Fig. 4: Depletion of LAPTM4B accelerates erastin-induced ferroptosis.

A Western blot analysis of LAPTM4B and SLC7A11 protein levels in A549 cells treated with 5 µM erastin for the indicated times. Left panel: Representative experiment. Right panel: Quantification of n = 3 experiments, presented as mean ± SEM. *p < 0.05. B Western blot analysis of LAPTM4B and SLC7A11 protein levels in H1299 cells treated with 5 µM erastin for the indicated times. Left panel: Representative experiment. Right panel: Quantification of n = 3 experiments, presented as mean ± SEM. *p < 0.05. C Cell viability measured by the Cell Counting Kit-8 (CCK8) assay in WT and LAPTM4B depleted A549 cells (Up Panel), and WT and LAPTM4B KO H1299 cells (Down Panel) incubated with 5 µM erastin for 0 h, 24 h, 48 h, and 72 h. Quantification of at least five experiments, presented as mean ± SEM. p(A549, A549 KO)_24h = 0.0477, p(A549, A549 KO)_48h = 9.927E−05, p(A549, A549 KO)_72h = 0.0016, p(H1299, H1299 KO)_24h = 0.0048, p(H1299, H1299 KO)_48h = 0.0059, p(H1299, H1299 KO)_72h = 0.0001. D 4 × 10³ WT or LAPTM4B KO A549 cells were seeded into a 6-well plate, treated with 5 μM erastin for 24 h and cultured at 37 °C for 10 days. Afterwards, the cells were fixed with methanol, stained with crystal violet, and subsequently imaged and quantified. Left panel: representative experiment. Right panel: quantification of n = 3 experiments, mean ± SEM. p(A549_DMSO, A549_Erastin) = 0.0115, p(A549_DMSO, A549 KO_DMSO) = 3.644E-07, p(A549 KO_DMSO, A549 KO_Erastin) = 0.0015, p(A549_Erastin, A549 KO_Erastin) = 1.339E−05. E 8 × 10³ WT or LAPTM4B KO A549 cells were seeded into 96-well plates. Following treatment with 5 μM erastin for 24 h, the cells were stained with DAPI (blue) and EdU (red) to visualize the proliferative cells. Representative experiments shown. F Quantification results of Edu experiments in WT and LAPTM4B KO A549 cells (Left panel), as well as in WT and LAPTM4B KO H1299 cells (Right panel). Quantification of n = 5 experiments, mean ± SEM. p(A549_DMSO, A549 KO_DMSO) = 0.0158, p(A549 KO_DMSO, A549 KO_Erastin)=0.0371, p(A549_Erastin, A549 KO_Erastin) = 0.0011, p(H1299_DMSO, H1299 KO_DMSO) = 0.0032, p(H1299 KO_DMSO, H1299 KO_Erastin) = 0.0176, p(H1299 _Erastin, H1299 KO_Erastin) = 9.69E−06. G Cell death was measured by PI assay in WT and LAPTM4B KO A549 cells (Left panel), as well as in WT and LAPTM4B KO H1299 cells (Right panel). Cells were treated with 5 μM erastin for 24 h. Quantification of n = 3 experiments, mean ± SEM. p(A549_DMSO, A549 KO_DMSO) = 0.0017, p(A549 KO_DMSO, A549 KO_Erastin)=0.0081, p(A549_Erastin, A549 KO_Erastin)=0.0221, p(H1299_DMSO, H1299 KO_DMSO) = 0.0006, p(H1299 KO_DMSO, H1299 KO_Erastin) = 0.0003, p(H1299_Erastin, H1299 KO_Erastin) = 0.0003. H Cell death was measured by PI assay in WT and LAPTM4B KO A549 cells (Left panel), as well as WT and LAPTM4B KO H1299 cells (Right panel). Cells were treated with 1 μM Ferrostatin-1, 1 μM Liproxstatin-1, 50 μM Deferoxamine (DFO), 10 μM Z-VAD-FMK, or 5 μM Bafilomycin A1 (BafA1) for 24 h. Quantification of n = 3 experiments, mean ± SEM. p(A549_DMSO, A549 KO_DMSO) = 0.0216, p(H1299_DMSO, H1299 KO_DMSO) = 0.0241.