Fig. 5.

Insc^ASYM induces ACDs in p53-KO mammary stem cells. a Insc transcript levels evaluated by RT–qPCR on mammospheres from wild-type FVB mice, or p53-KO mice infected with empty lentivirus (EV) or human full-length Insc (INSC) normalized to the Rpp0 control. Bars represent mean ± s.d. of three replicates. Statistics for 1 out of 2 independent experiments. b, c Images and quantification of the division mode of mammary epithelial cells from p53-KO mice evaluated by pair-cell assay. Cell infected with empty lentivirus (EV) or virus expressing full-length Insc (INSC) were stained with CD49f and DAPI. Scale bar, 10 μm. Quantification from 2 independent experiments with n > 10 cells for all conditions in each experiment. p = 0.005 according to the paired Student’s test. d Scheme of the SFE assay used to evaluate self-renewal of p53-KO MaSCs. Stem cells are in blue, progenitors in pink. e Number of mammospheres from p53-KO mice infected with an empty lentivirus (EV) or a virus expressing Insc (INSC) counted over three serial passages and shown by box-plots with mean of 10 wells count per each passage (green line). f–h SFE assays with p53-KO mammospheres infected with empty virus (EV) or viruses expressing Insc (FL), Insc^ASYM (aa 1–317, ASYM), or Insc^PEPT (aa 1–58, PEPT). For each passage, the number of spheres (f), the sphere area (g) and the GFP intensity (h) have been evaluated for 10 wells, and plotted as in e (each pair Student’s t-test). Statistics for 1 out of 3 independent experiments. i SFE assays performed with p53-KO mammospheres infected with empty lentivirus (EV) or viruses expressing Insc (FL), Insc^ASYM (ASYM), or Insc^ASYM-ΔαB (i.e. Insc^ASYM lacking aa 62–191). For each passage, the number of spheres has been counted for four wells, and plotted as above (each pair Student’s t-test). Statistics for one out of two independent experiments. j SFE assays performed with p53-KO mammospheres infected with empty lentivirus (EV) or viruses expressing Insc (FL), human LGN-GoLoco (aa 358–677) or both. For each passage, the number of spheres has been evaluated in three wells, and plotted as above (each pair Student’s t-test). In the figure, *P < 0.01, ***P < 0.001 by Wilcoxon’s test