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. 2017 Mar 20;36(29):4111–4123. doi: 10.1038/onc.2017.45

Figure 3.

Figure 3

BTP2 and drebrin knockdown inhibit PC-3 cell invasion in 3D assays in the presence of CXCL12 chemotactic gradients. (a, b) Micrographs of cresyl violet-stained PC-3 cells on the lower surface of the insert membrane from 3D invasion assays using control (Con) or BTP2 (5 μM) treated cultures. (c) Effects of BTP2 on PC-3 cell invasion in a 3D invasion assay. PC-3 cells were incubated in suspension with vehicle (Con) or with different concentrations of BTP2 for 1 h before re-addition of vehicle or BTP2 and then plating onto Matrigel in the Transwell insert. After 48 h, cells on the lower surface of the insert membrane were stained with cresyl violet and counted. Error bars are mean±s.e.m. from four independent experiments each of two replicates per condition. Significant differences: **P<0.01; ***P<0.001. (d) PC-3 cells transfected with GFP, wild-type drebrin-GFP or K270M, K271M drebrin-GFP show similar levels of protein expression. Immunoblots of transfected PC-3 cells were probed with antibodies against drebrin and GAPDH. The drebrin antibody recognizes endogenous drebrin (endo-dreb) and the expressed protein (dreb-GFP). (e) Effects of a BTP2-drebrin-binding mutant on BTP2 inhibition of PC-3 cell invasion in a 3D invasion assay. Cells were transfected with GFP, wild-type drebrin-GFP or K270M, K271M drebrin-GFP and incubated with BTP2 (10 μM) for 1 h followed by re-addition of BTP2 (10 μM) before seeding onto Matrigel in the Transwell insert. After 48 h, cells on the lower surface of the insert membrane were stained with cresyl violet and counted. Error bars are mean±s.e.m. from four independent experiments each of two replicates per condition. Significant differences: *P<0.05; ***P<0.001. (f) Immunoblots of PC-3 cells transfected with either control siRNA (Con siRNA) or drebrin siRNA 1 or drebrin siRNA 2 and probed with antibodies against drebrin and GAPDH, as a loading control. (g) Quantification of relative drebrin protein levels from immunoblots of PC-3 cells transfected with control siRNA (Con siRNA) or drebrin siRNA 1 or drebrin siRNA 2. Drebrin-specific siRNAs knockdown drebrin levels by >85% compared to control. Error bars are mean±s.e.m. from two independent experiments. Significant differences: **P<0.01, ***P<0.001. (h) Effects of siRNA knockdown of drebrin on PC-3 cell invasion in a 3D invasion assay. Cells were transfected with control siRNA (Con siRNA) or drebrin siRNA 1 or drebrin siRNA 2 before seeding onto Matrigel in the Transwell insert. After 48 h, cells on the lower surface of the insert membrane were stained with cresyl violet and counted. Error bars are mean±s.e.m. from four independent experiments each of two replicates per condition. Significant difference: ***P<0.001. (i) Effects of over-expression of drebrin on PC-3 cell invasion in a 3D invasion assay. Cells were transfected with drebrin-YFP or YFP alone before seeding onto Matrigel in the Transwell insert. After 48 h, cells on the lower surface of the insert membrane were stained with cresyl violet and counted. Error bars are mean±s.e.m. from three independent experiments each of two replicates per condition. Significant difference: **P<0.01.