Journal: ACS Omega

Article Title: Ellagic Acid Induces DNA Damage and Apoptosis in Cancer Stem-like Cells and Overcomes Cisplatin Resistance

doi: 10.1021/acsomega.3c08819

Figure Lengend Snippet: EA treatment alters mitochondrial dynamics, leading to the accumulation of intracellular ROS and DNA damage. Western blot analysis of the protein levels of Mfn2, Drp1, total-ERK, and p-ERK in the (A) A2780 monolayer, (B) A2780 spheroids, and (C) SKOV3 spheroids cells; A549-CD133 + (D,E) and SKOV3- spheroids (F,G) were treated with different concentrations of EA (0, 5, 10, 25 μM) for 24 h. Intracellular ROS accumulation was determined using flow cytometry. Representative histograms and corresponding bar diagrams showing ROS level in CSLCs; A549-CD133 + (H) and SKOV3- spheroid (I) cells were treated with different doses of EA for 48 h. γH2AX accumulation was detected through flow cytometry to evaluate the extent of DNA damage induced by EA. Representative contour plots obtained from flow cytometry analysis showing the percentage of γH2AX positive cells. Data was reanalyzed using FlowJo software. In all graphs, the results are represented as mean ± SD of triplicate experiments. ** P < 0.01, and *** P < 0.001.

Article Snippet: Washing was done twice with 1× PBS and staining was performed using the γH2AX (Miltenyi Biotec cat. no. 130-130-829) antibody.

Techniques: Western Blot, Flow Cytometry, Software

Journal: ACS Omega

Article Title: Ellagic Acid Induces DNA Damage and Apoptosis in Cancer Stem-like Cells and Overcomes Cisplatin Resistance

doi: 10.1021/acsomega.3c08819

Figure Lengend Snippet: Combinatorial treatment of EA and cisplatin impairs DNA damage repair in lung and ovarian CSLCs. The DNA repair kinetics study depicts the percent DNA damage accumulation following DMSO, EA, cisplatin, and EA + cisplatin treatment for 12 h followed by 3 h damage recovery in (A) A549-CD133 + and (B) SKOV3 spheroid cells. (C) The immunofluorescence staining shows the increasing accumulation of DNA damage in DMSO, EA, cisplatin, and EA + cisplatin-treated cells. The corresponding graph represents the mean fluorescence intensity of accumulated γH2AX. N=3, Bar, SD; * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Washing was done twice with 1× PBS and staining was performed using the γH2AX (Miltenyi Biotec cat. no. 130-130-829) antibody.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: The Botanical Drug PBI-05204, a Supercritical CO 2 Extract of Nerium Oleander, Is Synergistic With Radiotherapy in Models of Human Glioblastoma

doi: 10.3389/fphar.2022.852941

Figure Lengend Snippet: PBI-05204 augmented RT mediated DNA damage in U87 and U251 cells. (A) . Representative Western blots performed on total cell extracts collected from U87MG and U251 cells treated with RT (4 Gy), PBI-05204 (2.5 μg/ml) or a combination of RT and PBI-05204 for 1 and 18 h. γH2Ax bands were analyzed by image J software and adjusted densitometric Unit values (ADU) were normalized vs α-tubulin which were presented on the top of γH2Ax bands. (B) Time course ELISA detection for γH2Ax in U87MG and U251 cells treated with RT (4 Gy), PBI-05204 (5.0 μg/ml) or combinations for 1–48 h. Data are presented as mean ± SD ( n = 5). * p < 0.01 RT vs RT + PBI-05204. (C) Representative Immunofluorescence images for γH2Ax nuclear foci in U87MG cells. (D) Percentage of γH2Ax positive cells (defined as cells with at least five γH2Ax foci each) in at least 100 nuclei ( n = 3) in 1 h-treated U87MG and U251 cell lines. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01. (E) representative images of Comet assay in U87MG cells treated for 1 h with RT (4 Gy), PBI-05204 (5.0 μg/ml) or a combination of RT and PBI-05204. (F) . Percentage of DNA in the tails of U87MG and U251 cells after they were treated with RT, PBI-05204 and RT + PBI-05204 from Comet Assay ( n = 3). Data are presented as mean ± SD. ** p < 0.01.

Article Snippet: Antibodies against γH2AX (STJ90288), Ku70 (STJ29469), pDNA-PKc (STJ91334), caspase 3 (STJ11101177), cleaved caspase 3 (STJ90005), caspase 9 (STJ29774), cleaved caspase 9 (STJ90013), LC3A/LC3B (STJ117779) were purchased from St John’s Laboratory Ltd. (Knowledge Dock Business Centre Docklands Campus, London, United Kingdom).

