Fig. 7

HOXC8 knockdown increase CSC proliferation and transformed phenotype. a Knockdown of HOXC8 was induced by lentiviral transduction of MCF10A cells with pLKO-Tet-HOXC8 vector and gene expression measured by TaqMan® qRT-PCR. Results are presented as relative fold expression relative to RPLP0 and pLKO-Tet-Scrambled vector used as calibrator (n = 3). Results were analysed by Unpaired Student’s t-test (n = 3). ***P < 0.001. Bottom panel shows western blotting of nuclear lysates from cells transduced with pLKO-Tet-HOXC8 vector or pLKO-Tet-Scrambled vector was conducted to detect the expression of HOXC8 (34 kDa) and LAMIN A/C (41–50 kDa) as loading control. b Percentage of CD44⁺/CD24^low/-, CD44⁺/CD24⁺, CD44⁻/CD24⁺ cells after HOXC8 knockdown as measured by FACS analysis. Results were analysed by comparing cell populations levels in HOXC8 shRNA transduced cells compared to Scrambled shRNA (n = 3). Statistical analysis was performed by Unpaired Student’s t-test. *P < 0.05, **P < 0.01. Representative FACS profiles of cells double stained with CD44-FITC and CD244-PE to analyse the CSC and non-CSC populations are also shown. c Mammospheres morphology and number obtained by self-renewal of cell after HOXC8 knockdown compared to scrambled control vector after 3 generations. Mammospheres numbers were compared by Unpaired Student’s t-test (n = 3). **P < 0.01. Only spheres ≥ 100 μM were counted, images were taken at 10X magnification. d Colonies of HOXC8 shRNA and Scrambled shRNA cells grown in 3D matrigel. Top panels represents 10X magnification, bottom panel represents images at 40X magnification. e Colonies of cells with HOXC8 knockdown compared to scrambled control grown in soft agar for 2 weeks. Top images show the whole well stained with crystal violet, bottom images show a representative field of view at 10X magnification. f The number of soft agar colonies were calculated by counting 10 fields of view. Results were analysed by Unpaired Student’s t-test for each cell line (n = 3). ***P < 0.001