EBNA3C is degraded through autophagy-lysosomal pathway when proteasome is inhibited.

LCLs were either left untreated (DMSO control) or treated with 1 μM MG132, 0.1 μM bafilomycin (Baf) or MG132 plus Baf. 24 h post-treatment cells were subjected for either (A) western blot analysis or (B) immunostaining with the indicated antibodies. Each panel in (B) is representative picture of two independent experiments and nuclei were counterstained by DAPI before mounting the cells. Scale bars, 5 μm. (C) ~10 x 10⁶ HEK293 cells transfected with either empty vector (pEGFP-C1) or GFP-tagged EBNA3C expression plasmid, either left untreated (DMSO control), or treated with 20 μM MG132 alone or MG132 plus 50 μM chloroquine (CQ). (D) HEK293 cells stably transfected with pTripz-mCherry-Sh-Beclin1 construct expressing sh-Beclin 1 under doxycycline (Dox) inducible promoter were further transfected either empty vector (pA3M) or myc-tagged EBNA3C expressing construct. 36 h post-transfection cells were either left untreated or treated with 20 μM MG132. (C-D) 4 h post-treatment cells were harvested, washed with 1 x PBS, lysed in RIPA buffer and subjected for western blot analyses for the indicated antibodies. (E) ~5 x 10⁴ HEK293 cells stably expressing sh-Beclin 1 with or without doxycycline treatment were transfected with GFP-tagged EBNA3C expressing plasmid using Lipofectamine 3000. 24 h post transfection cells were either left untreated (DMSO control) or treated with 20 μM MG132 for another 4h and subjected to live cell confocal analysis after staining the nucleus with Hochest 33342. Doxycycline treatment induces both mCherry and sh-RNA expression. Scale bars, 5 μm. (F) A colony formation assay was conducted as described in the “Materials and Methods” section using a similar experimental setup in doxycycline inducible sh-Beclin1 stably expressing HEK293 cells transiently transfected with GFP-EBNA3C construct.