Figure 1

Loss of SOCS6 confers cells resistance to apoptotic insults. (a) The expressions of SOCS6 in HeLa cells stably harboring shRNAs targeting at luciferase (control-KD) and SOCS6 (SOCS6-KD) were assessed by RT-PCR and western blot analyses. Two individual shRNAs targeting SOCS6 were employed. (b–e) HeLa cells stably harboring shRNA directed against luciferase and SOCS6 were irradiated with UV at 90 J/m². After incubation for the designated times, cells were harvested for apoptosis assays. Data derived from three separate experiments are presented as the means±S.E.M. (b) Annexin V staining of phosphatidylserine exposed on the cell surface was measured by flow cytometric analysis. (c) Total cell lysates were prepared for western blot analysis of the designated proteins. (d) Cells were stained with propidium iodide and subjected to FACS analysis of sub-G1 population. (e) Cells were labeled with MitoTracker and immunostained using an anti-cytochrome c antibody followed by labeling with Alexa 488 and counterstaining with DAPI. Representative immunofluorescence images are shown. Cells that released cytochrome c, as indicated by arrows, were scored at the designated time points and presented as percentage of total cells in a bar graph. Scale bar: 10 μm. (f–j) Ectopic expression of SOCS6 promotes apoptosis and Bax activation. (f) AD293 cells were transfected with pcDNA3-SOCS6 (SOCS6) or the control vector (mock), UV irradiated, and subjected to FACS analysis of sub-G1 population at the designated times. Data derived from three separate experiments are presented as the means±S.E.M. (g) HeLa cells were transfected with a control vector or increasing amounts of pcDNA3-SOCS6. Cells were lysed in CHAPS buffer and immunoprecipitated using an anti-Bax monoclonal antibody, 6A7. Immunoprecipitates were subjected to western blotting using an anti-Bax polyclonal antibody (N-20). (h) Mock- and HA-SOCS6-transfected HeLa cells were treated with or without UV at 90 J/m². After 6 h, cells were labeled with MitoTracker and immunostained using an anti-Bax antibody. Representative images are shown. Cells with Bax accumulation in mitochondria, indicated by arrows, were scored and are presented as the percentage of total cells in a bar graph. Scale bar: 10 μm. Data derived from three separate experiments are presented as the means±S.E.M. *P<0.05 by Student’s t-test. (i) HeLa cells were transfected with the control vector or pcDNA3-SOCS6, and treated with increasing concentrations of STS for 6 h. Cells were lysed and examined for Bax conformational change as described above. (j) Mock- and HA-SOCS6-transfected HeLa cells were incubated with the crosslinker (DSS) and subjected to western blot analysis using a Bax polyclonal antibody