Fig. 9

Lactylation stabilizes DCBLD1 expression. A Western blot analysis of the changes induced by sodium L-lactate (25 mM) on DCBLD1 at different times. B Western blot analysis of the changes induced by Oxamate (25 mM) on DCBLD1 at different times. C qPCR was performed to determine MCT1 (SLC16A1) mRNA levels in SiHa cells treated with sodium L-lactate (25 mM). D Analysis of the correlation between DCBLD1 and MCT1 (SLC16A1) using TCGA database. E Western blot analysis of DCBLD1 in SiHa cells treated with or without L-lactate (25 mM) and subsequently exposed to cyclohexane (CHX, 10 μg/ml) for the specified duration. The graph on the right is a statistical plot of the western blot. F Western blot analysis of pan-lactylation and pan-ubiquitination in SiHa cells, with and without sodium L-lactate. G Co-IP assay in SiHa cells transfected with the His-DCBLD1 plasmid, with or without L-lactate treatment for 6 h. H LC–MS/MS was performed to confirm the Kla of the DCBLD1 protein and identify specific lactylated modification sites. Specific lactylated modification sites (K167, K172, K486, and K497) were shown. I Species (Home sapiens, Pan paniscus, Mus musculus, Danio rerio, and Astyanax mexicanus) conservation analysis of potential lactylation modification sequence sites for DCBLD1. J Overlap-PCR was used for the construction of mutation plasmids. Sanger sequencing was used to verify whether lysine was mutated to alanine (GCG or GCA). (K) The mutant plasmid was re-expressed in SiHa cells knocked down for DCBLD1, and the DCBLD1 lactylation level was detected using immunoblotting. The graph on the right is a statistical plot of the western blot. (L) The 3D structure of DCBLD1 was predicted using the AlphaFold tool. Auto-Dock 4.0 was used to predict DCBLD1 binding to L-lactate. (M) Western blot analysis of DCBLD1 in SiHa-DCBLD1 KD re-expressing DCBLD1-WT or DCBLD1 K172A cell treated with L-lactate (25 mM) and subsequently exposed to cyclohexane (CHX, 10 μg/ml) for the specified duration. Data are presented as mean ± SD. Statistical significance was assessed using an unpaired t test (C), a two-way ANOVA with Šídák's multiple comparisons test (E), and a one-way ANOVA with Dunnett's multiple comparisons test (K). (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05; ns, no significance)