Fig. 3: Truncated LKB1 leads to the degradation of XIAP in BID KO HeLa cells.
From: Truncated LKB1 nonenzymatically enhances Fas-induced apoptosis by acting as a surrogate of Smac
A Empty vector (EV) or LKB1 reconstituted BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for 4 h, then immunoprecipitated with the anti-XIAP antibody, and subjected to immunoblotting with the indicated antibodies. The band indicated by an asterisk is a non-specific band that does not change upon LKB1 expression. The arrowhead indicates tLKB1. S.E.: short exposure. L.E.: long exposure. B LKB1 reconstituted BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) and z-VAD (20 μM) for 0, 1, 2 or 4 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. C FLAG-LKB1, affinity-purified from 293 A cells overexpressing FLAG-LKB1, was reacted with recombinant active caspase-8 (0, 0.1, 0.25, 0.5, 1.0 units), and subjected to immunoblotting with the indicated antibodies. The band indicated with an asterisk is a non-specific band that is not affected by recombinant caspase-8. D Cell lysates from empty vector (EV) or LKB1-reconstituted BID KO HeLa cells were immunoprecipitated with protein G-Sepharose beads using the indicated antibodies, and subjected to immunoblotting with the indicated antibodies. E HEK293A cells were transfected with FLAG-LKB1 and/or HA-Caspase-8 p43 (C360A) plasmid for 24 h, and immunoprecipitated anti-FLAG-tagged agarose beads, and subjected to immunoblotting with the indicated antibodies. F FLAG-LKB1 (WT, D277E, D284E, D327/330E, D352/355/358/359E), affinity-purified from 293 A cells overexpressing FLAG-LKB1, were reacted with recombinant active caspase-8 (0.5 units), and subjected to immunoblotting with the indicated antibodies. The band indicated by an asterisk is a non-specific band that is not affected by recombinant caspase-8. G FLAG-LKB1 (WT or 4DE) reconstituted BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for 4 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. H Empty vector (EV) or LKB1 (WT or 4DE) reconstituted BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for 0, 2 or 4 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. I Empty vector (EV) or LKB1 (WT or 4DE) reconstituted BID KO HeLa cells were treated with Fc-FasL (0, 10, 50, 100 ng/mL) for 12 h. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. EV cells), ###p < 0.001 (vs. LKB1 WT cells). J Empty vector (EV) or LKB1 (WT/4DE) reconstituted BID KO HeLa cells were treated with Fc-FasL (0, 50, 100 ng/mL) for 12 h. Apoptotic cells were labeled with annexin V-FITC and PI for 15 min, and analyzed by FACS. Data is presented as FITC-PE fluorescence density plots. K Quantification of the percentage of Annexin V-positive cells shown in Fig. 3J. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. EV cells), ###p < 0.001 (vs. LKB1 WT cells). L Quantification of the percentage of PI and annexin V double-positive cells shown in Fig. 3J. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. EV cells), ###p < 0.001 (vs. LKB1 WT cells). M Quantification of the percentage of PI-positive/Annexin V-negative cells shown in Fig. 3J. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. EV cells), ###p < 0.001 (vs. LKB1 WT cells). All data are representative of at least three biologically independent replicates.
