Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Immunofluorescence microscopy quantification of DDR activation and histone marks in HeLa cells infected with control or Vpr-expressing virus. Vpr-infected HeLa cells were infected for 24 hours and then treated with vehicle or 3 mM caffeine for 24 hours ( n = 50 cells). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . (B) Immunofluorescence microscopy quantification of histone marks in HeLa cells infected with control or Vpr-expressing virus in the presence or absence of vehicle, 3 mM caffeine, 10 nM ATM i , or 10 μM ATR i ( n = 50 cells). Cells were infected for 24 hours prior to inhibitor treatment for 24 hours and preparation for immunofluorescence microscopy. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . (C) Immunofluorescence microscopy quantification of histone marks in HeLa cells infected with control or virus expressing the indicated Vpr subtype consensus sequence. Cells were infected for 24 hours and then treated with vehicle or with 3mM caffeine for 24 hours prior to preparation for immunofluorescence microscopy ( n = 50 cells). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . (D) Cell cycle analysis of DDR activation and histone marks in HeLa cells infected with control or Vpr-expressing viruses. Left, flow cytometric analysis of propidium iodide (PI) stained HeLa or MDM cells infected with the indicated virus. Right, flow cytometric analysis of immunolabeled and PI-stained HeLa cells infected with the indicated virus ( n = 4 experiment). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; ** p < 0.01. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ).

Article Snippet: For inhibitor experiments, cells were treated 24-hours post-infection with either 10 nM ATM inhibitor (Fisher, #AZD1390), 10 μM ATR inhibitor (Fisher, #NU6027), 3 mM caffeine, 16 μM DNA-PK inhibitor #1 (MedChemExpress, #NU7026), or 16 μM DNA-PK inhibitor #2 (MedChemExpress #NU7441) for 24 hours.

Techniques: Immunofluorescence, Microscopy, Activation Assay, Infection, Control, Expressing, Virus, Sequencing, Cell Cycle Assay, Staining, Immunolabeling

Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Top, schematic of proviruses used in these experiments. For these constructs, mCherry expression is driven off of the HIV promoter as opposed to being driven off of CMV. Bottom, flow cytometric analysis of HIV LTR activity in THP1 or HeLa cells infected with increasing MOI of the indicated viruses ( n = 3 experiments). For inhibitor treatments, cells were infected for 24 hours prior to a 24-hour treatment with 3 mM caffeine or combined ATM (10 nM)/ATR (10 μM) treatment. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (B) Flow cytometric quantification of HIV LTR activity in primary MDMs infected with Vpr WT virus in the presence or absence of DDR inhibition ( n = 7 experiments). MDMs were infected for 24 hours prior to treating with combined ATM (10 nM)/ATR (10 μM) inhibitors for 24 hours. Cells were detached and mCherry fluorescence was quantified via flow cytometry. ** p < 0.01. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (C) Flow cytometric quantification of LTR-mCh MFI in infected HeLa cells subjected to ATM (10nM), ATR (10 μM), or combined ATM and ATR inhibition 48 hours post-infection (n = 5 experiments). Cells were detached and mCherry fluorescence was quantified via flow cytometry. ns, not significant. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (D) Top, schematic of proviruses used in these experiments. For these proviruses, BFP was incorporated into the gag gene to generate Gag-BFP proteins from the viral promoter. Bottom left, live cell fluorescence microscopy images of HeLa cells 48-hours post-co-transfection with the indicated Gag-BFP proviruses and an eGFP control plasmid. Bottom middle, flow cytometric histogram of BFP fluorescence in eGFP-positive HeLa cells. Bottom right, quantification of BFP MFI in eGFP-positive cells with or without DDR inhibition ( n = 3 experiments). Cells were co-transfected for 24 hours prior to adding the indicated inhibitor for 24 hours. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; * p < 0.05. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (E) Diagram displaying the establishment of the HeLa latency model H-Lats (left) and representative live cell fluorescence microscopy images of H-Lat GFP expression when infected with indicated viruses. H-Lat cells were infected for 48 hours prior to being subjected to live cell fluorescence microscopy. Right, flow cytometric analysis of H-Lat GFP expression 48 hours post-infection with the indicated viruses. (F) Flow cytometric analysis of H-Lat reactivation following infection with increasing MOI of the indicated viruses in the presence or absence of DDR inhibition ( n = 3 experiments). Cells were infected for 24 hours prior to inhibitor treatment for 24 hours and preparation for flow cytometry. *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (G) Flow cytometric quantification of LTR-eGFP MFI in infected H-Lat cells subjected to ATM (10nM), ATR (10 μM), or combined ATM/ATR treatment. H-Lat cells were infected for 24 hours prior to a 24-hour inhibitor treatment and preparation for flow cytometry ( n = 5 experiments). ns, not significant; * p < 0.05. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (H) Flow cytometric analysis of CEM-GFP, J-Lat clone 10.6, and monocyte clone U1 latency models uninfected or infected with Vpr WT virus in the presence or absence of 3 mM caffeine ( n = 3 experiments). Cells were infected with increasing MOI of Vpr-expressing virus for 24 hours, treated with vehicle or 3 mM caffeine for 24 hours, and then subjected to flow cytometry. * p < 0.05; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ). (I) Flow cytometric quantification of LTR-eGFP MFI in H-Lat cells transiently expressing plasmids encoding Tat and/or Vpr and treated with 50 µM etoposide or vehicle ( n = 3 experiments). H-Lats were transfected with the indicated plasmid combination for 24 hours, treated with vehicle or etoposide for 24 hours, and then subjected to flow cytometry. Analyses performed using a one-way ANOVA; ** p < 0.01. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ).

