Role for TPD52-like proteins in cell migration and invasion. (A–G) RPE1 cell migration on a flat fibronectin substrate. (A, E, and F) Superplots showing migration speed of control vs TPD54- (A), TPD52- (E), or TPD53-depleted (G) cells. Dots represent individual cells, color-coded for experiments. Markers indicate mean speed for individual experiments. P value, Student’s t test; nexp = 4 (TPD54), 3 (TPD52 and TPD53); whereas ncell = 469 (TPD54), 564,573 (TPD52), and 588,597 (TPD53). (B) Boxplot to show the migration speed of cells that were treated with siCtrl or each one of three TPD54-targeting siRNAs. Boxes show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. P values from Dunnett’s post-hoc test using siCtrl as control; ncell = 88–99, nexp = 1. (C) Violin plot showing the average speed of siCtrl- or siTPD54-treated cells expressing GFP, GFP-TPD54 WT, or GFP-TPD54 1–155 as indicated. Dots, individual cells; markers, mean speed. P values from Dunnett’s post hoc test using siCtrl + GFP as control. ncell = 65–103, nexp = 1. (D and G) Superplots showing migration speed of cells expressing GFP, GFP + TPD54, or GFP + R159E (D) or GFP, GFP + TPD52, or GFP + TPD53 (E). P values in D from Dunnett’s post hoc test using GFP as control. D, ncell = 247–259, nexp = 3. G, ncell = 129–131, nexp = 2. (H–J) Invasion of A2780 cells stably expressing Rab25 in a 3D context. (H) Boxplot of migration speed of cells in CDM that were treated with siRNAs as indicated. P values, Dunnett’s post hoc test using siCtrl as control. nexp = 3. (I) Representative confocal images of A2780 cells stably expressing Rab25 treated with the indicated siRNAs migrating through fibronectin-supplemented collagen type-I matrix for 72 h. Scale bar, 250 µm. (J) Quantification of A2780 cell invasion in confocal sections ≥45 µm, normalized to siCtrl. Dots, individual wells. Box plots show IQR, bar represents the median, and whiskers show 9th and 91st percentiles. P value, Kruskal–Wallis test. nexp = 3.
