analysis immunofluorescence images  (Olympus)

Journal: The Journal of Clinical Investigation

Article Title: CRISPR/Cas9 screens identify LIG1 as a sensitizer of PARP inhibitors in castration-resistant prostate cancer

doi: 10.1172/JCI179393

Figure Lengend Snippet: ( A ) Immunoblot of PARP1, PAR, and H3 (used as loading control) in the chromatin-bound extract (CBE) of 22Rv1 transduced with the indicated sgRNAs and treated for 3 days with DMSO, as control, or OLA. ( B ) Comet tail moment measured by alkaline comet assay in 22Rv1 transduced with the indicated sgRNAs and treated as in A . The orange dots and bars indicate the mean value of each replicate and the mean ± SEM of the 3 experiments, respectively. P values were determined using 1-way ANOVA and Tukey’s multiple-comparisons test. Images are representative of the comet assays. Scale bars: 100 μm. ( C ) Quantification of cells with 5 or more γH2AX foci measured by immunofluorescence. Data are presented as mean ( n = 2 biological replicates). Images are representative of γH2AX (green) and nucleus (Hoechst, blue) immunostaining in 22Rv1 transduced with the indicated sgRNAs and treated as in A . Scale bars: 50 μm. ( D ) Immunoblot of LIG1, p-ATM, ATM, VINC (used as loading control), p-CHK1, CHK1, ACTB (used as loading control), γH2AX, and TUBB (used as loading control) in 22Rv1 transduced with the indicated sgRNAs and treated as in A . ( E ) Percentage of cells with 10 or more γH2AX foci measured by immunofluorescence after OLA washout. 22Rv1 transduced with the indicated sgRNAs were treated with 1 μM OLA for 3 days and then grown without treatment for 0, 1, 8 or 24 hours. Data are presented as mean ± SEM ( n = 3 biological replicates). P values were determined using 2-way ANOVA and Bonferroni’s multiple-comparisons test on control (sgNTC and sg EGFP ) and sg LIG1 samples. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: Immunofluorescence analysis was performed with ImageXpress Micro Confocal (Molecular Devices).

Techniques: Western Blot, Control, Transduction, Alkaline Single Cell Gel Electrophoresis, Immunofluorescence, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: CRISPR/Cas9 screens identify LIG1 as a sensitizer of PARP inhibitors in castration-resistant prostate cancer

doi: 10.1172/JCI179393

Figure Lengend Snippet: ( A ) Schematic diagram of the in vivo experiments. ( B ) Immunoblot analysis of LIG1 and ACTB (used as loading control) in 6 exemplary xenograft tumor samples collected after the in vivo experiments described in A . ( C ) Scatter plots of the tumor volume measured during treatment with vehicle or AZD5305. Data are presented as mean + SD. ( D ) Percentage of cells with 5 or more γH2AX foci measured by immunofluorescence in FFPE xenograft tumor samples collected after the in vivo experiments described in A . Images are representative of 3 FFPE xenografts tumor sections stained for γH2AX (red) and nucleus (DAPI, blue). Scale bars: 12 μm. Data are presented as mean ± SD. P values were determined using 2-tailed unpaired t test. * P ≤ 0.05; ** P ≤ 0.01; **** P ≤ 0.0001.

Article Snippet: Immunofluorescence analysis was performed with ImageXpress Micro Confocal (Molecular Devices).

Techniques: In Vivo, Western Blot, Control, Immunofluorescence, Staining