cmv promoter  (Addgene inc)

Journal: Scientific Reports

Article Title: A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress

doi: 10.1038/s41598-022-11704-9

Figure Lengend Snippet: NHEJ components Ku70 and Ku80 are novel ATG5 interactors. ( a ) HEK293T cells were co-transfected with plasmids encoding FLAG-tagged Ku70 and/or non-tagged full-length ATG5 proteins. ( b ) HEK293T cells were co-transfected with plasmids encoding FLAG-tagged Ku80 and/or non-tagged full-length ATG5 proteins. ( c ) HeLa cells were co-transfected with plasmids encoding FLAG-tagged Ku70 and/or non-tagged full-length ATG5 proteins. ( d ) HeLa cells were co-transfected with plasmids encoding FLAG-tagged Ku80 and/or non-tagged full-length ATG5 proteins. Cells were exposed to Etoposide, Doxorubicin and Cisplatin after 24 h post-transfection. 50 µM Etoposide, 12.5 µg/ml Cisplatin, 1 µm Doxorubicin; 25 µM Etoposide, 1 µg/ml Cisplatin, 100 nm Doxorubicin were used for HEK293T and HeLa cells, respectively. 48 h later, IPs were performed using FLAG beads. Anti-ATG5 and anti-FLAG antibodies were used for immunoblotting. Input, total cell extract. Molecular Mass was shown in kilodaltons (kDa).

Article Snippet: Flag and GFP tagged human Ku70 (Addgene, #46957) and Ku80 (Addgene, #46958), pmCherry-ATG5 (Addgene, #13095) plasmid were all provided by Addgene.

Techniques: Transfection, Western Blot

Journal: Scientific Reports

Article Title: A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress

doi: 10.1038/s41598-022-11704-9

Figure Lengend Snippet: ATG5 endogenously interacts with Ku70. ( a ) HEK293T cells were transfected with plasmids encoding non-tagged ATG5. Cells were exposed to either Etoposide or DMSO, as a vehicle, and IPs were performed with the anti-ATG5 antibody at 48 h post-transfection. ( b ) Endogenous IPs were performed from HEK293T cells treated with Etoposide for along 24 h. DMSO was used as a vehicle. Rabbit serum was used as a stickiness control. ( c ) GST Pull-down assay was performed. Glutathione-Sepharose beads were bound to GST-Ku70 recombinant protein or not incubated with His-ATG5 recombinant protein and washed (Input: Immunoblotting of recombinant proteins; GST-pull down: proteins following pull-down). ( d ) HEK293T cells were co-transfected with plasmids encoding FLAG-tagged Ku70 and/or non-tagged full-length ATG5 or plasmid encoding 1–192 a.a. of ATG5 proteins. Cells were exposed to either Etoposide or DMSO as a vehicle after 24 h post-transfection and IPs were performed using Flag beads. Anti-ATG5, anti-Ku70, anti-Ku80 and anti-FLAG antibodies were used for immunoblotting. Input, total cell extract. ACTB, anti-β-Actin was used as a loading control.

Article Snippet: Flag and GFP tagged human Ku70 (Addgene, #46957) and Ku80 (Addgene, #46958), pmCherry-ATG5 (Addgene, #13095) plasmid were all provided by Addgene.

Techniques: Transfection, Control, Pull Down Assay, Recombinant, Incubation, Western Blot, Plasmid Preparation

