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. Author manuscript; available in PMC: 2015 Jul 23.
Published in final edited form as: Cell Rep. 2015 Jul 9;12(3):511–524. doi: 10.1016/j.celrep.2015.06.044

Figure 2. N-Ras is required for efficient growth, transformation, and tumor formation of BLBC cells only.

Figure 2

(A) Cells expressing control or shRNAs targeting N-RAS or K-RAS4B were analyzed by Western blot for Ras proteins using isoform-specific antibodies. GAPDH was the loading control. The numbers below the blot indicate protein levels relative to the non-silenced control. (B) Cells were transduced by the shRNAs as in panel A and the growth rates were measured. (C) An N-RAS cDNA refractory to shRNA (N-RAS*) or a K-RAS4B cDNA was overexpressed in N-RAS-silenced SUM102PT cells. The growth rates of these cells were measured (top), and indicated proteins were analyzed by Western blot (bottom). The numbers below indicate protein levels relative to the controls. (D) Cells were transduced by shRNAs as in panel A and seeded either alone or together with HMFs (1:1) in soft agar. (E) N-RAS in indicated cells was similarly silenced as above with or without N-RAS* and examined for invasiveness (left). N-Ras in these cells was analyzed by Western blot (right). (F) Control or N-RAS-repressed SUM102PT cells were injected with HMFs into nude mice (n = 6). The control cells were also injected alone (n = 6) as an additional control. Kaplan-Meier curves of the portion of tumor-free animals are shown on the left while pictures of mice are shown in the middle. N-RAS silencing in the tissue was confirmed by RT-PCR (right). P-value (Gehan-Breslow-Wilcoxon test) was calculated between the green and red curves. (G) Control or N-RAS-repressed MDA-MB-468 cells carrying luciferase were injected into nude mice (2 mammary glands per mouse and 5 mice in each group, n = 10 tumors). Their luciferase activity (Figure S2I) was measured weekly (left) to calculate the tumor growth rates (between week-8 and week-13, middle). N-RAS silencing in the tissue was confirmed by RT-PCR (right). (H) SUM102PT cells carrying the inducible N-RAS shRNA were co-injected with HMFs (1:1) into nude mice (n = 9). When tumors became detectable (arrow), these mice were treated with doxycycline (DOX) or the vehicle control, and the tumor size was measured twice per week. See also Figure S2.