Figure 3: Long-term but not short-term REST knockdown promotes EMT and stemness properties of PCa LNCaP cells.

(A) Immunoblotting of REST, NE marker synaptophysin (SYP), mesenchymal marker Twist1 and stemness marker CD44 in LNCaP-TR-shREST cells receiving 1 μg/ml Dox for 3 days (P1) compared to no Dox control. n = 3. (B) Total cell lysates (TCLs) were prepared from LNCaP-TR-shREST treated as described in (A) for 3 (P1), 6 (P2), 9 (P3), 30 (P10), and 45 (P15) days and then immunoblotted with antibodies as indicated. n = 3. (C) LNCaP-TR-shREST cells were treated as described in (A) for 45 (P15) days, seeded on the transwell membrane, and after three days the cells were stained with Hoechst. Representative images (top) and quantification (bottom) of migrated LNCaP cells. (D) LNCaP-TR-shREST cells were treated as described in (C) and then cultured in defined serum-free sphere medium for 14 days. Representative images (top) and quantification (bottom) of sphere formation in LNCaP cells. (E) Immunoblotting showed the successful knockdown of Twist1 in LNCaP-TR-shREST cells receiving 1 μg/ml Dox for 45 days (P15). Representative images (top) and quantification (bottom) of cell migration in LNCaP cells described in (E). (F) Immunoblotting showed the successful knockdown of CD44 in LNCaP-TR-shREST cells receiving 1 μg/ml Dox for 45 days (P15). Representative images (top) and quantification (bottom) of sphere formation in LNCaP cells described in (F). n = 2. Data represent means ± SD. **p < 0.01, ***p < 0.001 by Student’s-t test.