Fig. 4

TPD54 knockdown leads to increased PDH E1α phosphorylation at serine 232 by PDK1 followed by increased ubiquitination and degradation. a PDH E1α protein levels were detected by western blot in control and TPD54 stably knocked down cells with either DMSO or MG132 (10 μM) treatment for 1 day. b control and TPD54 stably knocked down cells transfected with control siRNA or siPDK1 were treated with metformin for 2 days. Western blot assays were performed to detect indicated proteins. c The ratio of pS232 PDH E1α to total PDH E1α in Fig. 5b was quantified and plotted as a bar graph. d Immunoprecipitation (IP) of PDH E1α with lysate from control cell and TPD54 stably knocked down cells was performed followed by immunoblotting of indicated proteins. Input represents whole cell lysate. e immunoprecipitation (IP) of PDH E1α with lysate of TPD54 stably knocked down cells with or without transient PDK1 knockdown was performed followed by immunoblotting of indicated proteins. Total proteins were determined in the input. f Control and TPD54 stably knocked down cells were transiently transfected with control siRNA or SiPKD1, followed by metformin treatment for 2 days. Western blot was then performed to detect indicated proteins. g Cell survival was determined in cells transfected with control, siPDK1, shTPD54, or double knockdowns. Values are presented as mean ± SD, student t test was used to calculate p value for two-group comparisons at different concentrations of metformin treatment (shCon siCon vs. shCon siPDK1 or shTPD54 siCon vs. shTPD54 siPDK1 with ***p < 0.001, **p < 0.01, *p < 0.05). h Western blot was performed to detect transient transfected WT PDH E1α and PDH E1α S232A mutant using SNAP-tag antibody and its ubiquitination modification after immunoprecipitation PDH E1α in control cells or TPD54 stably knocked down cells. Ubiquitination status and PDH E1α in whole cell lysate was detected by western blot as demonstrated in Input. i Western blots detecting protein changes among wild-type PDH E1α and its mutants S232A, S293G, and S300A were performed using SNAP-tag antibody in MCF-7 cells with or without transient TPD54 knockdown. All data presented were representative experimental data that have been replicated by independent experiments