Fig. 6

PYK2 and metformin promote the enrichment of HER2+/ER−/PR- CD44^High/CD24^Low/− and ALDH^High breast cancer stem-like cells. a Images of representative spheres from 10 days sphere formation assay using SkBr3 control and PYK2 knockdowns cells metformin treated and untreated. Scale bar = 100 μm. b Graph representing sphere size quantification (μm) of spheres from SkBr3 control and PYK2 knockdowns cells metformin treated and untreated. Student t-test, ****P = < 0.0001, ***P = 0.0009 (Empty vector vs. PYK2 shRNA1), ***P = 0.0003 (untreated PYK2 shRNA1 vs. treated PYK2 shRNA1), ***P = 0.0002 (Treated PYK2 shRNA2vs. treated PYK2 shRNA2). N = 3 (3 replicates). c Graph representing average number of spheres from SkBr3 control and PYK2 knockdowns cells metformin treated and untreated. Student t-test, **P = 0.0014 (untreated empty vector vs. treated empty vector), *P = 0.0100 (untreated PYK2 shRNA1 vs. treated PYK2 shRNA1), *P = 0.0240 (untreated PYK2 shRNA2 vs. treated PYK2 shRNA2), **P = 0.0013 (Empty vector vs. PYK2 shRNA1), ***P = 0.0007 (Empty vector vs. PYK2 shRNA2). N = 3 (3 replicates). d Graph representing percentage of CD44^High/CD24^Low cells quantified by flow cytometry from SkBr3 control and PYK2 knockdowns cells metformin treated and untreated. Anova, *P = 0.0273 (Empty vector vs. PYK2 shRNA1), *P = 0.0182 (Empty vector vs. PYK2 shRNA2), **P = 0.0045 (Treated empty vector vs. treated PYK2 shRNA1), *P = 0.0211 (Treated empty vector vs. treated PYK2 shRNA2), **P = 0.0059 (untreated empty vector vs. treated empty vector), *P = 0.0208 (untreated PYK2 shRNA1vs. treated PYK2 shRNA1), **P = 0.0044 (untreated PYK2 shRNA2vs. treated PYK2 shRNA2). N = 3 (3 replicates). e Graph representing percentage of ALDH^High cells quantified by flow cytometry from SkBr3 control and PYK2 knockdowns cells metformin treated and untreated. Anova, *P = 0.0448 (Empty vector vs. PYK2 shRNA1), ***P = 0.0002 (Treated empty vector vs. treated PYK2 shRNA1), **P = 0.0033 (Treated empty vector vs. treated PYK2 shRNA1). N = 3 (3 replicates). f Cell invasion assay using SkBr3 control and PYK2 knockdowns cells metformin treated and untreated. and the corresponding data quantifying of number of invading cells (48 h with or without treatment). Anova ****P = < 0.0001. N = 3 (2 replicates). g Multivariate cox regression analysis of PYK2 and CD44 expression and their association with HER2+/ER−/PR- breast cancer patients’ survival over a 5-year term