Fig. 2: Loss of ZFHX3 increases cell proliferation, colony formation, and sphere formation in androgen-responsive prostate cancer cells.

a, b Isolation of clones with successful CRISPR-Cas9-mediated truncating mutations of ZFHX3 in C4-2B cells, as indicated by sequencing analysis of targeted locus (a) and expression detection of ZFHX3 by Western blotting (b). Wild-type sequence in parental cells is shown at the top of a. Wt, vector control cells; KO3, KO4, KO7, KO8, KO9, and KO10 are different clones of C4-2B cells with ZFHX3 mutations. c Loss of ZFHX3 increased cell proliferation in two-dimensional (2D) culture in C4-2B cells. Optical densities represent cell numbers. n = 4. d–g Loss of ZFHX3 increased sphere formation in Matrigel (d—upper, e, f) and colony formation in soft agar (d—lower, g) in C4-2B cells. Cells were grown for 10–14 days in Matrigel or soft agar. Shown are bright field images of spheres (d, upper) and colonies (d, lower), range of sphere sizes (e), the average number of spheres with a diameter >75 µm per well (f), and the average number of colonies with a diameter >100 μm (g). The ImageJ program was used to determine sphere/colony sizes. h–j Knockdown of ZFHX3 in LNCaP cells also increased sphere formation. The knockdown effect was validated by Western blotting (h), and bright field images of colonies (i) and the number of colonies with a diameter >100 μm (j) is shown. Scale bars in d, 100 μm. The n of e–g, j is 3. *P < 0.05; **P < 0.01; ***P < 0.001. ZFHX3, zinc-finger homeobox 3