Figure 9.

Colchicine a microtubule inhibitor induces HSPB8 protein expression and inhibits mCRPC migration. (a) Androgen-responsive LNCaP and androgen-independent C4-2B cells were treated with Vehicle (DMSO) or 5 µM of Enzalutamide. Total RNA was isolated, followed by qRT-PCR with HOXB13, c-MYC and HOTPAM9 primers. (b,c) C4-2B cells were transfected with control, HOXB13 or AR siRNAs, followed by treatment with Vehicle or Enzalutamide (5 µM) and expression of HOTPAM9 genes was determined by qRT-PCR. (d,e) Directed ChIP-quantitative PCR for HOXB13 recruitment to sites upstream of the HSPB8 gene with HOXB13 antibody or IgG in LNCaP (d) and C4-2B (e) cells. Chromosomal locations (Peak1: Chr:119,614,158- 119,614,438) and (Peak2: Chr:119,616,400- 119,616,900). Data are represented as mean ± SEM. ****p < 0.0001; ***p < 0.001; **p = 0. 0025, *p < 0.05t. C4-2B and 22Rv1 were treated with Vehicle (DMSO), 10 µM of Enzalutamide (Enz) or 1 µM of Colchicine (Col) for 24 h. (f-i) Total RNA was isolated, followed by qRT-PCR with HOXB13, HSPB8 and Actin primers. (j) Whole cell lysates were immunoblotted with anti-HSPB8, anti-HOXB13 and anti-AR antibody. Actin is a normalization control. (k) Transwell cell migration assay was performed on GFP-positive PC3 and 22Rv1 cells using fluroblock inserts. Scale bar 300 µm. n = 2 independent replicates. (l) Quantitation of cell migration shown in (k). Data are represented as mean ± SEM in (f-i and l). ****p < 0.0001, ***p < 0.0005, **p < 0.01, *p < 0.05, ns: not significant.