Fig. 4. Signaling in tumors derived from Snai2 WT^ErbB2+ and Snai2 KO^ErbB2+ mice.

(A and B) Comparison of the signaling in mammary glands and tumors derived from Snai2 WT^ErbB2 and Snai2 KO^ErbB2 mice, quantified by ELISA (Mann-Whitney U test); (A) pAKT1 (S473) and (B) ERK1/2(T185/Y187) (pERK1/2, after that). In panels A and B, N = 20 tumors per group and N = 5 mammary glands were studied per group. (C-G) Comparison of the signaling in tumors derived from Snai2 WT^ErbB2 and Snai2 KO^ErbB2 mice: (C) Total ERK1/2; (D) pERK1/2; (E) total AKT; (F) pAKT1 (T308); and (G) pAKT1 (S473). (H) Comparison of the signaling in tumors derived from Snai2 WT^ErbB2++ and Snai2 KO^ErbB2++ parous mice. In panels C-H, the levels of signaling molecules were quantified by ELISA and compared in N = 20 tumors per group by the Mann-Whitney U test. See also Table S7. (I) Detection of total AKT1 and pAKT1 (S473) in western blots of selected tumors (lower and higher expression) to confirm the sensitivity of the technique (N = 4 mice per group). (J) hCAFs were treated with control or SNAI2 siRNA for two days and then co-cultured with MCF7 cells or (K) MDA-MB-231 in transwell inserts, for two days before analyzing the ERK and AKT signaling pathways in the tumor cells by western blots.