cat 13082s confocal immunofluorescent analysis of hela cells chloroquine treated  (Cell Signaling Technology Inc)

Journal: Viruses

Article Title: Berberine Suppresses Influenza A Virus-Triggered Pyroptosis in Macrophages via Intervening in the mtROS-MAVS-NLRP3 Inflammasome Pathway

doi: 10.3390/v17040539

Figure Lengend Snippet: IAV induces NLRP3-caspase-1 mediated pyroptosis in macrophages in a time-dependent manner. J774A.1 cells uninfected and infected with the PR8 virus for 6 h, 12 h or 24 h. ( A ) The LDH levels in the culture supernatants from dead cells were measured using ELISA. ( B , C ) The expression levels of NLRP3 were detected by Western blot using β-actin as an internal control. ( D , E ) Flow cytometric analysis of the rates of pyroptosis mediated by caspase-1 using the FAM-FLICA Caspase-1 Kit. ( F ) The IL-1β levels in the culture supernatants were measured using ELISA. ( G , H ) The expression levels of GSDMD-N were detected by Western blot using β-actin as an internal control. ( I ) Representative images of immunofluorescence staining for GSDMD-N were observed by confocal laser scanning microscope. The nuclei are shown in blue using DAPI staining; while GSDMD-N is shown in red and detected by antibody. The white arrows indicate the fluorescent hub formed by GSDMD-N aggregation. ( J ) Bar diagram of the quantitative summary for GSDMD-N immunofluorescence. Around sixty cells in each group from three graphs were counted. * p < 0.05, ** p < 0.01 vs. the mock group.

Article Snippet: The expression and localization of targets were detected by immunofluorescence analysis under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan,) with oil immersion and 100 × objective in 1 mL PBS buffer.

Techniques: Infection, Virus, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Immunofluorescence, Staining, Laser-Scanning Microscopy

Journal: Viruses

Article Title: Berberine Suppresses Influenza A Virus-Triggered Pyroptosis in Macrophages via Intervening in the mtROS-MAVS-NLRP3 Inflammasome Pathway

doi: 10.3390/v17040539

Figure Lengend Snippet: BBR inhibited GSDMD-mediated pyroptosis and cytokine release in macrophages infected with IAV. ( A ) The IL-1β and ( B ) TNF-α release levels in the culture supernatants from J774A.1 cells uninfected, infected with PR8 for 24 h, and infected with PR8 combined with BBR treatment at concentrations of 4.2, 8.4, and 16.8 μM were measured using ELISA. ( C ) Immunofluorescence staining for GSDMD-N was observed by confocal laser scanning microscope. J774A.1 cells were treated with the PR8 virus while BBR was at a concentration of 16.8 μM for 24 h. The nuclei are shown in blue using DAPI staining; GSDMD-N is shown in red and detected by antibody. The white arrows indicate the fluorescent hub formed by GSDMD-N aggregation. ( D ) Bar diagram of the quantitative summary for GSDMD-N immunofluorescence. Around sixty cells in each group from three graphs were counted. ** p < 0.01 vs. the mock group. # p < 0.05, ## p < 0.01 vs. the PR8 group.

Article Snippet: The expression and localization of targets were detected by immunofluorescence analysis under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan,) with oil immersion and 100 × objective in 1 mL PBS buffer.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Laser-Scanning Microscopy, Virus, Concentration Assay

Journal: Viruses

Article Title: Berberine Suppresses Influenza A Virus-Triggered Pyroptosis in Macrophages via Intervening in the mtROS-MAVS-NLRP3 Inflammasome Pathway

doi: 10.3390/v17040539

Figure Lengend Snippet: BBR attenuates NLRP3 inflammasome activation to reduce macrophage pyroptosis triggered by IAV. J774A.1 cells were infected with the PR8 virus for 24 h in the presence of BBR (16.8 µM) treatment. As a positive control, cells were pretreated with the NLRP3 inhibitor MCC950 (50 μM) for 3 h and then infected with the PR8 virus for an additional 24 h. ( A ) The LDH levels in the culture supernatants from the dead cells were measured using ELISA. ( B , C ) The expression levels of NLRP3 were detected by Western blot using β-actin as an internal control. ( D ) Immunofluorescence staining for the NLRP3 inflammasome was observed by confocal laser scanning microscope. NLRP3 and ASC are shown in green and blue, detected by antibody, respectively, while the mitochondria are shown in red using Mito Tracker staining. The white arrows represent regions with ASC expression. ( E ) Bar diagram of the quantitative summary for the NLRP3 immunofluorescence. ( F ) Bar diagram of the quantitative summary for ASC immunofluorescence. Around sixty cells in each group from three graphs were counted. ( G , H ) Flow cytometric analysis of the rates of pyroptosis mediated by caspase-1 using the FAM-FLICA Caspase-1 Kit. ( I ) The IL-1β levels in the culture supernatants were measured using ELISA. ( J , K ) The expression levels of GSDMD-N were detected by Western blot using β-actin as an internal control. * p < 0.05, ** p < 0.01 vs. the mock group. # p < 0.05, ## p < 0.01 vs. the PR8 group.

Article Snippet: The expression and localization of targets were detected by immunofluorescence analysis under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan,) with oil immersion and 100 × objective in 1 mL PBS buffer.

Techniques: Activation Assay, Infection, Virus, Positive Control, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Immunofluorescence, Staining, Laser-Scanning Microscopy

Journal: Viruses

Article Title: Berberine Suppresses Influenza A Virus-Triggered Pyroptosis in Macrophages via Intervening in the mtROS-MAVS-NLRP3 Inflammasome Pathway

doi: 10.3390/v17040539

Figure Lengend Snippet: BBR suppresses mitochondrial ROS release in macrophages infected with IAV. ( A ) Immunofluorescence staining for MAVS was observed by confocal laser scanning microscope. MAVS and NLRP3 are shown in green and blue, detected by antibody, respectively, while the mitochondria are shown in red using Mito Tracker staining. The white arrows represent regions with higher NLRP3 expression. ( B ) Bar diagram of the quantitative summary for NLRP3 immunofluorescence. ( C ) Bar diagram of the quantitative summary for MAVS immunofluorescence. ( D ) Immunofluorescence staining for mtROS was observed by confocal laser scanning microscope. The ROS are shown in green marked by DCFH-DA, while the mitochondria are shown in red using Mito Tracker staining. The white arrows represent regions with higher NLRP3expression. ( E ) Bar diagram of the quantitative summary for ROS immunofluorescence. ( F , G ) Detection of the mtROS levels by flow cytometry with DCFH-DA. * p < 0.05, ** p < 0.01 vs. the mock group. ## p < 0.01 vs. the PR8 group. △△ p < 0.01 vs. the PR8 + NAC group.

Article Snippet: The expression and localization of targets were detected by immunofluorescence analysis under an FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan,) with oil immersion and 100 × objective in 1 mL PBS buffer.

Techniques: Infection, Immunofluorescence, Staining, Laser-Scanning Microscopy, Expressing, Flow Cytometry

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