Figure 5.

LASTR fosters cell fitness under stress conditions. (A) 2D colony formation of the indicated cell lines untreated and treated with doxycycline (1 μg/ml). Representative images of 2D colonies and quantification of the colony number. P-values were determined by two-tailed t-tests, n = 3. (B) Anchorage-independent growth of the indicated cell lines untreated and treated with doxycycline (1 μg/ml). P-values were determined by two-tailed t-tests, n = 3. (C) Cell growth of hypoxic MDA-MB-231/tet-shLASTR-2 cells untreated and treated with doxycycline (1 μg/ml) as measured by the CellTiter-Glo assay. Data are presented as mean ± s.e.m.; P-value was determined by two-way ANOVA, n = 3. (D) Tumor growth of MDA-MB-231/tet-shLASTR-2 xenografts. Cells were implanted into the breast pads of immunodeficient mice. When the average tumor volume reached 100 mm³, expression of the shRNA against LASTR was induced by adding doxycycline to the drinking water (2 mg/ml). Data are presented as mean ± s.e.m.; P-value was determined by two-way ANOVA, n = 9. (E) Tumor weight of MDA-MB-231/tet-shLASTR-2 xenografts, untreated or treated with doxycycline (2 mg/ml) at the end point. Data are presented as mean ± s.e.m.; P-value was determined by a two-tailed t-test. (F) RT-qPCR analysis of LASTR expression in MDA-MB-231/tet-shLASTR-2 xenografts. Data are presented as mean ± s.e.m.; P-value was determined by a two-tailed t-test. (G) Ki67 immunostaining of MDA-MB-231/tet-shLASTR-2 xenografts. Scale bar, 100 μm. Data are presented as mean ± s.e.m.; P-value was determined by a two-tailed t-test. (H) Colony formation assay of MCF10A-HER2 and MDA-MB-468 cells after exposure to increasing doses of ionizing radiation. The cells were pre-treated with the indicated GapmeRs 24 h prior irradiation. Survival is presented as percentage ± s.e.m. of colonies formed relative to untreated cells; P-values were determined by two-tailed t-tests, n = 3.