Fig. 7: TNF-α accelerates cystine uptake and GSH synthesis by upregulating ILF3 expression.

A SW620 cells were treated with different concentrations of TNF-α for 24 h, and protein expression levels of ILF3, SLC3A2, NF-κB p65, and p-NF-κB p65 were analyzed by western blot. B SW620 cells were treated with TNF-α (40 ng/mL) for 24 h, and the GSH/GSSG ratio was determined using a GSH and GSSG assay kit. C SW620 cells were treated with TNF-α (40 ng/mL) and RSL3 (10 μM) alone or in combination for 24 h, and lipid peroxidation levels were detected using a BODIPY (581/591) C11 probe. Scale bar = 10 μm. D SW620 cells were pretreated with TNF-α (40 ng/mL) for 12 h, followed by RSL3 treatment at different concentrations for 24 h. Cell viability was assessed using a CCK-8 assay. E, F Cystine uptake and GSH levels were measured in sh-Con and sh-ILF3#3 SW620 cells with or without TNF-α (40 ng/mL, 24 h). G SW620 cells were pretreated with TNF-α (40 ng/mL) for 24 h, then incubated with MG132 (20 μM, 4 h) or chloroquine (10 μM, 24 h), and ILF3 protein expression levels were analyzed by western blot. H SW620 cells were treated with TNF-α (40 ng/mL) for 24 h and co-treated with cycloheximide (60 μg/mL) for different durations before harvesting. ILF3 protein expression levels were analyzed by Western blot. I SW620 cells were treated with TNF-α (40 ng/mL) for 24 h, incubated with MG132 (20 μM) for 4 h before harvesting, and then subjected to in vivo ubiquitination analysis. J HEK293T cells were co-transfected with Myc-ILF3 and His-Ub plasmids for 48 h, treated with TNF-α (40 ng/mL, 24 h) and MG132 (20 μM, 4 h), and the protein mixture was purified using Ni-NTA magnetic beads and analyzed by western blot using an anti-Myc antibody. K HEK293T cells were co-transfected with Myc-ILF3 and His-Ub for 48 h, treated with TNF-α (40 ng/mL, 24 h) and MG132 (20 μM, 4 h), and subjected to in vivo ubiquitination analysis using an anti-His antibody.