Figure 2: Effect of MICU1 on cancer phenotypes.

(a) MICU1 silencing affects clonal growth of OvCa cells. CP20 and OV90 cells were transfected with MICU1 siRNA or control siRNA and 48 h after transfection cells were re-plated in 0.3% agar. Similarly, stable cell lines shCTL-OV90 and shMICU1- OV90 were also plated in 0.3% agar. After 10 (CP20) and 14 (OV90) days, colonies were stained and quantified. Values are represented as mean±s.d. (b) Silencing of MICU1 inhibits cell migration in OvCa cells (CP20 and OV90). Migration of siCTL and siMICU1 or shCTL and shMICU1 cells towards serum gradient was examined and number of cells per field was counted. Values are represented as mean±s.d. (c) Silencing of MICU1 inhibits cell invasion of fibronectin matrix by OvCa cells (CP20 and OV90). Invasion of siCTL and siMICU1 or shCTL and shMICU1 cells through fibronectin-coated filters was examined using Boyden chamber and number of cells per field was counted and values are represented as mean±s.d. (d) Cell migration assay performed in Boyden chamber followed by Calcein-AM staining and fluorescence measurement. (e) Cell invasion assay performed in Boyden chamber coated with fibronectin followed by Calcein-AM staining and fluorescence measurement. (f) Representative images of siCTL or siMICU1 transfected CP20 cells plated on Oregon Green 488 Gelatin coated coverslips for 48 h, fixed and stained with Alexa Fluor 555 Phalloidin. (g) Quantification of CP20 or OV90 cells degrading the gelatin matrix and values are mean±s.d. (h,i) CP20 (h) and OV90 (i) cells were transfected with siCTL or siMICU1 for 24 h and cells were re-plated in 96-well plates. Cells were treated with cisplatin at the indicated concentrations and proliferation was determined using CyQUANT NF cell proliferation assay after 48 h. Mean fold change±s.d. in cell proliferation was evaluated by comparing with respective cells receiving similar treatment. (j) shCTL-OV90 or shMICU1-OV90 cells were plated in 96-well plates. Cells were treated with cisplatin at the indicated concentrations and proliferation was determined as in i. *P<0.05 was statistically significant when compared to siCTL/shCTL (a–g) or similarly treated siCTL/shCTL (h–j). Two-sided Student’s t-test with unequal variances was used for statistical analysis. All the experiments were repeated independently at least three times and in triplicate. Scale bar (a) 2 mm and (b,c and f) 20 μm.