Figure 4.
INPP5K knockdown alters the structure and reduces signaling and cell proliferation of a human mutant CA oncogenic IL-7Rα chain expressed in BaF3 cells. (A-B) INPP5K (A) and p-STAT5Y694 (B) signals were analyzed using flow cytometry in plasma membrane–permeabilized CA–IL-7Rα BaF3 cells transduced with shINPP5K or control mock shRNA (mock). (A) Representative MFI histograms of INPP5K protein signals. C- is the background signal generated by the labeled secondary antibody alone. (B) Quantitative p-STAT5Y694 signal after MFI normalization to mock mean MFI. Results represent individual experiments and mean ± SEM. (C) Levels of p-STAT5A/BY694/699, STAT5A/B, p-AKT, AKT, p-S6, S6, p-ERK1/2, ERK1/2, and c-MYC were analyzed by western blotting in mock and shINPP5K CA–IL-7Rα BaF3 cells as described (Almeida et al6). (D) BCL-2 and BCL-XL antiapoptotic protein signals were analyzed using flow cytometry in plasma membrane–permeabilized mock and shINPP5K CA–IL-7Rα BaF3 cells. Representative MFI histograms of BCL-2 and BCL-XL protein signals are presented. (E) Cell cycle progression was analyzed using flow cytometry in mock and shINPP5K CA–IL-7Rα BaF3 cells after 15 minutes of incubation with DRAQ-5. The percentage of cells in cycles G0/G1 and G2 is indicated for each condition. (F) Mock and shINPP5K CA–IL-7Rα BaF3 cells were cultured in the absence of growth factors and counted after 48 hours. Results represent mean ± SEM. (G) The PtdIns(4,5)P2 signal was analyzed using flow cytometry in plasma membrane–permeabilized mock and shINPP5K CA–IL-7Rα BaF3 cells. Quantitative PtdIns(4,5)P2 signal after MFI normalization to mock mean MFI. Results represent individual experiments and mean ± SEM. (H) An indirect FRET strategy was used to analyze the proximity between the fluorescent plasma membrane DiO′ probe and p–IL-7RαY449 located at the carboxyterminal end of the mutant human IL-7Rα cytoplasmic domain in mock and shINPP5K CA–IL-7Rα BaF3 cells. FRET efficiency was calculated per the indirect FRET method validated by Guala et al.23 The quantitative energy transfer between the 2 fluorophores-labeled DIO′ and p–IL-7RαY449 (FRET efficiency) is presented. Results represent means ± SD (n = 30-50 cells per group). P values were calculated using unpaired nonparametric t test. NS: P > .05; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ERK, extracellular signal-regulated kinase; FRET, fluorescence resonance energy transfer; MFI, mean fluorescence intensity; NS, nonsignificant; SEM, strandard error of the mean.

INPP5K knockdown alters the structure and reduces signaling and cell proliferation of a human mutant CA oncogenic IL-7Rα chain expressed in BaF3 cells. (A-B) INPP5K (A) and p-STAT5Y694 (B) signals were analyzed using flow cytometry in plasma membrane–permeabilized CA–IL-7Rα BaF3 cells transduced with shINPP5K or control mock shRNA (mock). (A) Representative MFI histograms of INPP5K protein signals. C- is the background signal generated by the labeled secondary antibody alone. (B) Quantitative p-STAT5Y694 signal after MFI normalization to mock mean MFI. Results represent individual experiments and mean ± SEM. (C) Levels of p-STAT5A/BY694/699, STAT5A/B, p-AKT, AKT, p-S6, S6, p-ERK1/2, ERK1/2, and c-MYC were analyzed by western blotting in mock and shINPP5K CA–IL-7Rα BaF3 cells as described (Almeida et al6). (D) BCL-2 and BCL-XL antiapoptotic protein signals were analyzed using flow cytometry in plasma membrane–permeabilized mock and shINPP5K CA–IL-7Rα BaF3 cells. Representative MFI histograms of BCL-2 and BCL-XL protein signals are presented. (E) Cell cycle progression was analyzed using flow cytometry in mock and shINPP5K CA–IL-7Rα BaF3 cells after 15 minutes of incubation with DRAQ-5. The percentage of cells in cycles G0/G1 and G2 is indicated for each condition. (F) Mock and shINPP5K CA–IL-7Rα BaF3 cells were cultured in the absence of growth factors and counted after 48 hours. Results represent mean ± SEM. (G) The PtdIns(4,5)P2 signal was analyzed using flow cytometry in plasma membrane–permeabilized mock and shINPP5K CA–IL-7Rα BaF3 cells. Quantitative PtdIns(4,5)P2 signal after MFI normalization to mock mean MFI. Results represent individual experiments and mean ± SEM. (H) An indirect FRET strategy was used to analyze the proximity between the fluorescent plasma membrane DiO′ probe and p–IL-7RαY449 located at the carboxyterminal end of the mutant human IL-7Rα cytoplasmic domain in mock and shINPP5K CA–IL-7Rα BaF3 cells. FRET efficiency was calculated per the indirect FRET method validated by Guala et al.23 The quantitative energy transfer between the 2 fluorophores-labeled DIO′ and p–IL-7RαY449 (FRET efficiency) is presented. Results represent means ± SD (n = 30-50 cells per group). P values were calculated using unpaired nonparametric t test. NS: P > .05; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ERK, extracellular signal-regulated kinase; FRET, fluorescence resonance energy transfer; MFI, mean fluorescence intensity; NS, nonsignificant; SEM, strandard error of the mean.

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