Fig. 6: AARS1 functions as a lactyltransferase that facilitates NPM1 lactylation and induces ferroptosis in HK-2 cells.

a IP of NPM1 lactylation using an anti-NPM1 antibody in IR-CM-treated HK-2 cells following siRNA-mediated knockdown of enzymes (n = 3 group⁻¹). b Detection of NPM1-HA lactylation by anti-HA IP in HK-2 cells with AARS1 knockout (AKO) or overexpression (AOE) following sodium lactate (10 mM) treatment (n = 3 group⁻¹). c In vitro lactylation assay of purified WT and K253R NPM1 proteins incubated with AARS1, lactate (2 mM), and ATP (4 mM) for 2 h at 37 °C, followed by western blot (n = 3 group⁻¹). d GST pull-down assay to detect the direct interaction between GST-NPM1 and His-AARS1 in vitro; arrows represent the indicated proteins (n = 3 group⁻¹). e Co-IP assays of the endogenous NPM1 and AARS1 in HK-2 cells (n = 3 group⁻¹). Co-IP of NPM1-HA and AARS1-His in HK-2 cells using anti-HA (f) and anti-His (g) antibodies (n = 3 group⁻¹). h Co-IP of NPM1-HA and AARS1-His using an anti-HA antibody in HK-2 cells treated with diverse CM (n = 3 group⁻¹). i–l Lipid peroxidation level, MDA concentration, iron level, and GSH content in WT, AOE, and AKO HK-2 cells cocultured with NC-CM or IR-CM (n = 3 group⁻¹); iron level and GSH content were normalized according to negative control group results; one-way ANOVA followed by Tukey’s test. m Western blotting examining NPM1 and SLC7A11 expression in WT, AOE, and AKO HK-2 cells treated with the same experimental setup as described in (i–l) (n = 3 group⁻¹). n Transcriptional level of SLC7A11 in WT, AOE, and AKO HK-2 cells (n = 3 group⁻¹); normalized to negative control group results; one-way ANOVA followed by Tukey’s test. Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The pvalues are shown for the indicated comparisons. Source data are provided as a Source Data file.