Fig. 4: A four amino acid sequence of LKB1 resembling an IAP binding motif is required for Fas-induced apoptosis in BID KO HeLa cells.
From: Truncated LKB1 nonenzymatically enhances Fas-induced apoptosis by acting as a surrogate of Smac
A Alignment of an IAP binding motif-like sequence (IBM-LS) of LKB1 for the indicated species. aa76-79 of human LKB1 correspond to the IBM-LS. B Empty vector (EV) or LKB1 (WT or ΔIBM-LS) 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. C Cell lysates from LKB1 (WT/ΔIBM-LS) reconstituted BID KO HeLa cells were immunoprecipitated with protein G-Sepharose beads using the indicated antibodies, and subjected to immunoblotting with the indicated antibodies. D HEK293A cells were transfected with FLAG-LKB1 (WT/ΔIBM-LS) 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. E Empty vector (EV) or LKB1 (WT or ΔIBM-LS) reconstituted BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for 0, 1, 2 or 4 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. F Empty vector (EV) or LKB1 (WT or ΔIBM-LD) reconstructed BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for 3 h, and then the cell lysates were subjected to immunoblotting with the indicated antibodies. G Empty vector (EV) or LKB1 (WT or ΔIBM-LS) 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 < 0.05, ***p < 0.001, (vs. EV cells), ##p < 0.01, ###p < 0.001 (vs. LKB1 WT cells) (H) Empty vector (EV) or LKB1 (WT or ΔIBM-LS) reconstituted BID KO HeLa cells were treated with Fc-FasL (0, 10, 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. I Quantification of the percentage of annexin V-positive cells shown in Fig. 4H. 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 Quantification of the percentage of PI and annexin V double-positive cells shown in Fig. 4H. 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). K Quantification of the percentage of PI-positive/Annexin V-negative cells shown in Fig. 4H. 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.
