Fig. 1: LKB1 nonenzymatically promotes Fas-induced apoptosis in BID KO HeLa cells.

A Immunoblot analysis of LKB1 in LKB1 reconstituted HeLa WT or BID KO cells. Cell lysates were subjected to immunoblotting with the indicated antibodies. β-actin was used as a loading control. B Empty vector (EV) or LKB1 reconstituted BID KO HeLa cells were treated with Fc-FasL (100 ng/mL) for indicated periods, and then cell lysates were subjected to immunoblotting with the indicated antibodies. C Empty vector (EV) or LKB1 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. control cells). D Empty vector (EV) or LKB1 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. The data is presented as FITC-PE fluorescence density plots. E Quantification of the percentage of Annexin V-positive cells shown in Fig. 1D. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. control cells). F Quantification of the percentage of PI and annexin V double-positive cells shown in Fig. 1D. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. control cells). G Quantification of the percentage of PI-positive/Annexin V-negative cells shown in Fig. 1D. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; *p < 0.05, (vs. control cells). H Immunoblot analysis of LKB1 in LKB1 WT or K78M reconstituted HeLa BID KO cells. Cell lysates were subjected to immunoblotting with the indicated antibodies. I LKB1 WT or K78M reconstituted BID KO HeLa cells were cultured with glucose free medium for 0, 12 or 24 h, and then cell lysates were subjected to immunoblotting with the indicated antibodies. J Empty vector (EV) or LKB1 (WT or K78M) 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. 1J. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. control cells). L Quantification of the percentage of PI and annexin V double-positive cells shown in Fig. 1J. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. control cells). M Quantification of the percentage of PI-positive/Annexin V-negative cells shown in Fig. 1J. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t test; ***p < 0.001, (vs. control cells). All data are representative of at least three biologically independent replicates.