Article Snippet: For inhibitor experiments, cells were treated 24-hours post-infection with either 10 nM ATM inhibitor (Fisher, #AZD1390), 10 μM ATR inhibitor (Fisher, #NU6027), 3 mM caffeine, 16 μM DNA-PK inhibitor #1 (MedChemExpress, #NU7026), or 16 μM DNA-PK inhibitor #2 (MedChemExpress #NU7441) for 24 hours.

Techniques: Construct, Expressing, Activity Assay, Infection, Virus, Inhibition, Fluorescence, Flow Cytometry, Microscopy, Cotransfection, Control, Plasmid Preparation, Transfection

Journal: PLOS Biology

Article Title: DNA damage induced by HIV-1 Vpr triggers epigenetic remodeling and transcriptional programs to enhance virus transcription and latency reactivation

doi: 10.1371/journal.pbio.3003621

Figure Lengend Snippet: (A) Immunofluorescence microscopy quantification of NFκB translocation, phosphorylated SP1 residue Ser101, phosphorylated cJun residues Ser63 and Ser73, and phosphorylated RNA polymerase II residues Ser2 and Ser5 in differentiated THP1 cells infected with Vpr WT or control viruses in the presence or absence of ATM, ATR, or DNA-PK inhibition ( n = 50 cells). Cells were infected for 24 hours, treated with vehicle or the indicated inhibitor for 24 hours, and then subjected to immunofluorescence microscopy. Analyses were performed using a one-way ANOVA; ns, not significant; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . (B) Representative immunofluorescence microscopy images of R-loop abundance in primary MDM and HeLa cells infected with indicated viruses 48 hours post-infection ( n = 50 cells). Analyses performed using a student t test; *** p < 0.001. (C–E) Immunofluorescence microscopy quantification of R-loop abundance in HeLa cells infected with indicated viruses 48 hours post-infection. Samples were left untreated (C) or treated with RNaseH (D, left), triptolide (D, right), or with the indicated DDR inhibitors (E), respectively ( n = 50 cells). For RNaseH treatment, cells were infected for 48 hours prior to methanol fixation and addition of recombinant RNaseH to deplete RNA associated with R-loops. For inhibitor treatments, HeLa cells were infected for 24 hours, treated with the indicated inhibitor for 24 hours, and then subjected to immunofluorescence microscopy. Analyses were performed using a one-way ANOVA; ns, not significant; ** p < 0.01; *** p < 0.001. The data underlying this Figure can be found in . (F) Immunofluorescence microscopy quantification of DDR activation (left) and R-loop abundance (right) in HeLa cells infected with indicated viruses and transfected with RNaseH constructs ( n = 50 cells). Cells were infected for 24 hours before transfection of control or RNaseH-expressing plasmids for 24 hours prior to being prepared for immunofluorescence microscopy. Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001; * p < 0.05. The data underlying this Figure can be found in . (G) Flow cytometric histograms of HeLa cells infected with control or Vpr WT LTR-mCh viruses for 24 hours prior to transient expression of RNaseH for 24 hours and quantification of LTR-mCh MFI in transfected cells via flow cytometry ( n = 3 experiments). Analyses performed using a one-way ANOVA; ns, not significant; *** p < 0.001. The data underlying this Figure can be found in . Representative gating strategies are depicted in and raw FSC files can be found in the Figshare Data repository ( https://doi.org/10.6084/m9.figshare.c.8239897 ).

