Figure 6
Pharmacologic and genetic inhibition of procasp8 reduces the apoptotic effect of Lyn inhibition. (A) Freshly isolated B-CLL cells were cultured in the absence or presence of 10 μM PP2 with increasing concentrations of z-VAD-fmk (upper panels) or z-IETD-fmk (lower panels) for 48 hours, and cell apoptosis was analyzed by annexin V–propidium iodide (PI) flow cytometry. Quadrant analysis after flow cytometry was expressed as mean percentage ± SD of early and late apoptosis from 3 separate experiments performed in triplicate on samples from 16 B-CLL patients belonging to 2 different UM-CLL and M-CLL subsets (left and right panels, respectively) composed of 8 samples each. Compared with the effect of PP2 alone, changes were significant starting at 10 μM of z-VAD-fmk or z-IETD-fmk, respectively. (B) Freshly isolated B-CLL cells were transfected by nucleofection with either the negative control (lanes 1 and 3) or procasp8-siRNAs (lanes 2 and 4) and cultured for 48 hours in complete medium. Cell lysates then underwent Wb analysis with anti-procasp8 antibody to assess the efficacy of siRNA technology. Expression of procasp8 in the whole cell of procasp8 siRNA transfected cells compared with that from siRNA control transfected cells was significantly decreased (*P < .05, lanes 1, 2, and 3 vs 4). (C) Freshly isolated B-CLL cells were transfected by nucleofection with either the negative control (white bar) or procasp8-siRNAs (shaded bar), cultured for 48 hours in complete medium, and subsequently incubated for 0 or 24 hours in the absence (left panel) or presence (right panel) of 10 µM PP2. Cell apoptosis was then analyzed by annexin V–PI flow cytometry, with quadrant analysis being expressed as mean percentage ± SD of early and late apoptosis from 3 separate experiments performed in triplicate on samples from 16 B-CLL patients belonging to 2 different UM-CLL and M-CLL subsets (left and right panels, respectively) composed of 8 samples each. Compared with the effect of PP2, changes due to procasp8 siRNA were statistically significant (*P < .05 or higher degree of significance). (D) Annexin V–PI flow cytometry analysis of 2 samples representative of the UM-CLL and M-CLL groups from the right panel of C. FITC, fluorescein isothiocyanate.

Pharmacologic and genetic inhibition of procasp8 reduces the apoptotic effect of Lyn inhibition. (A) Freshly isolated B-CLL cells were cultured in the absence or presence of 10 μM PP2 with increasing concentrations of z-VAD-fmk (upper panels) or z-IETD-fmk (lower panels) for 48 hours, and cell apoptosis was analyzed by annexin V–propidium iodide (PI) flow cytometry. Quadrant analysis after flow cytometry was expressed as mean percentage ± SD of early and late apoptosis from 3 separate experiments performed in triplicate on samples from 16 B-CLL patients belonging to 2 different UM-CLL and M-CLL subsets (left and right panels, respectively) composed of 8 samples each. Compared with the effect of PP2 alone, changes were significant starting at 10 μM of z-VAD-fmk or z-IETD-fmk, respectively. (B) Freshly isolated B-CLL cells were transfected by nucleofection with either the negative control (lanes 1 and 3) or procasp8-siRNAs (lanes 2 and 4) and cultured for 48 hours in complete medium. Cell lysates then underwent Wb analysis with anti-procasp8 antibody to assess the efficacy of siRNA technology. Expression of procasp8 in the whole cell of procasp8 siRNA transfected cells compared with that from siRNA control transfected cells was significantly decreased (*P < .05, lanes 1, 2, and 3 vs 4). (C) Freshly isolated B-CLL cells were transfected by nucleofection with either the negative control (white bar) or procasp8-siRNAs (shaded bar), cultured for 48 hours in complete medium, and subsequently incubated for 0 or 24 hours in the absence (left panel) or presence (right panel) of 10 µM PP2. Cell apoptosis was then analyzed by annexin V–PI flow cytometry, with quadrant analysis being expressed as mean percentage ± SD of early and late apoptosis from 3 separate experiments performed in triplicate on samples from 16 B-CLL patients belonging to 2 different UM-CLL and M-CLL subsets (left and right panels, respectively) composed of 8 samples each. Compared with the effect of PP2, changes due to procasp8 siRNA were statistically significant (*P < .05 or higher degree of significance). (D) Annexin V–PI flow cytometry analysis of 2 samples representative of the UM-CLL and M-CLL groups from the right panel of C. FITC, fluorescein isothiocyanate.

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