anti-rabbit γh2ax  (Cell Signaling Technology Inc)

Journal: Journal of Nanobiotechnology

Article Title: Lyophilized apoptotic vesicles restore DNA damage and mitochondria dysfunction to ameliorate radiation enteritis

doi: 10.1186/s12951-025-03592-8

Figure Lengend Snippet: Lpl-apoVs repair DNA damage and alleviate oxidative stress in vivo. ( A ) Immunofluorescence images quantitative analysis of intestinal sections from control, irradiated, and Lpl-apoVs-treated groups, stained with γH2AX showing radiation-induced DNA damage ( n = 4). Scale bars, 100 μm. ( B ) Western blot analysis of γH2AX and p53 protein levels in intestinal tissue from control, irradiated, and Lpl-apoVs-treated groups. ( C ) Immunofluorescence images and quantitative analysis of 8-OHdG (red) showing oxidative stress-induced DNA damage in control, irradiated, and Lpl-apoVs-treated groups ( n = 4). Scale bars, 100 μm. ( D ) Fluorescence images and quantitative analysis of TOMM20 showing mitochondria damage in control, irradiated, and Lpl-apoVs-treated groups ( n = 4). Scale bars, 20 μm. Graph bars show the mean ± SD. * p < 0.05, *** p < 0.001. ns, not significant

Article Snippet: Anti-rabbit γH2AX (9718 S), anti-rabbit Annexin V (8555s), anti-rabbit Ku70 (4588 S), anti-mouse PCNA (2586 S), anti-rabbit PARP (9532 S), anti-rabbit calreticulin (12238 S) and anti-mouse P53 (2524 S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

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

Journal: Journal of Nanobiotechnology

Article Title: Lyophilized apoptotic vesicles restore DNA damage and mitochondria dysfunction to ameliorate radiation enteritis

doi: 10.1186/s12951-025-03592-8

Figure Lengend Snippet: Lpl-apoVs alleviate cell radiation injury after internalized by intestinal epithelial cells (IECs). ( A ) Time-dependent ex vivo fluorescence images of colon and cecum dissected after sacrificing the mice. ( B ) Representative images of intestinal slices from mice after enema administration of PKH26-Lpl-apoVs for 1 h. Scale bars, 50 μm for lower magnification, 5 μm for higher magnification. ( C ) Immunofluorescence images of Caco-2 cells after co-culture with PKH26-Lpl-apoVs for 24 h. Scale bars, 20 μm for lower magnification, 2 μm for higher magnification. ( D ) Immunofluorescence images and quantitative analysis of Caco-2 cells from control, irradiated, and Lpl-apoVs-treated groups, stained with γH2AX showing radiation-induced DNA damage ( n = 50). Scale bars, 5 μm. ( E ) Western blot analysis of protein levels related to DNA damage in Caco-2 cells from control, irradiated, and Lpl-apoVs-treated groups. ( F ) Flow cytometry analysis showing the reactive oxygen species (ROS) in Caco-2 cells from control, irradiated, and Lpl-apoVs-treated groups. ( G-H ) Immunofluorescence images ( G ) and Transmission electron microscopy (TEM) images ( H ) showing mitochondria morphology damage in Caco-2 cells from control, irradiated, and Lpl-apoVs-treated groups. Scale bars, 10 μm and 2 μm for lower magnification, 5 μm and 250 nm for higher magnification. ( I ) Immunofluorescence images showing mitochondria condition in Caco-2 cells from control, irradiated, and Lpl-apoVs-treated groups. JC-10 aggregates (red fluorescence) indicated healthy mitochondria and JC-10 monomers (green fluorescence) indicated depolarized mitochondria. Scale bars, 200 μm. Graph bars show the mean ± SD. *** p < 0.001

Article Snippet: Anti-rabbit γH2AX (9718 S), anti-rabbit Annexin V (8555s), anti-rabbit Ku70 (4588 S), anti-mouse PCNA (2586 S), anti-rabbit PARP (9532 S), anti-rabbit calreticulin (12238 S) and anti-mouse P53 (2524 S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Ex Vivo, Fluorescence, Immunofluorescence, Co-Culture Assay, Control, Irradiation, Staining, Western Blot, Flow Cytometry, Transmission Assay, Electron Microscopy

