Journal: International Journal of Molecular Sciences

Article Title: Less Severe Polymicrobial Sepsis in Conditional mgmt -Deleted Mice Using LysM-Cre System, Impacts of DNA Methylation and MGMT Inhibitor in Sepsis

doi: 10.3390/ijms241210175

Figure Lengend Snippet: The characteristics of bone marrow-derived macrophages (BMDM) from mgmt control (mgmt fl/fl ; LysM-Cre -/- ) or mgmt null (mgmt fl/fl ; LysM-Cre cre/- ) mice at 24 h after activation by lipopolysaccharide (LPS) as indicated by supernatant cell-free DNA ( A ), malondialdehyde (MDA; a reactive stress molecule) ( B ), and immunofluorescent stained for DNA break (phospho-histone H2A.X; green color) and actin filament (red color) in intensity score and representative pictures ( C , D ) are demonstrated. Triplicated independent experiments were performed. Mean ± SEM with the one-way ANOVA followed by Tukey’s analysis was used. #, p ˂ 0.05 vs mgmt control DMEM; *, p ˂ 0.05 between the indicated groups.

Article Snippet: Fixed samples were blocked with 2% bovine serum albumin in 1X TBS for 1 h at room temperature and then incubated overnight at 4 °C with phospho-histone H2A.X (Ser139) (20E3) rabbit mAb (Cell signaling).

Techniques: Derivative Assay, Activation Assay, Staining

Journal: bioRxiv

Article Title: Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations

doi: 10.1101/2023.03.17.533211

Figure Lengend Snippet: (A) Illustration of the hypothesis that escapees mount a replication stress response to manage aberrant S phases and tolerate DNA lesions. (B) Western blot analysis of γ-H2AX in the indicated cell lines after 96 h treatments. β-tubulin is used as a loading control. Bar plot displays quantification of γ-H2AX band intensity normalized to respective loading control band, and values are reported as fold change of control. (C) Representative immunofluorescence images of five human MAPK-mutant cancer cell lines after 72 h indicated treatments, stained for DNA content, EdU incorporation, and γ-H2AX. (D) Quantification of γ-H2AX nuclear fluorescence intensity after 72 h indicated treatments in EdU − and EdU + cells, displayed as split violins; p -values determined by Mann-Whitney U tests of MAPK-inhibited EdU + cells compared with DMSO-treated EdU + cells. (E) Top: Representative immunofluorescence images of phospho-CHK1 (S317) in A375 cells escaping BRAF inhibition (cycling status displayed by EdU intensity). Bottom: Histograms of phospho-CHK1 (S317) nuclear intensity in S phase cells (EdU + ) in the indicated cell lines after 96 h MAPK inhibition. (F) Distribution of EdU intensity in escapees (after 100 nM Osimertinib or 1 μM Encorafenib) co-treated with ATR inhibition (100 nM AZ20 or 100 nM AZD6738). 96 h treatments. Line drawn at the mean value; n = 190 cells plotted per condition; p -values determined by Mann-Whitney U test. (G) Quantification of cycling cells (determined by phospho-Rb S807/811 immunofluorescence) after 96 h of indicated treatment combinations with ATR inhibitors. AZ20 doses: 100 nM, 1 μM. AZD6738 doses: 100 nM, 1 μM. Mean ± std of 3 replicates; p -value determined by unpaired t-test.

Article Snippet: Primary antibodies used in this study include: phospho-Rb (S807/811) (D20B12) rabbit mAb (1:500, Cell Signaling Technology #8516); phospho-Rb (S780) mouse mAb (1:1000, BD Biosciences #558385); phospho-Histone H3 (S10) (D2C8) rabbit mAb (1:400, Cell Signaling Technology #3377); phospho-Histone H3 (S10) (6G3) mouse mAb (1:400, Cell Signaling Technology #9706); phospho-H2AX (S139) (20E3) rabbit mAb (1:400 for standard immunofluorescence, 1:100 for FFPE tissue staining, Cell Signaling Technology #9718); p21 (12D1) rabbit mAb (1:400, Cell Signaling Technology #2947); phospho-ATR (T1989) (D5K8W) rabbit mAb (1:250, Cell Signaling Technology #30632); phospho-CHK1 (S317) rabbit mAb (1:250, Abcam #ab278717); FANCD2 rabbit pAb (1:500, Novus Biologicals #100-182); Polκ mouse mAb (1:500, Abcam #ab57070; mouse mAb is discontinued and #ab115625 goat pAb is recommended by manufacturer as a suitable replacement); β-tubulin (D3U1W) mouse mAb (1:1000, Cell Signaling Technology #86298); Ki67 sheep pAb (1:200, R&D Systems #AF7617).