Techniques: Western Blot, Software, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Single Cell Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: ARID1A regulates DNA repair through chromatin organization and its deficiency triggers DNA damage-mediated anti-tumor immune response

doi: 10.1093/nar/gkae233

Figure Lengend Snippet: ARID1A promotes DSB repair pathways. ( A ) Clonogenic survival assay of U2OS cells transfected either with control siRNA (siCTR) or a pool of four siRNAs targeting ARID1A (siARID1A) and treated with the indicated dose of ionizing radiation. Data are presented as mean ± SEM, One-Way ANOVA with Bonferroni's multiple comparison test was performed. ( B ) Representative micrographs and quantification of IR-induced γH2AX foci in U2OS cells, ca. 500 cells were counted at indicated time points. Data are presented as mean ± SEM, One-Way ANOVA with Bonferroni's multiple comparison test was performed. ( C , D ) HR efficiency measured in U2OS-DR cells and NHEJ efficiency measured in U2OS-EJ5 cells using the indicated siRNAs, respectively. Data are presented as mean ± SD, one-way ANOVA with Tukey's multiple comparison test ws performed. ( E ) Enrichment of ARID1A at the indicated DSBs in WT AID-DIvA cells, measured by ChIP-qPCR. Data are presented as mean ± SEM, Student's t test was performed. ( F ) Quantification of ASiSI-induced γH2AX foci in AID-DIvA cells, ca. 500 cells were counted at indicated time points. Data are presented as mean ± SEM, one-way ANOVA with Bonferroni's multiple comparison test was performed. ( G ) Tornado plots showing BLISS signals in WT and ARID1A-KO cells at HR-prone DSBs at the indicated time points. ( H ) Quantification of BLISS signals in WT and ARID1A-KO cells at HR-prone DSBs at the indicated time points. ( I ) Tornado plots showing BLISS signals in WT and ARID1A-KO cells at NHEJ-prone DSBs at the indicated time points. ( J ) Quantification of BLISS signals in WT and ARID1A-KO cells at NHEJ-prone DSBs at the indicated time points. All data presented in this figure are from n = 3 independent experiments (biological replicates). Statistical significance is presented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: Mouse-anti-phospho-Histone H2A.X (S139) (Merck, Cat#05-636).

Techniques: Clonogenic Cell Survival Assay, Transfection, Comparison

Journal: Nucleic Acids Research

Article Title: ARID1A regulates DNA repair through chromatin organization and its deficiency triggers DNA damage-mediated anti-tumor immune response

doi: 10.1093/nar/gkae233

Figure Lengend Snippet: ARID1A is required for efficient chromatin loop formation. ( A – C ) 4C–seq normalized read counts at indicated viewpoints as well as differential 4C–seq track (log 2 +DSB/–DSB) in WT (grey) ARID1A-KO (blue) cells collected at indicated time points (T0 and T4). 4C–seq data were smoothed using 10-kb spans. ( D – F ) Box plots showing the differential 4C–seq (log 2 +DSB/–DSB) at indicated viewpoints. Four technical replicates from two independent experiments (biological replicates), data are presented mean ± SD, Student's t test. ( G – I ) Enrichment of γH2AX, RAD21 and CTCF, respectively, at the indicated DSBs in WT and ARID1A-KO cells, measured by ChIP-qPCR. n = 3 independent experiments (biological replicates); data are presented as mean ± SEM, Student's t test. Statistical significance is presented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: Mouse-anti-phospho-Histone H2A.X (S139) (Merck, Cat#05-636).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: CENP-A Reduction Induces a p53-Dependent Cellular Senescence Response To Protect Cells from Executing Defective Mitoses ▿

doi: 10.1128/MCB.01318-09

Figure Lengend Snippet: Senescencelike phenotypes induced by CENP-A depletion require p53. (A) Experimental design. (B) Immunoblotting of TIG3 cells expressing control, p53, or p16INK4a shRNA (first shRNA) in combination with control or CENP-A shRNA (second shRNA). TCE and soluble proteins were resolved by SDS-PAGE, followed by immunoblotting with the indicated antibodies. Histone H3 and actin were used as loading controls. (C and D) Quantitative RT-PCR of the cells indicated in panel B. The relative abundances of p21CIP1 mRNA (C) and p16INK4a mRNA (D) are shown after normalization using GAPDH mRNA. (E) Growth curves of TIG3 cells expressing control (left upper), p16INK4a (middle upper), or p53 shRNA (right upper) in combination with control or CENP-A shRNA. The number of cells on day 9 after the second shRNA infection was set at 1. The data are means ± the SD from three independent experiments. SA-β-Gal staining was performed on day 14 after the second shRNA infection (lower panels). Scale bars, 100 μm. (F) Immunofluorescence of CENP-A-depleted and ras-induced senescent TIG3 cells performed on day 10 after infection. Staining revealed γ-H2AX (red in overlay). DNA was counterstained with Hoechst (blue in overlay). As a positive control, cells were treated with UV. The numbers of cells (n = 300) containing 1 to 10 and >10 foci are shown. Scale bars, 10 μm. (G) Immunoblotting of CENP-A-depleted and UV-treated cells. Soluble proteins were resolved by SDS-PAGE, followed by immunoblotting with the indicated antibodies. Actin was used as a loading control.

Article Snippet: Cells were incubated with appropriate primary antibodies: anti-CENP-A (Upstate Biotechnology or GTX13939; GeneTex), anti-CENP-B (Upstate Biotechnology), anti-HP1β (Chemicon), anti-HP1γ (Chemicon), and anti-phospho-histone H2A.X (serine 139, catalog no. 05-636; Upstate Biotechnology).

Techniques: Western Blot, Expressing, shRNA, SDS Page, Quantitative RT-PCR, Infection, Staining, Immunofluorescence, Positive Control