Figure 2

BCR and cyclin D1 mediate the high constitutive NOXA mRNA levels via the PI3/AKT/mTOR pathway. (a) Inhibition of BCR signaling causes reduction of constitutive NOXA mRNA expression in MCL cell lines Mino and Rec1. Effect of RNAi-mediated silencing of CD79A on BCR downstream pathways (upper panel) and constitutive expression of NOXA mRNA (lower panel; *P<0.05). Cells were transfected with control siRNA or siRNA targeting CD79A. After 24 h, cells were harvested for western blot analysis and quantification of NOXA mRNA levels. NOXA mRNA expression was normalized to GAPDH. Data represent means±S.D. from three experiments. (b) Impact of inhibition of BCR downstream pathways on constitutive NOXA expression. Upper panel: inhibition of the MAPK pathway has only minor effects on NOXA mRNA levels. Cell lines Mino and Rec1 were treated with 1 μM of PD0325901 for 6 h then harvested for western blot analysis and quantification of NOXA mRNA. Middle panel: effect of p65 knockdown on constitutive NOXA expression in MCL cell lines Mino and Rec1. Cells were transfected with control siRNA or siRNA targeting p65. After 48 h, cells were harvested for western blot analysis and quantification of NOXA mRNA levels. Lower panel: inhibition of the PI3K/AKT/mTOR pathway significantly reduces NOXA mRNA and protein expression. Cell lines Mino and Rec1 were treated with 1 μM of Bez235 for 6 h then harvested for western blot analysis and quantification of NOXA mRNA levels (***P<0.001). NOXA mRNA expression was normalized to GAPDH. All data represent means±S.D. from three experiments. (c) Knockdown of cyclin D1 attenuates PI3K/AKT/mTOR signaling and reduces constitutive NOXA mRNA levels in MCL cell lines Mino and Rec1. Cells were transfected with control siRNA or siRNA targeting cyclin D1 (CCND1). After 16 h, cells were harvested for western blot (upper panel) analysis and quantification of NOXA mRNA levels (lower panel; *P<0.05, ***P<0.001). NOXA mRNA expression was normalized to GAPDH. Data represent means±S.D. from three experiments. (d) Upper panel: comparison of PI3K/AKT/mTOR signaling in MCL cell lines, primary MCL samples and PBMCs of healthy donors. Phosphorylation of PI3K downstream kinases was measured by western blot. Lower panel: effect of PI3K/AKT/mTOR pathway inhibition on constitutive NOXA mRNA expression in MCL patients. Primary MCL cells were treated with 1 μM of Bez235 for 6 h then harvested for western blot analysis and quantification of NOXA mRNA levels (**P<0.01). NOXA mRNA expression was normalized to GAPDH. Data represent means±S.D. from three technical replicates