Figure 5.

The SEC23B c.1781T>G (p.Val594Gly) Variant Confers Neoplastic Phenotypes upon ER Stress

(A) Representative images of invaded Nthy-SEC23B-WT (left) and Nthy-SEC23B-V594G (right) cells in a Transwell assay after overnight serum starvation. Invaded cells were counted after 24 hr in quadruplicate fields of view in triplicate membranes. Data represent mean values ± SEM. ^∗∗∗p < 0.001 (two-sided Student’s t test).

(B) Gelatin zymography was performed with conditioned media from wild-type and mutant cells and showed more MMP-2 and MMP-9 activity in supernatant originating from Nthy-SEC23B-V594G (right lanes) than in supernatant from Nthy-SEC23B-WT (left lanes) cells. Data represent triplicate lanes with band intensity normalized to protein mass. Data represent mean values ± SEM. ^∗∗∗p < 0.001, ^∗p < 0.05 (two-sided Student’s t test).

(C) Wild-type and mutant cells were treated with 1 μM thapsigargin (TG) for the induction of ER stress. Large colonies of cells expressing altered SEC23B formed, after which both Nthy-SEC23B-WT and Nthy-SEC23B-V594G cells were fixed and stained with Giemsa, and colonies were counted. Stained colonies represent cells 14 days after treatment, and a colony is standardly defined as a cluster of ≥50 cells. Data shown represent three independent experiments done in triplicate for each genotype. Data represent mean values ± SEM. ^∗∗∗p < 0.001 (two-sided Student’s t test).

(D) Treatment of wild-type and mutant cells with 0.1 μg/ml tunicamycin (TM), another ER-stress-inducing agent, resulted in more growth of mutant cells than of wild-type cells. Data shown represent two independent experiments done in triplicate for each genotype at each time point. Data represent pooled mean values ± SEM. ^∗∗∗p < 0.001, ^∗∗p < 0.01 (two-sided Student’s t test).