Figure 3.

XIAP induces Beclin 1 transcription via p65/NFκB activation. (A) Schematic diagram of XIAP-mediated NFκB activation. BIR1 and RING domains of two XIAPs interact leading to dimerization that recruits TAB1. TAK1 is consequently activated by interaction with this complex in the form of dimers, which triggers NFκB signalling. This occurs after the phosphorylation of IκB and its resultant ubiquitination and proteasomal degradation. In this way, the p50/p65 heterodimer is translocated to the nucleus, where gene transcription occurs. (B) For overexpression (OE) bars, HeLa cells were co-transfected with a luciferase reporter containing the NFκB promoter plus empty vector (C-) or XIAP expression constructs for 48 h. For knockdown (KD) bars, HeLa cells were transfected with a control (C-) or XIAP siRNA. Twenty-four hours later, cells were transfected with a luciferase reporter containing the NFκB promoter for 48 h. In all cases, cells were treated or not with 2 µg/ml TGFβ for 2 h. The mean values of the relative luciferase activity are reported in the histograms. (C) HeLa cells were transfected with empty vector (C-) or XIAP expression constructs for 48 h (left panel) or with a control (C-) or XIAP siRNA for 72 h (right panel). The western blots in both panels are representative of at least three independent experiments performed in triplicate. (D) (left panel) HeLa cells were co-transfected for 48 h with a luciferase reporter that contains a promoter with p65 binding site plus empty vector (C-) or XIAP expression constructs. In both cases (C- and XIAP), an empty vector or a p65 expression constructs was included in the co-transfection; (right panel) HeLa cells were transfected for 48 h with a control (C-) or XIAP siRNA. Twenty-four hours later, cells were co-transfected with a luciferase reporter that contains a promoter with p65 binding site plus empty vector (C-) or p65 expression constructs. In both panels, values of the relative luciferase activity are reported in the histograms. (E) (left panel) HeLa cells were co-transfected for 48 h with CHET4-luciferase reporter containing the Beclin 1 promoter plus empty vector (C-) or XIAP expression constructs. In both cases (C- and XIAP), an empty vector or a p65 expression constructs was included in the co-transfection; (right panel) HeLa cells were transfected for 48 h with a control (C-) or XIAP siRNA. Twenty-four hours later, cells were co-transfected with CHET4-luciferase reporter containing the Beclin 1 promoter plus empty vector (C-) or p65 expression constructs. In both panels, values of the relative luciferase activity are reported in the histograms. The values shown in all the histograms represent the mean ± standard deviation from at least three independent experiments performed in triplicate samples/condition. The P-values were determined using Student's t-test. See also Supplementary Material, Figure S3.