Article Snippet: For inhibitor experiments, cells were treated 24-hours post-infection with either 10 nM ATM inhibitor (Fisher, #AZD1390), 10 μM ATR inhibitor (Fisher, #NU6027), 3 mM caffeine, 16 μM DNA-PK inhibitor #1 (MedChemExpress, #NU7026), or 16 μM DNA-PK inhibitor #2 (MedChemExpress #NU7441) for 24 hours.

Techniques: Immunofluorescence, Microscopy, Translocation Assay, Residue, Infection, Control, Inhibition, Recombinant, Activation Assay, Transfection, Construct, Expressing, Flow Cytometry

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Oxidized ataxia telangiectasia mutated promotes triple-negative breast cancer-cancer stem cells enrichment and stemness maintenance. A: Representative images showing inhibition of mammosphere formation by the ataxia telangiectasia mutated inhibitor Ku60019; B: Quantification of mammosphere size; C: Quantification of mammosphere number; D-F: Effects of ataxia telangiectasia mutated knockdown on cancer stem cell enrichment (D), mammosphere size (E), and mammosphere number (F); G and H: Western blot analysis of stemness-associated proteins (c-Myc, sex-determining region Y-box 2, Kruppel-like factor 4, octamer-binding protein 4) following Ku60019 treatment (G) or ataxia telangiectasia mutated knockdown (H). Scale bar: 200 μm. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2; p-ATM: Phosphorylated ataxia telangiectasia mutated; shATM: Ataxia telangiectasia mutated knockdown.

Article Snippet: The ATM inhibitor Ku60019 was purchased from MedChem Express.

Techniques: Inhibition, Knockdown, Western Blot, Binding Assay

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Hypoxia and oxidized ataxia telangiectasia mutated facilitate metabolic remodeling in triple-negative breast cancer-cancer stem cells. A: Volcano plot showing differentially regulated metabolites (115 upregulated, 129 downregulated) in MDA-MB-231 cancer stem cells under hypoxia compared with normoxia; B: Pathway enrichment analysis of differentially regulated metabolites; C: Kyoto Encyclopedia of Genes and Genomes classification of altered metabolic pathways; D: Volcano plot showing metabolic changes in hypoxic cancer stem cells after Ku60019 treatment; E: Pathway enrichment analysis after Ku60019 treatment; F: Kyoto Encyclopedia of Genes and Genomes classification of altered metabolic pathways after Ku60019 treatment.

Article Snippet: The ATM inhibitor Ku60019 was purchased from MedChem Express.

Techniques:

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Oxidized ataxia telangiectasia mutated promotes serine hydroxymethyltransferase 2 and methylenetetrahydrofolate dehydrogenase 2 expression through c-Myc. A and B: Western blot analysis of phosphorylated ataxia telangiectasia mutated, c-Myc, serine hydroxymethyltransferase 2 (SHMT2), and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in Hs578T and MDA-MB-231 cells after treatment with Ku60019 and ataxia telangiectasia mutated knockdown; C: Consensus c-Myc binding motif; D: Schematic representation of predicted c-Myc binding sites in the human MTHFD2 and SHMT2 promoter regions; E: Luciferase assay showed SHMT2 and MTHFD2 relative luciferase activity; F: Representative chromatin immunoprecipitation (ChIP)-polymerase chain reaction (PCR) showing c-Myc occupancy at the MTHFD2 and SHMT2 promoters; input and immunoglobulin G served as controls; G and H: ChIP-quantitative PCR analysis demonstrating c-Myc enrichment at the MTHFD2 (G) and SHMT2 (H) promoters in Hs578T and MDA-MB-231 cells. ChIP-quantitative PCR enrichment expressed as % input relative to immunoglobulin G. Data were presented as mean ± SD ( n = 3). a P < 0.05; b P < 0.01. p-ATM: Phosphorylated ataxia telangiectasia mutated; MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; IgG: Immunoglobulin G.