Journal: Journal of Nanobiotechnology

Article Title: Lyophilized apoptotic vesicles restore DNA damage and mitochondria dysfunction to ameliorate radiation enteritis

doi: 10.1186/s12951-025-03592-8

Figure Lengend Snippet: Lpl-apoVs restore irradiation-induced cell dysfunction in IECs by repairing DNA damage. ( A ) Immunofluorescence images and quantitative analysis of irradiated Caco-2 cells receiving Lpl-apoVs treatment with or without PARP inhibitor (OLP), stained with γH2AX showing radiation-induced DNA damage ( n = 50). Scale bars, 5 μm. ( B ) Western blot analysis of protein levels related to DNA damage in Caco-2 cells. ( C ) Flow cytometry analysis showing the reactive oxygen species (ROS) in Caco-2 cells. ( D ) Immunofluorescence images showing radiation-induced mitochondria morphology damage in Caco-2 cells, and respective mitochondrial morphology analysis of cells in mean area, perimeter, branch length, aspect ratio per mitochondrion ( n = 10). Scale bars, 10 μm for lower magnification, 5 μm for higher magnification. ( E ) Immunofluorescence images showing mitochondria condition in Caco-2 cells. JC-10 aggregates (red fluorescence) indicated healthy mitochondria and JC-10 monomers (green fluorescence) indicated depolarized mitochondria. Scale bars, 200 μm. Graph bars show the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, not significant

Article Snippet: Anti-rabbit γH2AX (9718 S), anti-rabbit Annexin V (8555s), anti-rabbit Ku70 (4588 S), anti-mouse PCNA (2586 S), anti-rabbit PARP (9532 S), anti-rabbit calreticulin (12238 S) and anti-mouse P53 (2524 S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Irradiation, Immunofluorescence, Staining, Western Blot, Flow Cytometry, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Lyophilized apoptotic vesicles restore DNA damage and mitochondria dysfunction to ameliorate radiation enteritis

doi: 10.1186/s12951-025-03592-8

Figure Lengend Snippet: Lpl-apoVs eliminate dysfunctional mitochondria and restore DNA damage in IECs through mitophagy. ( A ) Immunofluorescence images of Caco-2 cells showing mitochondrial (green)-lysosomal (red) co-localization. Caco-2 cells were subjected to no irradiation, irradiation, Lpl-apoVs treatment with or without Mdv intervention after irradiation. Quantification of co-localization intensity is shown on the right. Scale bars, 10 μm. ( B ) TEM images showing mitochondria and mitophagosome (green arrows) in Caco-2 cells. Scale bars, 500 nm for lower magnification, 20 nm for higher magnification. ( C ) Western blot analysis of expression level related to mitophagy pathway. ( D ) Flow cytometry analysis showing the ROS in Caco-2 cells. ( E ) Immunofluorescence images and quantitative analysis of irradiated Caco-2 cells receiving Lpl-apoVs treatment with or without Mdv-intervention, stained with DNA damage marker γH2AX ( n = 50). Scale bars, 5 μm. ( F ) Western blot analysis of expression levels related to DNA damage in Caco-2 cells. Graph bars show the mean ± SD. ** p < 0.01, *** p < 0.001. ns, not significant. Mdv: Mdivi-1

Article Snippet: Anti-rabbit γH2AX (9718 S), anti-rabbit Annexin V (8555s), anti-rabbit Ku70 (4588 S), anti-mouse PCNA (2586 S), anti-rabbit PARP (9532 S), anti-rabbit calreticulin (12238 S) and anti-mouse P53 (2524 S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunofluorescence, Irradiation, Western Blot, Expressing, Flow Cytometry, Staining, Marker

Journal: Science Advances

Article Title: The RNA demethylase FTO promotes glutamine metabolism in clear cell renal cell carcinoma through the regulation of SLC1A5

doi: 10.1126/sciadv.adv2417

Figure Lengend Snippet: ( A and B ) Cells were cultured in media with vehicle or 5 μM FB23-2 and supplemented with pyrimidines (cytidine 5 μM, thymidine 5 μM, and uridine 5 μM), NAC (4 mM), or a combination of pyrimidines and NAC for 72 hours. Immunofluorescence analysis of γH2AX foci in 786-M1A cells and UMRC2 cells (A). Bar graphs show the percentage of nuclei with >50 γH2AX foci (B). At least 50 nuclei for each biological replicate were analyzed. ( C and D ) Colony formation assays examined the growth and survival of 786-M1A and UMRC2 cells treated with FB23-2 (5 μM), FB23-2 (5 μM) + pyrimidines (5 μM), FB23-2 (5 μM) + NAC (4 mM), and FB23-2 (5 μM) + pyrimidines (5 μM) + NAC (4 mM). ( E and F ) 786-M1A and UMRC2 shCtrl and shFTO cells were pretreated with Dox (2 μg/ml) for 5 days and then the cells were supplemented with pyrimidines (5 μM), NAC (4 mM), or a combination of pyrimidines and NAC for 72 hours. Immunofluorescence analysis of γH2AX foci (E). Percentage of nuclei with greater than 50 γH2AX foci (F). At least 50 nuclei for each biological replicate were analyzed. ( G and H ) The 786-M1A and UMRC2 shCtrl and shFTO cells were pretreated with Dox (2 μg/ml) for 5 days, then with or without supplementation with 5 μM pyrimidines, 4 mM NAC, or the combination. Cell growth and survival were determined by 2D colony formation assays. Each column represents the mean ± SD. Biological replicates ( n = 3) were analyzed in each group. Statistically significant differences are indicated: * P < 0.05; ** P < 0.01; *** P < 0.001; determined by the Student’s two-tailed t test.

