Figure 7: Effect of MICU1 knockdown on tumour growth and drug resistance.

(a)To assess the effects of knockdown of MICU1 on tumour growth, treatment with cisplatin was initiated 1 week after i.p. injection (1.0 × 10⁶ shCTL- or shMICU1-OV90) of tumour cells. Mice were divided into four groups (n=10 mice per group): (i) shCTL, (ii) shCTL +cisplatin (160 μg per mouse i.p. twice weekly), (iii) shMICU1 and (iv) shMICU1+cisplatin (160 μg per mouse i.p. twice weekly). Treatment was continued until 4 weeks after tumour inoculation before the killing. (a) Scattered plot of tumour weight (n=5) in HBSS-treated (sham) or 160 μg per mouse i.p. cisplatin-treated animals post 30 days of treatment. Statistical analysis was performed using one-way ANOVA followed by Newman-Keuls multiple comparison test. (b) Kaplan-Meier curves were plotted for the four groups of animals (n=5). Log-rank test P value was reported. (c) Representative histology of tumours from mice xenografts of shCTL-OV90 or shMICU1-OV90 cells with Ki67 expression and acquired at × 20 magnification. Scale bar represents 100 μm. (d) Quantification of Ki67 staining in the mouse xenografts respectively and values represent mean±s.d. (e) Representative histology of tumours from mice xenografts of shCTL-OV90 or shMICU1-OV90 cells with TUNEL staining and acquired at × 20 magnification. (f) Quantification of TUNEL staining in the respective mouse xenografts and values represent mean±s.e.m. (g) Immunoblotting for the expression of glycolytic enzyme lactate dehydrogenase (LDH) and pPDH(ser293) in tissues from mice tumour from shCTL-OV90 and shMICU1-OV90 cells. GAPDH was used as a loading control. (h) PDH activity measured in tumour tissues from shCTL-OV90 and shMICU1-OV90 cells and values represent mean±s.d. *P<0.05 was statistically significant when compared to the respective controls, using two-sided Student’s t-test with unequal variances (d,h) or ANOVA with Bonferroni’s correction (f). Horizontal line represents statistical comparison between respective groups. All the experiments were repeated independently at least three times and in triplicate.