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. Author manuscript; available in PMC: 2016 Feb 11.
Published in final edited form as: Sci Signal. 2015 Aug 11;8(389):ra80. doi: 10.1126/scisignal.aab1624

Fig. 3. Survivin regulation of mitochondrial Complex II.

Fig. 3

(A) PC3 cells treated with vehicle (Veh) or YM155 were permeabilized with digitonin and analyzed for Complex II activity in the presence of succinate and rotenone. Oligo, oligomycin. Graphs shows means±SEM from three independent experiments. **, p<0.01. (B) PC3 cells transfected with control (Ctrl) or survivin-directed (SVV) siRNA were analyzed by Western blotting. Blots are representative of two independent experiments. (C) Mitochondrial Complex II was immunoprecipitated from siRNA-transfected PC3 cells as in (B) and analyzed for enzymatic activity. Right, quantification. Graph shows means±SEM from three independent experiments. *, p=0.037. (D) Mitochondria from PC3 cells treated with vehicle (Veh) or YM155 were analyzed by Western blotting. The position of oxidative phosphorylation complex subunits is indicated. d, days. Blots are representative of two independent experiments. (E and F) PC3 cells treated with vehicle (Veh) or YM155 (E), or MCF-7 cells transfected with vector or mt-SVV cDNA (F) were analyzed by Western blotting. In (F), the position of endogenous (SVV) or transfected (HA) survivin is indicated. Blots in (E) and (F) are representative of two independent experiments. (G) PC3 cells were transfected with the indicated siRNAs and proteins remaining insoluble at increasing detergent concentrations (CHAPS) were analyzed by Western blotting. The extra band in the SDHC lane corresponds to non-specific reactivity with a molecular weight marker. Blots are representative of two independent experiments. (H) Mitochondrial extracts from PC3 cells were immunoprecipitated (IP) with IgG or an antibody to survivin, and pellets were analyzed by Western blotting. Blots are representative of two independent experiments. (I) Aliquots of GST-TRAP-1 (top) or GST (bottom) were incubated with recombinant survivin (SVV), and bound proteins were analyzed by Western blotting. The molar ratio of survivin to TRAP-1 was 0, 0.1, 0.5 and 1. Blots are representative of two independent experiments. (J) PC3 mitochondrial extracts were immunoprecipitated (IP) with IgG or an antibody to survivin, and pellets were analyzed by Western blotting. Blots are representative of two independent experiments. (K and L) PC3 cells were transfected with control (Ctrl) or TRAP-1-directed siRNA (K), or treated with mitochondrial-targeted small molecule Hsp90 inhibitor, Gamitrinib (Gam) (L), and extracts were analyzed after 48 h (K) or at the indicated time intervals (L), by Western blotting. Blots are representative of two independent experiments for (K) and (L). (M and N) PC3 cells were treated with vehicle (Veh) or Gamitrinib (Gam), then cycloheximide, and analyzed by Western blotting (M), with quantification of survivin half-life by densitometry (N). The quantification from two independent experiments (Expt.) is shown.