Figure 4.

BAX translocation induced by MIEF1 loss is mediated by BCL2L1 retranslocation. (a) HeLa cells were transfected with siRNA as indicated for 48 h. Then cells were treated with 20 ng/ml TNF plus 3 μg/ml CHX for further 16 h. Cells were hereafter fixed and immunostained for CYCS (red) and endogenous BAX (green). Hoechst, blue. Scale bar: 10 µm. Arrowheads indicate cells with BAX translocation on mitochondria. (b) The translocation of endogenous BAX was quantified from 3 independent experiments as in (a). Error bars represent SD. ***P < 0.001. n > 150 cells for each group from 3 independent experiments. (c) Cells were transfected with control or MIEF1 siRNA for 72 h. Cytosolic and mitochondrial fractions were extracted and BCL2 family members were detected by immunoblotting. Asterisk indicates a non-specific band. (d) The quantification of BCL2L1 in (c) is shown. BCL2L1 was normalized to TUBB (cytosol) or TIMM23 (mitochondria). Cytosolic or mitochondrial BCL2L1 was expressed as a percentage of total BCL2L1. Data are means ± SD (n = 3). ***P < 0.001. (e) HeLa cells were co-transfected with the indicated plasmids. After 24 h, cells were extracted and co-immunoprecipitated with anti-Flag beads followed by immunoblotting with anti-MYC and anti-Flag antibodies respectively. (f) HeLa cells were transfected with control or MIEF1 siRNA for 24 h. Cells were then transfected with mCherry-tagged vector or BCL2L1 (red) plus GFP-BAX (green) for further 24 h. Cells were fixed and immunostained with anti-CYCS antibody (cyan). Hoechst, blue. Scale bar: 10 µm. (g) Scoring of the GFP-BAX translocation onto mitochondria as in (f). Bar represents mean ± SD. ***P < 0.001. n > 100 cells for each group from 3 independent experiments. (h) HeLa cells were transfected with control or MIEF1 siRNA for 24 h before transfection with MYC-tagged vector or BCL2L1 for further 24 h. Hereafter, cells were treated with DMSO or 0.3 µM actinomycin D for 16 h. Lysates were analyzed by SDS-PAGE and immunoblotting.