Fig. 3

BBI608 and YM155 inhibited STAT3 phosphorylation and G9a and ERBB3 expression in EGFR-positive lung cancers. a To validate the inhibitory capacity of BBI608 and YM155 against the formation of cancer stemness, HCC827-derived stem-like cancer tumorspheres (HCC827CSC) were treated separately with 1 μg/mL of afatinib, BBI608, and YM155 over a 7-day incubation. We demonstrated that BBI608 and YM155 could reduce the formation of HCC827-derived tumorspheres compared with afatinib as observed under an inverted microscope. b The tumorsphere sizes of HCC827CSC were measured and compared. Results showed that BBI608 and YM155 significantly reduced HCC827CSC formation, whereas afatinib exhibited no effect at a concentration of 1 μg/mL. c There were lower levels of HER3 in A549shSTAT3 compared with A549shLuc, indicating that STAT3 facilitated HER3 expression. d In addition, BBI608 and YM155 caused a similar reduction in the viability of A549 cells in a dose-dependent manner. e Gene cluster profiling from the RNAseq analysis revealed a similar pattern between BBI608- and YM155-treated A549 cells (Additional file 2: Table S2 and Additional file 3: Table S3). f There were 309 common differentially expressed genes between BBI608- and YM155-treated A549 cells (Additional file 6: Table S6), g which were consequently analyzed by NetworkAnalyst to identify key potential genes mediated by BBI608 and YM155. Results revealed and validated reductions in ERBB2 and ERBB3 expression, whereas BIRC5 is an inhibitory target of YM155. h In addition, BBI608 and YM155 substantially reduced endogenous and EGF-mediated STAT3 phosphorylation, resulting in G9a and HER3 (ERBB3) reductions. *p < 0.05, **p < 0.01. Scale bar, 100 μm