Fig 1: Hsp90/CDC37 chaperone determined the apoptotic and necroptotic function of RIPK3 kinase.(A) The cell lysates from cultured HT29, HeLa, MCF7, and KGN cells were analyzed by western blotting using antibodies as indicated. (B, C) Cultured HeLa-RIPK3, MCF7/TO-RIPK3, and KGN/TO-RIPK3 cells were treated with the indicated stimuli for 36 hr. Cell viability was determined by measuring cellular ATP levels. The data are represented as the mean ± SD of triplicate wells. ***p<0.001. p-values were determined by two-sided unpaired Student’s t-tests. 17AAG, Hsp90 inhibitor. (D, E) HeLa/TO-RIPK3 cells were treated with the indicated stimuli for 36 hr. Cell viability was determined by measuring cellular ATP levels in (D). The data are represented as the mean ± SD of triplicate wells. ***p<0.001. p-values were determined by two-sided unpaired Student’s t-tests. 24 hr after treatment, the cell lysates were analyzed by western blotting using antibodies against phospho-serine 164/threonine 165 of RIPK3, RIPK3, and β-actin as indicated in (E). (F, G) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with DMSO or Dox for 36 hr. Cell viability was determined by measuring cellular ATP levels in (F). The data are represented as the mean ± SD of triplicate wells. ***p<0.001. p-values were determined by two-sided unpaired Student’s t-tests. 24 hr after treatment, the cell lysates were analyzed by western blotting using antibodies against phospho-serine 164/threonine 165 of RIPK3, RIPK3, Hsp90, CDC37, and β-actin as indicated in (G). (H) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hr. The cells were then harvested, and RIPK3 was immunoprecipitated from the cell lysates using anti-Flag resin. The cell lysates and immunocomplexes were analyzed by western blotting using antibodies as indicated. (I) Cultured HeLa/TO-RIPK3 cells were treated with Dox or Dox plus 17AAG for 24 hr. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel. (J) Cultured MCF7/TO-RIPK3 cells co-transfected with HSP90 and CDC37 as indicated were treated with Dox for 24 hr. Immunofluorescence of the cells with Flag-RIPK3 (red) antibody. Counterstaining with DAPI (blue). Scale bar, 10 μm. Higher-power views (right panels) were acquired from the selected boxed areas from the left panel.