Article Snippet: The ATM inhibitor Ku60019 was purchased from MedChem Express.

Techniques: Expressing, Western Blot, Knockdown, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: World Journal of Stem Cells

Article Title: Hypoxia facilitates triple-negative breast cancer stem cells enrichment and stemness maintenance through oxidized ataxia telangiectasia mutated-induced one-carbon metabolism

doi: 10.4252/wjsc.v18.i1.112278

Figure Lengend Snippet: Oxidized ataxia telangiectasia mutated sustains triple-negative breast cancer-cancer stem cells stemness via serine hydroxymethyltransferase 2- and methylenetetrahydrofolate dehydrogenase 2-mediated one-carbon metabolism. A: NADPH/NADP+ ratio in cancer stem cells (CSCs) under hypoxia compared with normoxia; B and C: NADPH/NADP+ ratio decreased in CSCs after Ku60019 treatment (B) or shc-Myc knockdown (C); D-F: Effects of one-carbon metabolite supplementation on mammosphere size (D) and number (E and F) in CSCs with serine hydroxymethyltransferase 2 (SHMT2) knockdown or methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) knockdown; G: Western blot analysis of MTHFD2, SHMT2, Kruppel-like factor 4, and sex-determining region Y-box 2 expression following SHMT2 or MTHFD2 knockdown under hypoxia. Data were presented as mean ± SD ( n = 3). a P < 0.05. MTHFD2: Methylenetetrahydrofolate dehydrogenase 2; SHMT2: Serine hydroxymethyltransferase 2; KLF4: Kruppel-like factor 4; SOX2: Sex-determining region Y-box 2.

Article Snippet: The ATM inhibitor Ku60019 was purchased from MedChem Express.

Techniques: Knockdown, Western Blot, Expressing

Journal: NAR Cancer

Article Title: Androgen receptor inhibition extends PARP inhibitor activity in prostate cancer models beyond BRCA mutations and defects in homologous recombination repair

doi: 10.1093/narcan/zcaf035

Figure Lengend Snippet: ( A ) Combination activity between olaparib and enzalutamide in a panel of prostate cancer cell lines. Each dot represents a cell line (see ). ( B ) Combination activity between olaparib and enzalutamide in LNCAP and two different isogenic LNCAP ATM KO clones. ( C ) Combination activity between olaparib and enzalutamide in VCAP and a VCAP ATM KO pool. ( D ) Combination activity between the PARPi, olaparib or veliparib, and enzalutamide in LNCAP ATM KO (left panel) and VCAP ATM KO (right panel) backgrounds. ( E ) Clonogenic survival assay of C4-2 cells treated with a dose range of different PARPi – olaparib (left panel), talazoparib (middle panel), and veliparib (right panel) – combined with DMSO or a fixed dose of enzalutamide (0.3 μM). IC50 values for the combinations are provided at the bottom of each graph. ( F ) IC50 fold change for the PARPi used in panel (E) between DMSO and enzalutamide combinations. ( G ) RNA-seq differential expression levels in the AR hallmark gene signature in LNCAP treated with enzalutamide, olaparib, or their combination (top panel) and leading-edge analysis (bottom panels). In all panels, a minimum of two biological replicates for each condition are presented.

Article Snippet: ATM , ataxia telangiectasia mutated , 472 , DDR , Hs01112355_g1.

Techniques: Activity Assay, Clone Assay, Clonogenic Cell Survival Assay, RNA Sequencing, Quantitative Proteomics