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. 2023 May 15;9:165. doi: 10.1038/s41420-023-01463-5

Fig. 4. Activation of SGK1 by lactate reduced interaction of NEDD4L with GPX4.

Fig. 4

A A549 and H1299 cells were treated with lactate for the indicated concentrations, western blot was used to determine p-NEDD4L, NEDD4L, GPX4 protein levels. B After transfection with SGK1 cDNA for 36 h, A549 and H1299 cells were treated with lactate (0, 10, and 20 mM) for 3 h. Western blot was carried out for analysis of p-NEDD4L, NEDD4L, GPX4, SGK1 levels. C Cells were transfected with wild-type HA-NEDD4L cDNA or HA-NEDD4L (S448A) cDNA for 48 h, western blot was carried out for analysis of GPX4 levels. D After transfection with wild-type HA-NEDD4L cDNA or HA-NEDD4L (S448A) cDNA for 36 h, cells were untreated, or treated with lactate for 3 h. Cell lysates were immunoprecipitated with anti- GPX4 antibody and then Western blotted with anti- HA-NEDD4L. E Cells were treated with lactate for the indicated concentrations, Western blot was used to determine p-p38 protein levels. F After transfection with p38β cDNA for 48 h, A549 and H1299 cells were treated with lactate (0, 10 and 20 μM) for 3 h. Western blot was carried out for analysis of GPX4, NEDD4L, SGK1 levels. G After transfection with p38β cDNA for 36 h, A549 and H1299 cells were treated with Etoposide (0, 25 and 50 μM) for 12 h. Western blot was carried out for analysis of p-NEDD4L, NEDD4L, GPX4, SGK1 levels. H H1299 cells were treated with Etoposide, Etoposide plus NEDD4L (WT) cDNA and Etoposide plus NEDD4L (S448A) cDNA. Cell viability assay showed that NEDD4L cDNA significantly decreased H1299 cell viability. In H1299 cells, the IC 50 (half maximal inhibitory concentration) of etoposide was 19.87 μM, the cell survival was decreased in the presence of NEDD4L (WT) cDNA or NEDD4L (S448A) cDNA. (*p < 0.05, ##p < 0.01 for difference from etoposide-treated control by ANOVA with Dunnett’s correction for multiple comparisons). I H1299 cells were treated with Etoposide, Etoposide plus NEDD4L (S448A) cDNA in the presence and absence of Ferroptosis inhibitors Fer-1 or DFO. Cell viability assay showed that NEDD4L cDNA significantly decreased H1299 cell viability. Inhibition of ferroptosis with Fer-1 and DFO rescues S448A NEDD4L-increased sensitivity to etoposide. (**p < 0.01, for difference from etoposide-treated control, ##p < 0.01, for difference from etoposide plus NEDD4L (S448A) cDNA-treated control, by ANOVA with Dunnett’s correction for multiple comparisons).