA).

Techniques: In Vivo, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Biological Chemistry

Article Title: Hsp90 (Heat Shock Protein 90) Inhibitor Occupancy Is a Direct Determinant of Client Protein Degradation and Tumor Growth Arrest in Vivo

doi: 10.1074/jbc.m110.141580

Figure Lengend Snippet: FIGURE 8. Hsp90 occupancy correlates with antitumor activity of IPI-504 in a xenograft model of NSCLC. H1650 tumor bearing mice were treated with vehicle (F), 25 mg/kg (), 50 mg/kg (), or 100 mg/kg (E) of IPI-504 (IP, twice weekly) and tumor growth was assessed by caliper measurement (A). Data are expressed as means and standard error (n 10 per arm). A separate group of H1650 tumor bearing mice was treated with a single dose of 25, 50, or 100 mg/kg of IPI-504 and sacrificed 2 h post dose. Hsp90 occupancy was determined as described and plotted against tumor size (mm3) measured on day 50 of drug treatment (B). Data are expressed as averages with standard deviation (n 2 (x-axis)) and as means with stan- dard error (n 10 (y-axis)). The points were fit to a linear least squares re- gression equation with a calculated R2 0.98.

Article Snippet: Anti-Hsp90 antibody (clone 68) was from BD Biosciences; anti-Hsp90 antibody (clone H-114) and anti-HER2 (C-18) from Santa Cruz Biotechnology; and anti-EGFR, Akt (protein kinase B), and cRaf antibodies were from Cell Signaling (Beverly, MA).

Techniques: Activity Assay, Standard Deviation

Journal: Nature Communications

Article Title: Isonicotinylation is a histone mark induced by the anti-tuberculosis first-line drug isoniazid

doi: 10.1038/s41467-021-25867-y

Figure Lengend Snippet: a – e INA, SIN, and INH stimulate HepG2 histone K inic . a Chemical structures of isonicotinic acid (INA), sodium isonicotinate (SIN), and isoniazid (INH). b Core histones acid-extracted from HepG2 cells that treated with increasing concentration of INA for 24 h and tested using pan-K inic , pan-K ac , and H3 antibodies by Western blot analysis. Total histones were visualized with Coomassie blue staining. “INA” indicates isonicotinic acid. c Core histones acid-extracted from HepG2 cells that treated with increasing concentration of SIN for 24 h and tested using pan-K inic , pan-K ac , and H3 antibodies by Western blot analysis. Total histones were visualized with Coomassie blue staining, “SIN” indicates sodium isonicotinate. d Core histones acid-extracted from HepG2 cells that treated with increasing concentration of INH for 24 h and tested using pan-K inic , pan-K ac , and H3 antibodies by Western blot analysis. Total histones were visualized with Coomassie blue staining, “INH” indicates Isoniazid. e HepG2 cells were treated with 10 mM INA, SIN, and INH respectively for 24 h and stained with pan-K inic rabbit (green) and pan-K ac mouse (red) antibodies. Nuclei were stained with DAPI (blue), followed by visualization with confocal microscopy. Scale bar, 20 μm. f INH stimulates mice liver K inic levels. Liver samples from mice treated with normal saline (NS) or 50 mg/kg/day of INH for ten days were collected ( n = 10 mice each group). And core histones and non-histones were extracted and tested using pan-K inic , GAPDH and H3 antibodies by Western blot. Total histones were visualized with Coomassie blue staining. “NS” indicates normal saline treated mice, “INH” indicates isoniazid treated mice. g INH is like to stimulate histone K inic directly. Schematic diagram of INH metabolism in vivo (left), core histones acid-extracted from normal HepG2 cells, 1 mM ABT (1-Aminobenzotriazole), 5 mM APAP (Acetaminophen) or 15 mM BNPP (Bis-p-nitrophenyl phosphate) pretreated HepG2 cells respectively, and also treated with 10 mM INH for 24 h, then tested using pan-K inic , pan-K ac and H3 antibodies by Western blot analysis. Total histones were visualized with Coomassie blue staining (right). h – i INH stimulates the production of Inic-CoA. h CoA was extracted from HepG2 cells treated with or without D4-INH (PH = 7.4) for 24 h, then tested the Ac-CoA (left) and D4-Inic-CoA (right) levels using HPLC-MS/MS assay, “CON” indicates untreated HepG2 cells; “D4-INH” indicates D4-isoniazid treated HepG2 cells, quantifications of the chromatographic peak area, data presented are the mean ± SEM from three biological replicates ( n = 3), as determined by unpaired two-tailed Student’s t- test. i CoA was extracted from HepG2 cells treated with increasing concentration of INH (PH = 7.4) for 24 h, and tested the Inic-CoA levels using HPLC-MS/MS assay, quantifications of the chromatographic peak area, data presented are the mean ± SEM from three biological replicates ( n = 3), as determined by one-way ANOVA followed by Bonferrroni’s multiple comparisons test.

Article Snippet: A485 (HY-107455), SGC-CBP30 (HY-15826), Acetaminophen (APAP, HY-66005), 1-Aminobenzotriazole (ABT, HY-103389), Trichostatin A (TSA, HY-15144) and Nicotinamide (NAM, HY-B0150) were purchased from MedChemExpress.

Techniques: Concentration Assay, Western Blot, Staining, Confocal Microscopy, Saline, In Vivo, Tandem Mass Spectroscopy, Two Tailed Test

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