Fig. 5
RNA sequencing analysis of the gene expression changes induced by ADAM12 knockdown in SUM159PT cells. a Diagram summarizing the design and the outcome of the RNA-Seq experiment. b Heatmaps of differentially expressed genes in response to ADAM12 knockdown. Compared are SUM159PT_shADAM12 cells without versus with doxycycline treatment (left) and SUM159PT_ shControl versus SUM159PT_ shADAM12 cells treated with doxycycline (right). c ADAM12 gene expression signature score versus ADAM12 mRNA expression in 421 breast invasive carcinomas from the TCGA database (Cell 2015 dataset). ADAM12 signature scores were generated based on the expression of 45 ADAM12-regulated genes shown in panel b. d CSC signature score versus ADAM12 score in 421 breast invasive carcinomas from the TCGA database (Cell 2015 dataset). CSC signature scores were generated based on ref. [22], as described in Methods. e Results of the IPA Upstream Regulator analysis. Potential upstream regulators with an overlap P‐value < 0.05 and an activation |z score| > 2 are shown. f Immunoblot analysis of SUM159PT cells after incubation for 24 h in complete media in the presence or absence of 1 μM erlotinib, an EGFR inhibitor, followed by 30 min treatment with 20 ng/ml EGF, as indicated. g, h EGFR inhibition mimics the effect of ADAM12 knockdown on the CD44hi/CD24-/lo cell population. SUM159PT cells grown in complete medium were treated for 3 days with DMSO or 1 μM erlotinib. g CSC-containing CD44hi/CD24-/lo population (green) was identified by flow cytometry. h Percentage of CD44hi/CD24-/lo populations was determined in three independent experiments