Fig. 2
Generation and RRAS2-dependency of a murine TNBC cell line derived from a spontaneous tumor growing in Rosa26-RRAS2fl/flxWap-Cre mice. a, Immunohistochemical characterization of the CBM-MBC21 murine breast cancer cell line. Hematoxilin and eosin counterstains of immunoperoxidase stainings carried out with the indicated rabbit (estrogen receptor; ErbB2) and mouse (progesterone receptor; R-RAS2) antibodies. Control stainings with unspecific antibodies of mouse and rabbit origin were carried out in parallel. b, Immunoperoxidase staining of tissue paraffin sections from human breast cancer samples positive for ERα (luminal A), PR (luminal A), or ERBB2 (HER2-enriched) were stained with the same antibodies and used as positive controls of Fig. 2a stainings. ERα staining is clearly concentrated in the nuclei of the positive cells, ERBB2 staining produced a plasma membrane staining, and PR staining resulted in a mixed, mostly cytoplasmic pattern. c, A RRAS2 knockdown cell line derived from CBM-MBC21 cells was derived using a shRNA construct. The bar plot shows the mean ± s.e.m. of RRAS2 mRNA expression in the original and the knockdown CBM-MBC21 cell lines determined by RT-qPCR. Significance was assessed using a non-parametric Mann-Whitney test. d, Western blot analysis of R-RAS2 protein expression in CBM-MBC21 and knockdown cells. Immunoblot (IB) loading and specificity controls were carried out by incubation with an anti-actin and an anti-pan-RAS (classical) antibodies. The bar plots to the right shows the quantification of the R-RAS2 band referred to the actin band, as well as a quantification of total classical RAS protein detected with the pan-RAS antibody and referred to the actin band. Data were generated by densitometry of western blots run in triplicate. Data is represented as the mean ± s.e.m. Significance was assessed using a non-parametric Mann-Whitney test. e, In vitro proliferation of RRAS2 knockdown CBM-MBC21 and control (CBM-MBC21 transduced with a scrambled shRNA construct) cells was assessed by counting the number of live cells at the indicated time points. The line plot shows the the mean ± s.e.m. of triplicates. Significance was assessed using a two-way ANOVA test. f, Analysis of the cell cycle in control and RRAS2 knockdown CBM-MBC21 cells after a 10-hour incubation with BrdU and double staining with anti-BrdU-APC antibody and 7-AAD. The positions of cell populations at different stages of the cell cycle are indicated with colored rectangles. The presence of a cell population with high incorporation of BrdU but little amplification of the DNA (low 7-AAD) is indicated with a yellow square as a population in early S-phase. The bar plot to the right shows the quantification of cells at different stages of the cell cycle as the mean ± S.D. of triplicates. Significance was assessed using a two-way ANOVA test. ****, p < 0.0001. g, In vivo proliferation in orthotopic (breast) location was assessed after inoculating 5 × 106 cells into the left inguinal mammary gland of C57BL/6 female mice and by measuring the volume of the tumor protrusion at skin level at the indicated time points. Datapoints show the mean ± s.e.m. for n = 5 implanted mice per cell line. Significance was assessed using a two-way ANOVA test. h, Tumor weight was measured at day of termination (day 33). A box and whiskers plot showing the median, the mean (+) and all datapoints is displayed. Significance was assessed using an unpaired t-test with Welch’s correction