Fig. 3: Deletion of Nf1, Tsc1, or Tgfbr2 increased metastatic colonization.

A Western blot evaluation of individual sgRNAs induced Nf1, Tsc1, and Tgfbr2 deletion, as well as Nf2 KO. 3–4 sgRNAs were selected for each gene from the sgRNA library and used for validation. The Western blots were repeated for the cells with most significant KO for each TS gene and showed similar results. B Representative H&E staining of the lungs and number of metastatic nodules. Lung surface metastasis normalized to tumor mass of nude mice. n = 7 per group. C Primary tumor growth in nude mice transplanted with 4T1-C1 cells with sgRNA knockdown of Nf1, Tsc1, or Tgfbr2 (n = 9 per group). D, E Western blot for shRNA knockdown of Nf1, Tsc1, or Tgfbr2 (D), and representative Indian ink staining of the lungs (E left) and metastatic nodule counts (E right) (n = 10 mice each group). F Number of lung metastatic nodules (normalized to primary tumor weight) comparing nude and Cas9 transgenic mice that received MFP injection of tumor cells with sgRNA mediated Nf1, Tsc1, or Tgfbr2 deletion. G–I TSAE1+mHer2 mouse model: Cas9 transgenic mice received TVI of TSAE1 tumor cells with sgRNA mediated Nf1, Tsc1, or Tgfbr2 deletion: Western for Her2 overexpression in TSAE1 tumor cells (G); Western for sgRNA KO of Nf1, Tsc1, or Tgfbr2 (H); metastatic nodule counts from lungs (n = 10 mice each group) I. Statistical significance was determined by one-way ANOVA followed by Sidak’s test for (B); two-tailed Student’s t-test for (E), (F), and (I). All graphs show mean ± s.d. *p < 0.05; **p < 0.01; ***p < 0.001. Exact p-values are provided in a source data file for (F).