Fig. 5: Several LKB1 mutants found in PJS failed to promote Fas-induced apoptosis in BID KO HeLa cells.

A BID KO HeLa cells were reconstituted with LKB1 WT, G135R, D176N, or D194N. Cell lysates were subjected to immunoblotting with the indicated antibodies. B Cell lysates from LKB1 (WT/G135R/D176N/D194N) reconstituted BID KO HeLa cells were immunoprecipitated with protein G-Sepharose beads using the indicated antibodies, and subjected to immunoblotting with the indicated antibodies. C LKB1 (WT, G135R, D176N, or D194N) reconstituted BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for 3 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. D Empty vector (EV) or LKB1 (WT or G135R) reconstructed BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for 0, 3 or 6 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. E Empty vector (EV) or LKB1 (WT, D176N, or D194N) reconstituted BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for 0, 3 or 6 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. F Empty vector (EV) or LKB1 (WT, G135R, D176N, or D194N) reconstituted BID KO HeLa cells were treated with Fc-FasL (0, 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) (G) Empty vector (EV) or LKB1 (WT, G135R, D176N, or D194N) 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. H Quantification of the percentage of V-positive cells shown in Fig. 5G. 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). I Quantification of the percentage of PI and annexin V double-positive cells shown in Fig. 5G. 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-positive/Annexin V-negative cells shown in Fig. 5G. 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.