Figure 5
Figure 5. Reconstitution of p66Shc expression in CLL B cells restores the balance of antiapoptotic and proapoptotic Bcl-2 family members. (A) Quantification by real-time RT-PCR of the relative levels of p66Shc, Bcl-2, Bcl-xL, Bax, and Bak mRNA in PBLs from 7 patients with CLL (29M, 30M, 31M, 32M, 30U, 31U, 32U). Cells were cotransfected with either empty vector or the same vector encoding human p66Shc. A GFP reporter was included in all samples as a transfection control. Transcript levels were normalized to the expression level of GAPDH. For each transfected sample, Syber green runs were performed on duplicate samples of cDNAs from 2 independent reverse transcription reactions, each carried out on 400 ng of total RNA. RNA from the control sample (transfection with empty vector) of patient 29M was included in duplicate in each run as reference to normalize the data obtained in the individual experiments. The ΔΔCT method was applied as a comparative method of quantification, using as a reference the average of all the Ct data obtained on the samples transfected with empty vector. (B top) Immunoblot analysis of p66Shc, Bcl-2, Bcl-xL, Bax, and Bak expression in PBL lysates from a representative patient with CLL of 3 analyzed (29M, 30M, 30U). Cells were cotransfected with either empty vector or the same vector encoding human p66Shc. Each gel included a nontransfected PBL lysate from the same patient, as well as lysates of purified normal B cells. Stripped filters were reprobed with anti-actin antibodies as loading control. (Bottom) Quantification by laser densitometry of the data obtained on the 3 patients with CLL and 4 healthy controls. (C) Quantification by real-time RT-PCR of the relative levels of p66Shc, Bcl-2, Bcl-xL, Bax, and Bak mRNA in purified splenic B cells from wild-type (+/+) or p66Shc−/− (−/−) mice. Two pools of B cells, each from 3 wild-type or 3 p66Shc−/− mice, were used. Transcript levels were normalized to the expression level of GAPDH. For each sample, Sybr green runs were performed on duplicate samples of cDNAs from 2 independent reverse transcription reactions, each carried out on 400 ng of total RNA. RNA from the same pool of B cells from wild-type mice was included in duplicate in each run as reference to normalize the data obtained in the individual experiments. The ΔΔCT method was applied as a comparative method of quantification, using as a reference the average of all the Ct data obtained on the 2 pools of B cells from wild-type mice. ***P ≤ .001, **P ≤ .01, * P ≤ .05. Error bars indicate SD.

Reconstitution of p66Shc expression in CLL B cells restores the balance of antiapoptotic and proapoptotic Bcl-2 family members. (A) Quantification by real-time RT-PCR of the relative levels of p66Shc, Bcl-2, Bcl-xL, Bax, and Bak mRNA in PBLs from 7 patients with CLL (29M, 30M, 31M, 32M, 30U, 31U, 32U). Cells were cotransfected with either empty vector or the same vector encoding human p66Shc. A GFP reporter was included in all samples as a transfection control. Transcript levels were normalized to the expression level of GAPDH. For each transfected sample, Syber green runs were performed on duplicate samples of cDNAs from 2 independent reverse transcription reactions, each carried out on 400 ng of total RNA. RNA from the control sample (transfection with empty vector) of patient 29M was included in duplicate in each run as reference to normalize the data obtained in the individual experiments. The ΔΔCT method was applied as a comparative method of quantification, using as a reference the average of all the Ct data obtained on the samples transfected with empty vector. (B top) Immunoblot analysis of p66Shc, Bcl-2, Bcl-xL, Bax, and Bak expression in PBL lysates from a representative patient with CLL of 3 analyzed (29M, 30M, 30U). Cells were cotransfected with either empty vector or the same vector encoding human p66Shc. Each gel included a nontransfected PBL lysate from the same patient, as well as lysates of purified normal B cells. Stripped filters were reprobed with anti-actin antibodies as loading control. (Bottom) Quantification by laser densitometry of the data obtained on the 3 patients with CLL and 4 healthy controls. (C) Quantification by real-time RT-PCR of the relative levels of p66Shc, Bcl-2, Bcl-xL, Bax, and Bak mRNA in purified splenic B cells from wild-type (+/+) or p66Shc−/− (−/−) mice. Two pools of B cells, each from 3 wild-type or 3 p66Shc−/− mice, were used. Transcript levels were normalized to the expression level of GAPDH. For each sample, Sybr green runs were performed on duplicate samples of cDNAs from 2 independent reverse transcription reactions, each carried out on 400 ng of total RNA. RNA from the same pool of B cells from wild-type mice was included in duplicate in each run as reference to normalize the data obtained in the individual experiments. The ΔΔCT method was applied as a comparative method of quantification, using as a reference the average of all the Ct data obtained on the 2 pools of B cells from wild-type mice. ***P ≤ .001, **P ≤ .01, * P ≤ .05. Error bars indicate SD.

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