Rab13 and DENND2B promote epithelial cell invasion. (A) MCF10A cells were transduced with lentivirus driving expression of RFP, RFP-Rab13 T22N, or Q67L. Cells were fixed and imaged using DIC microscopy. Arrows indicate the presence of plasma membrane protrusions. Boxed region is magnified on the right. Bars: (low power view) 50 µm; (magnified view) 10 µm. (B) Protrusion length of transfected MCF10A cells was quantified (mean ± SEM from a minimum of three independent experiments measuring a minimum of 40 cells per condition per experiment; one-way ANOVA with Dunnett’s post-test; **, P < 0.01; ***, P < 0.001). (C) Transfected MCF10A cells were quantified as in B (mean ± SEM from three independent experiments measuring a minimum of 45 cells per condition per experiment). (D) Invasion of MCF10A spheroids transduced with RFP or RFP-Rab13 Q67L into collagen matrix. Bar, 50 µm. Arrows indicate invading cells. The images correspond to Video 1. (E and F) Quantification of the invasion from D (mean ± SD from three independent experiments measuring a minimum of 10 spheroids per condition per experiment; Student’s t test; ***, P < 0.001). (G) Flag-DENND2B expressing HEK-293T cells were transduced with control shRNAmiR or two different shRNAmiRs targeting DENND2B, and the indicated proteins were detected by blotting. (H) Quantification of Flag-DENND2B levels from G normalized to Hsc70 and expressed relative to the control shRNAmiR cells (mean ± SD from three independent experiments; statistical analysis used as in B). (I) MCF10A cells transduced with the indicated shRNAmiRs were plated on Matrigel-coated membranes for 14 h, and cells that invaded through the membranes were fixed and stained with crystal violet. Bar, 300 µm. (J) Quantification of MCF10A cell invasion from I normalized to control shRNAmiR cells (mean ± SEM from four independent experiments measuring 24 fields of view per condition per experiment; statistical analysis used as in B).