Fig. 2

RARRES2-OE inhibits metastasis of breast cancer to the brain. a–c MDA-MB-231 cells were stably transduced with lentivirus encoding short hairpin RNAs targeting RARRES2 (shRARRES2-1 and -2) or negative control RNA (shControl). Then, the cells were assayed for RARRES2 expression (a), proliferation (b) and invasion (c). Significance was assessed in panel (b) using two-way ANOVA with Geisser-Greenhouse correction (^**P = 0.0023) or in panel c using one-way ANOVA followed by Dunnett’s multiple-comparisons test. d-f The experiments in panels a–c were repeated using cultures of 4T1 cells. In panel e, ^*P = 0.0177. g–i Immunoblot of lysate confirming overexpression of RARRES2 (g), cell proliferation assay (h) and invasion assay (i) from MDA-MB-231 cells infected with control or encoding RARRES2 lentivirus (RARRES2-OE). Two-way ANOVA with Geisser-Greenhouse correction (h), ^***P = 0.0005. Two-tailed unpaired sample t-test (i), ^**P = 0.0025. j, k Same as g, h for 4T1 cells, P = 0.1053 (k). l Bioluminescence imaging (BLI) at 12 d after intracranial injection of RARRES2-OE and Control 4T1 cells. The dots on the right represent the photon flux in brain for each mouse at day 12 (n = 6 for each group). ^*P = 0.0303 (two-sided Mann–Whitney test). m BLI at 15 d after intracardiac injection of RARRES2-OE and Control 4T1 cells. The dots on the right represent the photon flux in brain for each mouse at day 15 (n = 5 for Control, n = 6 for RARRES2-OE). Two-tailed unpaired-samples t test. n BLI of brain and liver from mice receiving intracardiac injection in the experiments. The dots on the right represent the photon flux in brain or liver for each mouse at day 15 (n = 5 for Control, n = 6 for RARRES2-OE). ^*P = 0.0401 (two-tailed unpaired-samples t test). RARRES2 retinoic acid receptor responder 2, RARRES2-OE RARRES2 overexpression, ns non-significant