FIGURE 5.

Requirement of intact mTORC2 for the generation of IFNγ-biological effects. A, U937 cells were transfected with either control siRNA or siRNA specifically targeting Rictor, as indicated. The cells were subsequently plated in methylcellulose in the absence or presence of human IFNγ, and leukemic CFU-L colony formation was assessed. Data are expressed as the percentage of colony formation of untreated control siRNA-transfected cells, and error bars represent mean ± S.E. of four independent experiments. Statistical analysis was performed using Student's t test as indicated. *, p < 0.05. B, normal human CD34⁺ bone marrow-derived cells were transfected with control siRNA or Rictor siRNA and incubated in clonogenic assays in methylcellulose in the presence or absence of human IFNγ as indicated. CFU-GM and BFU-E progenitor colonies were scored after 14 days in culture. Data are expressed as the percentage of colony formation of untreated control siRNA-transfected cells, and error bars represent mean ± S.E. of three independent experiments. Statistical analyses were performed using Student's t test as indicated. *, p < 0.05. C, Rictor^+/+ and Rictor^−/− MEFs were incubated with the indicated doses of mouse IFNγ and subsequently challenged with EMCV. Virus-induced CPEs were quantified 4 days later. Data are expressed as percent protection from EMCV CPEs and are representative of three independent experiments. Values are mean + S.E. of quadruplicate assays. D, Rictor^+/+ MEFs were pretreated with the indicated doses of mouse IFNγ for 16 h and then challenged with EMCV. In parallel, Rictor^+/+ MEFs were pretreated for 60 min with rapamycin (20 nm) and then with the indicated doses of mouse IFNγ for 16 h in the continuous presence of rapamycin (20 nm) and subsequently challenged with EMCV. Virus-induced CPEs were quantified 24 h post-infection. Data are expressed as percent protection from EMCV CPEs. Values are mean + S.E. of quadruplicate assays for two independent experiments.