Fig. 5: LIMK2-mediated downregulation of SPOP promotes oncogenic phenotypes and enzalutamide-resistance in CRPC cells.

a WT and 3A-SPOP inhibits cell proliferation in C4-2 cells. C4-2, LIMK2-C4-2, SPOP-C4-2 and 3A-SPOP-C4-2 cells were cultured in 96-well plates for 24, 48 and 72 h followed by MTT assay. b LIMK2 depletion substantially reduces cell growth in SPOP-C4-2 and 3A-SPOP-C4-2 cells. * and # indicate statistically significant difference compared to respective controls; P < 0.05. c LIMK2 overexpression augments cell proliferation in SPOP-C4-2 and 3A-SPOP-C4-2 cells. * and # indicate statistically significant difference compared to respective controls; P < 0.05. d Expression of WT and 3A-SPOP impaired cell proliferation in 22Rv1 cells. 22Rv1, LIMK2-22Rv1, SPOP-22Rv1 and 3A-SPOP-22Rv1 cells were cultured in 96-well plates for 24, 48 and 72 h and subjected to MTT assay. e LIMK2 knockdown decreases cell growth in SPOP-22Rv1 and 3A-SPOP-22Rv1 cells. * and # indicate statistically significant difference compared to respective controls; P < 0.05. f LIMK2 overexpression enhances cell proliferation in SPOP-22Rv1 and 3A-SPOP-22Rv1 cells. * and # indicate statistically significant difference compared to respective controls; P < 0.05. g, h SPOP prevents colony formation in a soft agar assay in C4-2 and 22Rv1 cells. *P < 0.05 compared to vector-expressing control. i Both WT-SPOP and 3A-SPOP inhibit cell migration in C4-2 cells. Representative images of chemotaxis assay. Magnification, ×200. j Chemotaxis assay was performed in C4-2, SPOP-C4-2 and 3A-SPOP-C4-2 cells using Boyden chambers. Histogram shows mean ± SEM of three experiments conducted independently. *P < 0.05 compared to vector control. k, l LIMK2 overexpression increases cell motility in both SPOP-C4-2 and 3A-SPOP-C4-2 cells, although to a higher extent in the former as compared to the latter. m, n LIMK2 depletion inhibits cell motility in SPOP-C4-2 and 3A-SPOP-C4-2 cells with more significant decrease in the latter. o Representative images and p quantitative data of chemotaxis assays measuring migration of 22Rv1, WT-SPOP-22Rv1 and 3A-SPOP-22Rv1 cells. q, r LIMK2 overexpression increases cell motility in both SPOP-22Rv1 and 3A-SPOP-22Rv1 cells. s, t LIMK2 depletion inhibits cell motility in both SPOP-22Rv1 and 3A-SPOP-22Rv1 cells. u 3A-SPOP-expressing cells are more sensitive to enzalutamide (1 μM, treated for 48 h), compared to WT-SPOP-C4-2 cells.