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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: Nat Cell Biol. 2022 Aug 1;24(8):1291–1305. doi: 10.1038/s41556-022-00962-4

Extended Data Fig. 4. EGFR ligand overexpression prolongs survival, reduces invasiveness and increases proliferation in orthotopic glioblastoma mouse model.

Extended Data Fig. 4

a, Immunoblot of the indicated proteins in GBM9 stably transfected with empty (GBM9V) or TGFα expression vector (GBM9TGFα). b, Immunoblot of EGFR expression in cells stably transfected with empty, TGFα, or BIN3 expression vectors. c, EGFR copy numbers in cells described in b. d, Immunoblot of EGFR expression in multiple lines cultured in 10% serum for the indicated times. e, EGFR copy numbers in cellsdescribed in d. f, Matrigel invasion assay of cells cultured in 10% serum for 8 weeks in the presence or absence of EGF. g, Kaplan–Meier survival curves of mice with GBM9V and GBM9TGFα tumours (n=8/group). h-i, H&E staining, SMI-31 immunostaining (black arrow)and quantification of SMI-31 countsin mouse tumours. j-k, Ki67 immunostaining and quantification of Ki67 positive cellsin mouse tumours. l, Immunoblot of EGFR expression in various tumours.. m, EGFR copy numbers in mouse tumours compared to fresh explants cultures. n, Immunoblot of BIN3 expression in cells stably overexpressing BIN3 or empty vector. o, Matrigel invasion assay of multiple lines. p, Kaplan–Meier survival curves of mice with GBM12V and GBM12BIN3 tumours (n=8/group). q-r, H&E staining, SMI-31immunostaining and quantification of SMI-31 counts in mouse tumours. s, ELISA for EGF in GBM12 stably transfected with EGF overexpressing or empty vector. t, Immunoblot of the indicated proteins in EGF-overexpressing GBM12 clones. u, Kaplan–Meier survival curves of mice with GBM12V and GBM12EGF (GBM12EGF_02) tumours (n=6/group). v, H&E staining, SMI-31 immunostaining and quantification of SMI-31 countsin mouse tumours. x-y, Ki67 immunostaining and quantification of Ki67 postive cells in mouse tumours. z-aa, Representative TUNEL staining (black arrows) and quantification of TUNEL positive cells in GBM12 orthotopic tumours from vehicle orEGF treated mice. bb-cc, Representative TUNEL staining and quantification of TUNEL positivecells in mouse tumours. Scale bars: 50 μM. Western blot images are representative of three independent biological replicates. Actin served as the loading control. Data are represented as mean ± SEM from three independent experiments. Statistical significance was determined by one-way ANOVA adjusted by Bonferroni’s correction (c, e, m, s), or by two-tailed one-sample Student’s t-test (f, o), or two-tailed unpaired Student’s t-test (i, k, r, w, y, aa, cc). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.001, n.s. not significant. Numerical source data, statistic, exact P values and unprocessed blots are available as Source Data.