Autocrine induction of HRG drives survival of resistant cells. (A) Immunofluorescence microscopy of HRG expression in the indicated cell lines by using a primary rabbit anti-HRG antibody and visualized with an anti-rabbit IgG Alexa Fluor 555 (red) conjugated secondary antibody. Cell nuclei were visualized by DAPI staining (blue). These results are representative of other fields on the slide. (B) Western blot analysis of HRG type 1 (115 kDa) and type 2 (40 kDa) steady-state protein levels in lapatinib-resistant (rSKBR3; rAu565; rBT474) maintained in 1 μM lapatinib, and untreated parental cell counterparts (SKBR3; BT474; Au565); actin served as a control for equal loading of protein. (C) Western blot analysis of survivin and cleaved PARP product after HRG knockdown in rAu565 and rSKBR3 cells. Cells transfected with scrambled siRNA construct (NSC) served as controls. Actin served as a control for equal loading of protein. (D) Effects of siRNA-mediated knockdown of HRG on tumor cell growth in rAu565 and rSKBR3 cell lines. Results represent the mean ± standard error of triplicate samples, and are representative of three independent experiments. P < 0.009 (rSKBR3) and P < 0.0023 (rAu565). (E) Western blot analysis of ADAM17 protein level in parental BT474 and SKBR3 ± lapatinib treatment as indicated in the figure. Resistant cells (rBT474 and rSKBR3) were growing in the presence of 1 μM lapatinib. Hela cell extract was used as a positive control. Two bands from 75 kDa to 100 kDa can be detected by a specific ADAM17 antibody. These results are representative of three independent experiments.