Figure S1.
CDC42EP5 is required for melanoma migration and invasion. (A) Efficacy of RNAi silencing of Borg genes in 690.cl2 cells. Graph shows fold normalized expression of Cdc42ep1-5 (Ep1-5) against control cells (siCtr) cells when individual genes were targeted (siEp1-5). Bars indicate mean (n = 2 independent experiments). (B) Diagram showing the targeting sequence for endogenous Cdc42ep5 CRISPR/CAS9 knockout in murine wild-type 690.cl2 cells. Underneath is the sequence of the same locus in the 690.cl2KO cells. (C) Western blot showing Cdc42ep5, GFP, and Gapdh expression in parental 690.cl2 cells (wild-type) and in 690.cl2KO cells expressing GFP or GFP-Cdc42ep5. (D) Graph shows fold migration ability of wild-type 690.cl2 (WT) cells compared with 690.cl2KO cells expressing GFP or GFP-Cdc42ep5. Bars indicate mean ± SEM (n = 3 experiments; one-way paired ANOVA Tukey’s test; *, P < 0.05; **, P < 0.01). (E) Graph shows migration ability of parental 690.cl2 cells ectopically expressing GFP or GFP-CDC42EP5. Bars indicate mean ± SEM (n = 3 experiments; paired t test). (F) Graph shows fold normalized expression of Cdc42EP5 in human WM266.4 and MDA-MB-231-LM2 cells after transfection with control (siCtr) and CDC42EP5 (siEP5) siRNAs. Bars indicate mean (n = 2 independent experiments). (G and H) Graphs show fold migration ability (G) and 3D invasion (H) of WM266.4 and MDA-MB-231 cells after transfection with control (siCtr) and CDC42EP5 (siEP5) siRNAs. Bars indicate mean ± SEM (n = 3 experiments [G] and 4 [H]; paired t tests). (I and J) Graphs show expression of Borg genes (CDC42EP1-5, EP1-5) in the indicated cell lines (I) and databases (J). Expression levels in 690.cl2, WM266.4, and MDA-MB-231-LM2 were normalized against expression of Rplp1 and GAPDH, as indicated. Expression levels in melanoma and breast cancer were obtained from The Cancer Genome Atlas and expressed in RSEM (RNA-seq by expectation maximization). (K) Cell proliferation curves of 690.cl2KO cells ectopically expressing GFP (KO) or GFP-Cdc42EP5 (KO-EP5). Lines indicate mean ± SEM (n = 3 experiments). (L) Graph shows quantification of volumes at day 7 post-injection of subcutaneous tumors induced by injection of 690.cl2KO cells expressing GFP or GFP-Cdc42ep5. Bars indicate mean ± SEM (n = 6 mice; paired t test). KO, knockout.

CDC42EP5 is required for melanoma migration and invasion. (A) Efficacy of RNAi silencing of Borg genes in 690.cl2 cells. Graph shows fold normalized expression of Cdc42ep1-5 (Ep1-5) against control cells (siCtr) cells when individual genes were targeted (siEp1-5). Bars indicate mean (n = 2 independent experiments). (B) Diagram showing the targeting sequence for endogenous Cdc42ep5 CRISPR/CAS9 knockout in murine wild-type 690.cl2 cells. Underneath is the sequence of the same locus in the 690.cl2KO cells. (C) Western blot showing Cdc42ep5, GFP, and Gapdh expression in parental 690.cl2 cells (wild-type) and in 690.cl2KO cells expressing GFP or GFP-Cdc42ep5. (D) Graph shows fold migration ability of wild-type 690.cl2 (WT) cells compared with 690.cl2KO cells expressing GFP or GFP-Cdc42ep5. Bars indicate mean ± SEM (n = 3 experiments; one-way paired ANOVA Tukey’s test; *, P < 0.05; **, P < 0.01). (E) Graph shows migration ability of parental 690.cl2 cells ectopically expressing GFP or GFP-CDC42EP5. Bars indicate mean ± SEM (n = 3 experiments; paired t test). (F) Graph shows fold normalized expression of Cdc42EP5 in human WM266.4 and MDA-MB-231-LM2 cells after transfection with control (siCtr) and CDC42EP5 (siEP5) siRNAs. Bars indicate mean (n = 2 independent experiments). (G and H) Graphs show fold migration ability (G) and 3D invasion (H) of WM266.4 and MDA-MB-231 cells after transfection with control (siCtr) and CDC42EP5 (siEP5) siRNAs. Bars indicate mean ± SEM (n = 3 experiments [G] and 4 [H]; paired t tests). (I and J) Graphs show expression of Borg genes (CDC42EP1-5, EP1-5) in the indicated cell lines (I) and databases (J). Expression levels in 690.cl2, WM266.4, and MDA-MB-231-LM2 were normalized against expression of Rplp1 and GAPDH, as indicated. Expression levels in melanoma and breast cancer were obtained from The Cancer Genome Atlas and expressed in RSEM (RNA-seq by expectation maximization). (K) Cell proliferation curves of 690.cl2KO cells ectopically expressing GFP (KO) or GFP-Cdc42EP5 (KO-EP5). Lines indicate mean ± SEM (n = 3 experiments). (L) Graph shows quantification of volumes at day 7 post-injection of subcutaneous tumors induced by injection of 690.cl2KO cells expressing GFP or GFP-Cdc42ep5. Bars indicate mean ± SEM (n = 6 mice; paired t test). KO, knockout.

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