Figure 1.

TMEM176B knockdown decreased the proliferation, wound healing, and the migration of MDA-MB-231 cells in vitro. (A) TMEM176B mRNA expression was assessed by qRT-PCR in control (Scrb) and TMEM176B-silenced (176Bsh1, 176Bsh2) MDA-MB-231 cells. (n = 3 per group, with two independent experiments). (B,C) Western blot analysis examining TMEM176B expression in control and TMEM176B-silenced cells (n = 3 per group, with three independent experiments). (D) Results of proliferation assay for control and TMEM176B-silenced cells grown for 72 h (n = 6 for Scrb; n = 3 for 176Bsh1 and 176Bsh2 per group, with three independent experiments). (E) Representative images of wound healing assay. Images were taken at time points T0 (0 h), T24 (24 h), and T48 (48 h) after performing the scratch at the same coordinates for each image. Scale bars: 200 μm. (F) Quantification of wound healing assay. “Scratch area %” indicates the percent of area remaining compared with time 0. (n = 3 per group, with two independent experiments). (G) Representative images of transwell migration assay after 20 h, stained with Giemsa solution. Scale bars, 50 μm. (H) Quantification of transwell migration assay. Stained area is expressed as a percent of control (Scrb) cells. (n = 6 for Scrb; n = 2 for 176Bsh1; n = 3 for 176Bsh2 per group, with two independent experiments). Data are presented as means ± SEM. Differences between groups were evaluated by the one-way (A,C,D,F,H) ANOVA test with the Bonferroni post hoc test. (# p < 0.05, ## p < 0.01, ### p < 0.001 between Scrb vs. 176Bsh1; * p < 0.05, ** p < 0.01, *** between Scrb vs. 176Bsh2) dataset (Supplementary Figure S1B) [28], and in histological grade 3 breast cancer compared with lower-grade breast cancers (Supplementary Figure S1C) [29]. High TMEM176B mRNA expression was also associated with decreased relapse-free survival in a majority of breast cancer studies examined (Supplementary Figure S1D–F) [30].