Fig 2: 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-?H2A.X. Anti-ß-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-?H2A.X. Anti-ß-Actin was used as a loading control.

Fig 3: 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-ß-Actin was used as a loading control.

Fig 4: 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-ß-Actin were used as nuclear and cytosolic fractionation control, respectively.