Techniques: Western Blot, Immunofluorescence, Mutagenesis, Staining, Fluorescence, MANN-WHITNEY, Inhibition

Journal: bioRxiv

Article Title: Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations

doi: 10.1101/2023.03.17.533211

Figure Lengend Snippet: (A) Top: Long-term culture of MAPK-mutant cell lines with indicated MAPK inhibitors and clinical ATR inhibitor (5 μM AZD6738). Cell count was quantified longitudinally by imaging H2B-mIFP fluorescence. Unfettered proliferation in control groups causes contact inhibition within first 2 weeks of imaging. Mean ± std of 4 replicate wells; p -value determined by unpaired t-test of final time point. Bottom: Representative wells from the final day of imaging. Inverted H2B-mIFP fluorescence shown. (B) Formalin-fixed paraffin-embedded (FFPE) tissue sections longitudinally biopsied before and after treatment from patients with BRAF V600E melanoma (see for patient and tumour details). Immunofluorescence of Ki67 (proliferation marker) and γ-H2AX (DNA damage marker) displayed. (C) Quantification of all cells (shown in blue coloring) and Ki67-positive cells (shown in red coloring) in FFPE sections with elevated γ-H2AX intensity. Line drawn at the mean; n > 235 cells scored before Ki67 gating; p-value determined by Mann-Whitney U test. (D) Kaplan-Meier survival analyses based on data generated from the Harmonized Cancer Datasets within the Genomic Data Commons (GDC) research database (as of April 28, 2022; https://portal.gdc.cancer.gov ). Patients were included with tumours harboring ≥ 1 EGFR, KRAS, or BRAF mutation and ≥ 1 TP53 mutation. Cohort comparisons were made between tumour subgroups containing wild-type or mutant forms of the indicated genes. Left-most plot examines a cohort that has ≥ 1 mutation among a list of FA-associated genes (see for FA gene list). Displayed p -values were determined by log-rank test.

Article Snippet: Primary antibodies used in this study include: phospho-Rb (S807/811) (D20B12) rabbit mAb (1:500, Cell Signaling Technology #8516); phospho-Rb (S780) mouse mAb (1:1000, BD Biosciences #558385); phospho-Histone H3 (S10) (D2C8) rabbit mAb (1:400, Cell Signaling Technology #3377); phospho-Histone H3 (S10) (6G3) mouse mAb (1:400, Cell Signaling Technology #9706); phospho-H2AX (S139) (20E3) rabbit mAb (1:400 for standard immunofluorescence, 1:100 for FFPE tissue staining, Cell Signaling Technology #9718); p21 (12D1) rabbit mAb (1:400, Cell Signaling Technology #2947); phospho-ATR (T1989) (D5K8W) rabbit mAb (1:250, Cell Signaling Technology #30632); phospho-CHK1 (S317) rabbit mAb (1:250, Abcam #ab278717); FANCD2 rabbit pAb (1:500, Novus Biologicals #100-182); Polκ mouse mAb (1:500, Abcam #ab57070; mouse mAb is discontinued and #ab115625 goat pAb is recommended by manufacturer as a suitable replacement); β-tubulin (D3U1W) mouse mAb (1:1000, Cell Signaling Technology #86298); Ki67 sheep pAb (1:200, R&D Systems #AF7617).

Techniques: Mutagenesis, Cell Counting, Imaging, Fluorescence, Inhibition, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Marker, MANN-WHITNEY, Generated

Journal: PLoS ONE

Article Title: The Level of Ets-1 Protein Is Regulated by Poly(ADP-Ribose) Polymerase-1 (PARP-1) in Cancer Cells to Prevent DNA Damage

doi: 10.1371/journal.pone.0055883

Figure Lengend Snippet: ( A ) Western blot analysis of phosphorylated H2AX (γH2AX) in transfected HeLa cells treated with PJ-34. pcDNA3 expression vectors without insert (lanes 1 and 3) and encoding Ets-1 (lanes 2 and 4) were transfected in HeLa cells at 50–80% confluence. At 24 h after transfection, cells were treated for 20 h with PJ-34 (10 µM; lanes 3 and 4). Cell lysates (30 µg total proteins) were analysed by Western blot using the C-20 anti-Ets-1 antibody and the anti-γH2AX antibody. ( B ) Immunofluorescence of γH2AX in HeLa cells treated with PJ-34. HeLa cells were treated with PJ-34 (10 µM) for 20 h. Ets-1 is visualised in green (Alexa Fluor® 488) and γH2AX in red (Alexa Fluor® 594). ( C ) Immunofluorescence of γH2AX and Ets-1 in transfected HeLa cells treated with PJ-34. HeLa cells were transfected with peGFP-C1-Ets-1 expression vector (1 µg) and treated with PJ-34 for 20 h. γH2AX is visualised in red (Alexa Fluor® 594) and Ets-1 in green (eGFP). In (B) and (C), nuclei were visualised with DAPI staining. Cells were examined by fluorescence microscopy at ×40 magnification. Scale bar = 20 µm.

Article Snippet: Western blot analyses were performed according to Baillat et al. . Primary antibodies used were rabbit C-20 and mouse C-4 anti-Ets-1, rabbit H-250 anti-PARP-1, mouse F-2 anti-PARP-1, H-79 anti-c-Jun, DO-1 anti-p53 and C-14 anti-Erk-2 from Santa Cruz Biotechnology (Heidelberg, Germany), 10HA anti-PAR from Trevigen (Gaithersburg, MD, USA), (Ser139) 20E3 anti-phospho-H2AX from Cell Signaling (Danvers, MA, USA) and ab8227 anti-β-Actin from Abcam (Paris, France).

Techniques: Western Blot, Transfection, Expressing, Immunofluorescence, Plasmid Preparation, Staining, Fluorescence, Microscopy