MOESM2 of p66ShcA functions as a contextual promoter of breast cancer metastasis

Additional file 2: Figure S2. p66ShcA is overexpressed in lung and liver metastatic triple negative breast cancer cells. (A) Whole cell lysates were generated from parental 4T1 cells along with explants isolated either from breast tumors (BT: 148,152) or that are enriched for their ability to metastasize to lung (526, 537), liver (2776, 2792) or bone (590, 592). Immunoblot analysis using ShcA- and Tubulin-specific antibodies. (B) Individual clones from 4T1 parental tumors were analyzed by immunoblot using a ShcA-specific antibody. (C) p66ShcA mRNA levels (transcripts per million) in RNAseq TCGA datasets that include primary breast tumors and metastases. This includes 7 metastases within the luminal A/B and basal subtypes along with 767 primary tumors (also luminal A/B and basal). (D) p66ShcA was deleted from 4T1-537 cells by Crispr/Cas9 genomic editing. Individual clones were screened by immunblot analysis using ShcA- and Tubulin-specific antibodies. p66ShcA-null clones identified in red font were pooled to generate p66-CR cells used for further analysis. (E) 4T1-537, p66-CR cells were transfected with either empty vector (VC) or p66ShcA-WT or p66ShcA-S36A expression vectors. Immunoblot analysis using ShcA- and Tubulin-specific antibodies.