Fig. 5: Overexpression of LAPTM4B enhances SLC7A11 stability and protects against ferroptosis in NSCLC.

A Protein levels of SLC7A11 in LAPTM4B stably expressing A549 and H1299 cells were assessed by Western blotting. Left panel: Representative experiment. Right panel: Quantification of n = 3 experiments, mean ± SEM, data normalized to “A549 Ctrl” or “H1299 Ctrl”. For SLC7A11 levels, p(A549 Ctrl, A549 LAPTM4B) = 0.0359, p(H1299 Ctrl, H1299 LAPTM4B) = 0.0003. For LAPTM4B levels, p(A549 Ctrl, A549 LAPTM4B) = 2.067E−06, p(H1299 Ctrl, H1299 LAPTM4B) = 0.009. B A549 cells stably expressing LAPTM4B and control cells were treated with 50 µg/mL CHX for the indicated times, and SLC7A11 protein levels were assessed by Western blotting. Upper panel: Representative experiment. Lower panel: Quantification of n = 3 experiments, mean ± SEM. * p < 0.05. C LAPTM4B stably expressing A549 and H1299 cells, as well as control cells, were subjected to immunoprecipitation. Cells were treated with 20 µmol/L MG-132 for 12 h before harvesting. Immunoprecipitation was performed with SLC7A11 antibody, and the lysates were immunoblotted with an antibody against Ubiquitin. Upper panel: Representative experiment. Lower panel: Quantification of n = 3 experiments, mean ± SEM, data normalized to “A549 Ctrl” or “H1299 Ctrl”. p(A549 Ctrl, A549 LAPTM4B) = 0.00012, p(H1299 Ctrl, H1299 LAPTM4B) = 0.0099. D Stably expressing LAPTM4B A549 cells and the control cells were transfected with the indicated siRNA, and subsequent western blotting was performed to determine SLC7A11 protein levels. Upper panel: Representative experiment. Lower panel: Quantification of n = 3 experiments, presented as mean ± SEM. * p < 0.05. E Stably expressing LAPTM4B A549 cells and the control cells were transfected with indicated siRNA. Immunoprecipitation of the cell lysate using SLC7A11 antibody, followed by immunoblotting with Ubiquitin antibody. Left panel: Representative experiment. Right panel: Quantification of at least three experiments, presented as mean ± SEM. * p < 0.05. F LAPTM4B stably expressing A549 cells and control cells were harvested to measure lipid peroxidation. Left panel: Representative experiment. Right panel: Quantification of n = 3 experiments, mean ± SEM. p(A549 Ctrl, A549 LAPTM4B) = 0.0016. G LAPTM4B stably expressing A549 and H1299 cells, as well as control cells, were harvested to measure MDA. Quantification of n = 3 experiments, mean ± SEM. p(A549 Ctrl, A549 LAPTM4B) = 0.0002, p(H1299 Ctrl, H1299 LAPTM4B) = 0.0003. H LAPTM4B overexpressing A549 cells and control cells were seeded at a density of 8 × 10³ cells per well in 96-well plates. After treatment with 5 μM erastin for 24 h, cells were stained with DAPI (blue) and EdU (red) to visualize proliferative cells. Quantification data from n = 3 experiments, presented as mean ± SEM. Statistical analysis revealed p(A549 Ctrl_DMSO, A549 Ctrl_Erastin) = 0.0481 and p(A549 Ctrl_DMSO, A549 LAPTM4B_DMSO) = 0.0467, p(A549 Ctrl_Erastin, A549 LAPTM4B_Erastin) = 0.0106. I LAPTM4B stably expressing A549 cells and control cells were transfected with SLC7A11 siRNA. After 72 h, cells were harvested to measure lipid peroxidation. The left panel shows a representative experiment, while the right panel presents quantification data from n = 3 experiments, displayed as mean ± SEM. Statistical analysis revealed p(A549 Ctrl_NC siRNA, A549 Ctrl_SLC7A11 siRNA)=0.0097, p(A549 Ctrl_NC siRNA, A549 LAPTM4B_NC siRNA)=0.003, and p(A549 LAPTM4B_NC siRNA, A549 LAPTM4B_SLC7A11 siRNA) = 0.0186. J LAPTM4B stably expressing A549 cells and control cells were transfected with SLC7A11 siRNA and subsequently seeded into 96-well plates. After 72 h, the cells were stained with DAPI and EdU to visualize proliferative cells. Quantification data from n = 3 experiments are shown as mean ± SEM. Statistical analysis revealed p(A549 Ctrl_NC siRNA, A549 Ctrl_SLC7A11 siRNA)=0.0012, p(A549 Ctrl_NC siRNA, A549 LAPTM4B_NC siRNA)=0.0002, and p(A549 LAPTM4B_NC siRNA, A549 LAPTM4B_SLC7A11 siRNA) = 2.542E−06.