Figure 6

Cells lacking CSN2, but not CSN5, are associated with autophagy. K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and re-cultured. (A) Jenner-Giemsa staining of shVC and shCSN2 cytospins day 6 post transfection. (B) shVC and shCSN2 cells were stained with the autophagosome marker monodansylcadaverine day 6 post transfection. All images shown are representative of n = 3 transfections. (C) Cells were harvested day 6 (shCSN2, top panels) or day 4 (shCSN5, bottom panels) post transfection and the level of LC3-II protein determined by western blot. Even loading was determined by β-actin signal. (D) Cell counts were taken daily and the cumulative growth of shVC cells and shCSN2 cells +/- 3-MA calculated. The insert has had the untreated shVC data removed in order to demonstrate the similarity between the shVC +3-MA and shCSN2 cumulative growth profiles. Data shown is the mean of n = 3 ± s.e.m. (E) Jenner-Giemsa staining of shVC +/- 1 μM bafilomycin A1 cytospins day 7 post transfection. Images shown are representative of n = 3 transfections. (F) Cells were harvested day 7 post transfection and the level of LC3-II and β-actin protein determined by western blot.