Supplementary Figure 2: Identification of ROR1-associated proteins, characterization of ROR1 phosphorylation and examination of the role of p-HER3 (Tyr1307) in breast and lung cancer.
From: A ROR1–HER3–lncRNA signalling axis modulates the Hippo–YAP pathway to regulate bone metastasis
(a) A list of top ROR1-associated proteins identified by protein pull-down and MS in MDA-MB-231 cells. (b) IB detection of p-HER3 (Tyr1307) in MDA-MB-231 cells treated with or without NRG1 using antibodies pre-incubated with indicated blocking peptides. Antibodies generated from two independent rabbits were tested. (c and d) Immunoprecipitation (IP) and IB detection of indicated proteins in MDA-MB-231 cells transfected with indicated plasmids (c) or treated with indicated small compound inhibitors (d) followed by NRG1 treatment. (e) IP and IB detection of indicated proteins in 32D cells transfected with indicated expression vectors followed by NRG1 stimulation. (f) IB detection of indicated proteins in parental/HER3 KO MDA-MB-231 single cell clones. (g and h) Cell proliferation assay of individual (g) and pooled (h) clones of HER3 KO MDA-MB-231 cells. (i) Cell invasion assay of individual clones of HER3 KO MDA-MB-231 cells with overexpression of indicated plasmid (left, Scale bars, 200 μm; right, quantification). (j and k) Cell migration (j) and invasion (k) assay of pooled clones of HER3 KO MDA-MB-231 cells (left, Scale bars, 200 μm; right, quantification). (l) Immunohistochemical (IHC) staining of p-HER3 (Tyr1307) in normal lung tissues, lung adenocarcinomas, non-metastatic (TnN0M0) and metastatic (TnN > 0M ≥ 0) lung adenocarcinomas (left, Scale bars, 40 μm; right, n = 75, 75, 43 and 32 lung tissues/adenocarcinomas respectively, median, one-way ANOVA) (m) Kaplan-Meier survival analysis of p-HER3 (Tyr1307) low and high lung adenocarcinoma patients (n = 20 and 55 patients respectively, log rank test). For g–k, mean ± s.e.m. were derived from n = 3 independent experiments (∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9 Statistics source data for i-l are in Supplementary Table 8.
