Cell proliferation assay

Cell proliferation described in Fig. 1 was measured using The CyQUANT® NF assay (Molecular Probes™ {"type":"entrez-nucleotide","attrs":{"text":"C35007","term_id":"2371148","term_text":"C35007"}}C35007) and following manufacturer recommendations. Fluorescence intensity was measured using a fluorescence microplate reader TECAN ULTRA fluorescence spectrophotometer with excitation at ~ 485 nm and emission detection at ~ 530 nm (Infinite® 200 PRO). Cell proliferation described in Fig. 3 was performed using the xCELLigence system, and cell index was measured following manufacturer recommendations. The results were analysed using RTCA software (Real-time cell analysis software Xcelligence).

Effect of metformin on cell proliferation and apoptosis of breast cancer cell lines representing different phenotypes of breast cancer. a, b Effect of different concentrations of metformin on cell proliferation of BT-474, MCF-7, MDA-MB-231, MDA-MB-468 and SkBr3, 24 h and 48 h post-treatment. N = 3 (6 replicates). c, d Effect of different concentrations of metformin on apoptosis of BT-474, MCF-7, MDA-MB-231, MDA-MB-468 and SkBr3, 24 h and 48 h post-treatment. N = 3 (2 replicates). The statistical values are provided as Additional file 4: Data S1. e Heatmap of microarray analysis showing upregulated (in red) and downregulated genes (in blue) in untreated vs. treated cells. Bonferroni corrected P value ≤0.05. N = 6 (6 replicates). f RT-PCR analysis of relative gene expression of selected IRF-9 and PYK2 (Upregulated), and c2orf42 and DHFR2 (downregulated). N = 3 (3 replicates). g, h Immunoblot images representing PYK2 expression in metformin treated and untreated SkBr3 and MDA-MB-453 cell lines. Densitometric ratio is measured by Arbitrary Units (AU). Student t-test, **P = 0.0030 and ***P = 0.0006. N = 3 (3 replicates)

Effect of PYK2 knockdown on cell proliferation and invasion of the HER2+/ER−/PR- breast cancer cell lines SkBr3 and MDA-MB-453. a Cell invasion assay using SkBr3 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, **P = 0.0025 (Treated empty vector vs. treated PYK2 shRNA1), ***P = 0.0032 (Treated empty vector vs. treated PYK2 shRNA2). N = 3 (2 replicates). b Cell invasion assay using MDA-MB-453 metformin treated and untreated cells, and the corresponding data quantifying number of invading cells (48 h with or without treatment). Anova ****P = < 0.0001, **P = 0.0032 (Empty vector vs. treated empty vector), **P = 0.0030 (Empty vector vs. PYK2 shRNA1), **P = 0.0017 (Empty vector vs. PYK2 shRNA1), ***P = 0.0005 (Treated empty vector vs. treated PYK2 shRNA2), ***P = 0.0005 (Treated empty vector vs. treated PYK2 shRNA1). N = 3 (2 replicates). c Cell proliferation assay using SkBr3 metformin treated and untreated cells, and the corresponding data quantified as cell index (48 h with or without treatment). Anova, ***P = 0.0001 (Empty vector vs. PYK2 shRNA1), ***P = 0.0004 (Empty vector vs. PYK2 shRNA2), ***P = 0.0002 (Treated empty vector vs. treated PYK2 shRNA1), **P = 0.0003 (Treated empty vector vs. treated PYK2 shRNA2). N = 3 (2 replicates). d Cell proliferation assay using MDA-MB-453 metformin treated and untreated cells, and the corresponding data quantified as cell index (48 h with or without treatment). Anova, **P = 0.0034 (Empty vector vs. treated empty vector), **P = 0.0010 (Empty vector vs. PYK2 shRNA1), ***P = 0.0003 (Empty vector vs. PYK2 shRNA2), **P = 0.0060 (Treated empty vector vs. treated PYK2 shRNA1), **P = 0.0022 (Treated empty vector vs. treated PYK2 shRNA1), ***P = 0.0007 (Untreated PYK2 shRNA1vs. treated PYK2 shRNA1), ***P = 0.0004 (Untreated PYK2 shRNA2vs. treated PYK2 shRNA2). N = 3 (2 replicates). e Schematic representation of the dual role of PYK2 in proliferation, migration and invasion of HER2+/ER−/PR- breast cancer in response to metformin