Fig. 4

STOML2 interacted with PINK1 and contributed to its stability. a Total cell lysate was extracted from STOLM2 Flag-expressing or control cells treating with CCCP (10 μM), purified and resolved on SDS-PAGE. Silver stained gel showed differential bands, then the bands were retrieved and analyzed by MS. Identified PINK1 peptides are shown. b SMMC-7721 cells were transfected with STOML2-Flag or empty vector and subjected to immunoprecipitation using anti-Flag mAb. Co-immunoprecipitated PINK1 was detected using anti-PINK1 antibody (up panel). Endogenous STOML2 in HCCLM3 cells was immunoprecipitated using anti-STOML2 antibody with rabbit IgG as nonspecific control (down panel). Co-immunoprecipitated PINK1 was detected using anti-PINK1 antibody. c The co-localization between STOML2 (green) with PINK1 (red) was analyzed by confocal microscopy in SMMC-7721-STOML2 and HCCLM3 with CCCP (10 μM) stimulation. (Scale bar: 10 μm) d The expression and co-localization between STOML2 and PINK1 were analyzed in HCC and peri-tumor liver tissue of HCC patients by confocal microscopy. (Scale bar: 25 μm) e Under the treatment of CCCP (10 μM), total and mitochondrial protein levels of PINK1and Parkin were analyzed by Western blot. GAPDH and COX IV were used as loading controls. f Western blot analysis for PINK1, Parkin, LC3B I/II in 48 HCC tissues and peri-tumorous tissues patients (left panel), correlation analysis of the relative protein expression showed positive correlation between STOML2 and PINK1 in HCC patients (right panel). g Overexpression of STOML2 in SMMC-7721 increased accumulation of polyubiquitinated PINK1 when treated with MG132. PINK1 was pulled down and anti-ubiquitin antibody was used to detect polyubiquitinated PINK1 (left panel). Knockdown of STOML2 in HCCLM3 reduced polyubiquitinated PINK1 (right panel). h The upregulation of STOML2 reduces CCCP-induced PINK1 degradation. Western blot detected the alteration of PINK1 in SMMC-7721 and HCCLM3 with co-treatment of 10 μM CCCP and 10 μg/ml CHX for the indicated times (left panel). Densitometric analysis of PINK1 blots from three independent experiments is shown (right panel). GAPDH was used as a loading control