(a–f) BT474, AU565, HCC1419, ZR75, MCF7, and HCC1569 breast cancer cell lines were assayed for proliferation in the response to lapatinib ±10 nM NRG as described in Figure 1a. BT474 and AU565 represent lapatinib-sensitive lines. HCC1419 and ZR75 represent partially-sensitive lines. MCF7 and HCC1569 represent lapatinib-insensitive lines. (g) Western blot analysis of endogenous EGFR family protein levels in SKBR3, BT474, AU565, HCC1419, ZR75, MCF7 and HCC1569 cell lines. (h–i) SKBR3 and BT474 cells were treated for 72 hr with a titration of lapatinib ±10 nM EGF, after which proliferation was measured using CellTiter-Glo. (j) CellTiter-Glo proliferation assay of SKBR3 cells with transient siRNA knockdown of HER3 using single oligonucleotides. Western blot denotes knockdown efficiency of HER3 si11 and HER3 si13 oligonucleotides. (k) CellTiter-Glo endpoint analysis of proliferation of SKBR3 spheroid cultures after 8 days of lapatinib ±NRG. (l) Western blot analysis of SKBR3 spheroid cultures in conditions matched to Figure 1c, Figure 1—figure supplement 1k. One representative example of three independent experiments is shown. All proliferation data represented as mean ±SEM of three independent experiments each performed in triplicate, except for (j), which represents six independent experiments each performed in triplicate. Corresponding data and statistics available as Figure 1—figure supplement 1—source data 1.