Journal: Scientific Reports

Article Title: A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress

doi: 10.1038/s41598-022-11704-9

Figure Lengend Snippet: Dynamic nature of ATG5-Ku70 interaction during genotoxic stress. ( a – c ) HEK293T cells were exposed to Etoposide ( b ) or Doxorubicin ( c ) treatment for 24 h. DMSO ( a ) was used as a vehicle. Total cell extracts were subjected to gel filtration chromatography. Collected fractions were analyzed by immunoblotting using anti-ATG5 and anti-Ku70 antibodies. Molecular weights of fractions were marked. L, cell lysates before fractionation. ( d ) HEK293T cells were cultured on coverslides and co-transfected with GFP-tagged Ku70 (green) and Cherry-tagged ATG5 (red) constructs. Cells were exposed to Etoposide for 24 h after transfection. DMSO was used as a vehicle. 48 h post-transfection cells were fixed and analyzed under a confocal microscope at 63× magnification. Nuclei were stained with Hoechst (blue). Merge, overlay green and red signals. ( e ) Confocal images were analyzed by counting 100 cells and % of ATG5-Ku70 colocalization in the nucleus was represented in a graph (mean ± S.D. of independent experiments, n = 3, *, p < 0.05). ( f ) Cellular fractionation was performed after Doxorubicin treatment. DOX (+), Doxorubicin; (−), DMSO. Nucleus, nuclear fraction; Cytosol, cytosolic fraction; Lysate, the whole-cell lysate was subjected to immunoblotting. Anti-ATG5, anti-Ku70, anti-Ku80, anti-Lamin A/C and anti-β-Actin were used as nuclear and cytosolic fractionation control, respectively.

Article Snippet: Flag and GFP tagged human Ku70 (Addgene, #46957) and Ku80 (Addgene, #46958), pmCherry-ATG5 (Addgene, #13095) plasmid were all provided by Addgene.

Techniques: Filtration, Chromatography, Western Blot, Fractionation, Cell Culture, Transfection, Construct, Microscopy, Staining, Cell Fractionation, Control

Journal: Scientific Reports

Article Title: A novel ATG5 interaction with Ku70 potentiates DNA repair upon genotoxic stress

doi: 10.1038/s41598-022-11704-9

Figure Lengend Snippet: Role of ATG5-Ku70 interaction in DNA damage sensing and repair. ( a ) HeLa cells were cultured on coverslides and exposed to Etoposide for 1 h. DMSO was used as a vehicle. Etoposide was washed out after 1 h and cells were cultured for along 6 h to recover. t = 0, 1 h etoposide treated cells. t = 6, 6 h recovered cells. Cells were fixed and indirect immunofluorescent analysis was utilized. Alexa-488 (green) and Alexa-568 (red) were used as secondary antibodies against rabbit anti-ATG5 and mouse anti-Ku70, respectively. Cells were visualized under a confocal microscope at 63× magnification. Nuclei were stained with Hoechst (blue). Merge, overlay green and red signals. ( b ) Colocalization coefficients of 50 cells were calculated from confocal images and data represented as a graph (mean ± S.D. of independent experiments, n = 3, *, p < 0.05). ( c ) HeLa WT and ATG5 KO cells were treated with Etoposide for 1 h. After treatment etoposide was washed out and cells remained in the culture for along 6 h, 24 h and 48 h for recovery. CNT, DMSO treated cells as a vehicle. Then proteins were collected and analyzed by immunoblotting using anti-ATG5, anti-Ku70, and anti-γH2A.X. Anti-β-Actin was used as a loading control. ( d ) A rescue experiment was performed. HeLa ATG5 KO cells were cultured and transfected with ATG5 expressing plasmid. 48 h post-transfection cells were treated with etoposide for 1 h. After treatment etoposide was washed out and cells remained in the culture for along 6 h, 24 h and 48 h for recovery. CNT, DMSO treated cells as a vehicle. Then cells were collected and analyzed by immunoblotting using anti-ATG5, anti-Ku70, and anti-γH2A.X. Anti-β-Actin was used as a loading control.

Article Snippet: Flag and GFP tagged human Ku70 (Addgene, #46957) and Ku80 (Addgene, #46958), pmCherry-ATG5 (Addgene, #13095) plasmid were all provided by Addgene.

Techniques: Cell Culture, Microscopy, Staining, Western Blot, Control, Transfection, Expressing, Plasmid Preparation