Article Snippet: Following three rinses with ice-cold PBS and fixation with 4% paraformaldehyde for 15 min at room temperature, the cells were washed again with ice-cold PBS for 5 min. Next, 0.3% Triton X-100 was added to each well and incubated for 10 min at room temperature, followed by another wash with ice-cold PBS for 5 min. To block nonspecific binding, the cells were incubated with 2% BSA for 30 min. After blocking, the primary rabbit anti-γH2AX antibody (Cell Signaling Technology, catalog no. 9718, 1:400) was incubated overnight at 4°C.

Techniques: Cell Culture, Immunofluorescence, Two Tailed Test

Journal: Science Advances

Article Title: The RNA demethylase FTO promotes glutamine metabolism in clear cell renal cell carcinoma through the regulation of SLC1A5

doi: 10.1126/sciadv.adv2417

Figure Lengend Snippet: ( A ) Western blot analysis of FTO, SLC1A5, and B-actin in shCtrl, shCtrl + SLC1A5, shFTO, and shFTO + SLC1A5 786-M1A cells. 786-M1A shCtrl and shFTO cells were pretreated with Dox (2 μg/ml) for 5 days, then transfected with pcDNA-3.1 vector or pcDNA-3.1-SLC1A5 plasmid for 48 hours before extracting proteins. ( B ) U-13C glutamine tracing in cells. The fractions of m + 3 aspartate, malate, and fumarate were analyzed using LC-MS. ( C ) Relative GSH/GSSG ratio in shCtrl and shFTO 786-M1A cells pretreated with Dox (2 μg/ml) for 5 days, transfected with pcDNA-3.1 vector or pcDNA-3.1-SLC1A5 plasmid for 48 hours. ( D and E ) Immunofluorescence analysis of γH2AX foci of 786-M1A shCtrl and shFTO cells pretreated with Dox (2 μg/ml) for 5 days, then transfected with pcDNA-3.1 vector or pcDNA-3.1-SLC1A5 plasmid for 48 hours. Bar graphs show the percentage of nuclei with >50 γH2AX foci per nucleus. At least 50 nuclei for each biological replicate were analyzed. ( F and G ) Colony formation assays examined the growth and survival of shCtrl, shCtrl + SLC1A5, shFTO, and shFTO + SLC1A5 786-M1A cells. Macroscopic images of representative colonies formed in each group (F). Quantification of total colony number in each group (G). Each column represents the mean ± SD. Biological replicates ( n = 3) were analyzed in each group. Statistically significant differences are indicated: * P < 0.05; ** P < 0.01; *** P < 0.001; determined by the Student’s two-tailed t test.

Article Snippet: Following three rinses with ice-cold PBS and fixation with 4% paraformaldehyde for 15 min at room temperature, the cells were washed again with ice-cold PBS for 5 min. Next, 0.3% Triton X-100 was added to each well and incubated for 10 min at room temperature, followed by another wash with ice-cold PBS for 5 min. To block nonspecific binding, the cells were incubated with 2% BSA for 30 min. After blocking, the primary rabbit anti-γH2AX antibody (Cell Signaling Technology, catalog no. 9718, 1:400) was incubated overnight at 4°C.

Techniques: Western Blot, Transfection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Two Tailed Test

Journal: Science Advances

Article Title: The RNA demethylase FTO promotes glutamine metabolism in clear cell renal cell carcinoma through the regulation of SLC1A5

doi: 10.1126/sciadv.adv2417

Figure Lengend Snippet: ( A to C ) Immunofluorescence analysis of γH2AX foci of 786-M1A and UMRC2 cells pretreated with 5 μM FB23-2 and/or 1 μM PARPi (talazoparib) for 72 hours. Bar graphs show the percentage of nuclei with >50 γH2AX foci per nucleus. At least 50 cells for each biological replicate were analyzed. ( D to F ) 786-M1A and UMRC2 cells were treated with vehicle or 5 μM FB23-2 in the presence or absence of 1 μM PARPi (talazoparib) in colony formation assays. Each column represents the mean ± SD. Biological replicates ( n = 3) were analyzed in each group. Statistically significant differences are indicated: * P < 0.05; ** P < 0.01; *** P < 0.001, **** P < 0.0001; determined by the Student’s two-tailed t test.

Article Snippet: Following three rinses with ice-cold PBS and fixation with 4% paraformaldehyde for 15 min at room temperature, the cells were washed again with ice-cold PBS for 5 min. Next, 0.3% Triton X-100 was added to each well and incubated for 10 min at room temperature, followed by another wash with ice-cold PBS for 5 min. To block nonspecific binding, the cells were incubated with 2% BSA for 30 min. After blocking, the primary rabbit anti-γH2AX antibody (Cell Signaling Technology, catalog no. 9718, 1:400) was incubated overnight at 4°C.

Techniques: Immunofluorescence, Two Tailed Test