Figure 6: MICU1 silencing restores pyruvate dehydrogenase activity.

(a) Immunoblot analysis for phospho pyruvate dehydrogenase (pPDH[ser293]), pyruvate dehydrogenase (PDH), pyruvate dehydrogenase phosphatase (PDP) and pan-pyruvate dehydrogenase kinase (pan-PDK) in OSE and various OvCa cell lines. GAPDH was used as a loading control. (b) MICU1 silencing increases PDP-PDH interaction. Immunoprecipitation of PDP followed by immunoblot for PDH and PDP in siCTL-OV90 and siMICU1-OV90 cells. (c) Quantification for PDH band intensity measurement using ImageJ software normalized against PDP. Values represent mean±s.d. (d) MICU1 silencing causes diminished phosphorylation status of PDH. Immunoblot for pPDH in whole cell lysates from siCTL-OV90 and siMICU1-OV90 cells and compared against PDH. (e) Densitometry analysis of (d) by ImageJ, pPDH band intensity is normalized to corresponding PDH levels and represented as mean fold change±s.d. (f) Pyruvate dehydrogenase (PDH) activity was measured in MICU1 siRNA treated OV90 or CP20 cells and MICU1 knockdown OV90 cells and values represent mean±s.d. (g) MICU1 overexpression decreases PDP and PDH interaction. Immunoprecipitation of PDP followed by immunoblot for PDH and PDP in Flag-MICU1 expressing OSE cells. (h) Quantification for PDH band intensity measurement using ImageJ software normalized against PDP and values represent mean±s.d. (i) In Flag-MICU1 expressing OSE cells, PDH activity was measured and values represent mean±s.d. (j) MICU1 silencing increases PDP and PDH interaction upon cisplatin (10 μM) treatment. Immunoprecipitation of PDP followed by immunoblot for PDH and PDP in siCTL-OV90 and siMICU1-OV90 cells. (k) Quantification for PDH band intensity measurement using ImageJ software normalized against PDP. (l) Immunohistochemical staining of a tissue microarray of drug resistant epithelial OvCa samples for MICU1 (1:150) or pPDH (1:200). Representative images taken at × 4 magnification are shown of (i) weak, (ii) moderate and (iii) high staining. Inset shows magnified areas of individual IHC stains taken at × 20 magnification. The bar represents 100 μm. *P<0.05 statistically significant when compared to the respective controls, using two-sided Student’s t-test with unequal variances. Horizontal bar represents statistical comparison between respective groups. All the experiments were repeated independently at least three times and in triplicates.