CIN85 knockdown enhances the survival, growth, and differentiation of primary B cells. (A) Control and CIN85-knockdown primary B cells were incubated for 24 hours in medium containing F(ab′)2 goat anti–human IgG/IgA/IgM (αIg, 20 μg/mL) or αIg plus CpG (1μM), and CIN85, BcLxL, A1, cyclin D2, and myc mRNA levels were quantified by real-time PCR. The data are normalized to the expression of 18S rRNA. The results shown are representative of 3 independent experiments (*P < .05, **P < .01 vs controls). (B) Control and CIN85-knockdown primary B cells were incubated for 24 hours in the absence or presence of F(ab′)2 goat anti–human IgG/IgA/IgM (αIg, 20 μg/mL). The cell lysates were subsequently separated on a SDS-PAGE gel and analyzed by Western blotting with anti-BclxL mAb, anti-cyclin D2 pAb, or anti–β-actin mAb. The resulting values are expressed as fold changes in protein expression compared with nonstimulated control cells. The values are the mean ± SD of 3 independent experiments (**P < .01 vs controls). (C) Control and CIN85-knockdown primary B cells were incubated for 48 hours in the absence or presence of F(ab′)2 goat anti–human IgG/IgA/IgM (αIg, 20 μg/mL). After culture, the cells were stained with PE-labeled annexin V and analyzed using flow cytometry. The percentages of annexin-positive cells are shown. A representative histogram of 3 independent experiments is shown. (D) Control and CIN85-knockdown primary B cells were incubated for 48 hours in the absence or presence of F(ab′)2 goat anti–human IgG/IgA/IgM (αIg, 20 μg/mL). After culture, the cells were pulsed with BrdU, and its incorporation was detected by incubation with anti-BrdU mAb, followed by rhodamine-conjugated anti–mouse Ab. A representative histogram of 3 independent experiments is shown (**P < .01 vs controls). (E) Control and CIN85-knockdown primary B cells were incubated for 48 hours in the absence or presence of F(ab′)2 goat anti–human IgG/IgA/IgM (αIg, 20 μg/mL) and CpG (1μM), and quantitation of Blimp-1 and Xbp-1 mRNA by real-time PCR was carried out. The data are normalized to the expression of 18S rRNA. The results shown are representative of 3 independent experiments (*P < .05 vs controls). (F) Control and CIN85-knockdown primary B cells were incubated for 48 hours with or without F(ab′)2 goat anti–human IgG/IgA/IgM (αIg, 20 μg/mL) in the absence or presence of CpG (1μM). The cell lysates were subsequently separated on a SDS-PAGE gel and analyzed by Western blotting with anti–Blimp-1 mAb or anti–β-actin mAb. The resulting values are expressed as fold changes in protein expression compared with unstimulated control cells. The values are the mean ± SD of 3 independent experiments (**P < .01 vs controls).
