Fig. 3: A20 downregulates PFKL protein levels by post-transcriptional modification.

a A20 decreases the PFKL protein level. Huh7 and LM3 cells were transfected with pcDNA3-A20 or pMKO-shA20 plasmids. The protein levels of PFKL were determined by standard western blotting (left panel). The relative PFKL protein compared with the β-actin level was quantified (right panel). b A20 reduces PFKL protein level in a dose-dependent manner. Huh7 cells were transfected with the PFKL vector together with varying amounts of A20 or empty control (Con) vectors, and the protein levels of PFKL were determined by standard western blotting (left panel). The relative PFKL protein compared with the β-actin level and relative A20 protein compared with the β-actin level was quantified (right panel). c LPS treatment increases the endogenous A20 protein level and decreases the PFKL protein level. Huh7 cells were treated with 9 and 18 μg/mL LPS for 4 h. Untreated cells were used as controls. The expression level of A20 protein was determined by western blotting. d–f A20 decreases PFKL expression at the post-transcriptional level. PFKL mRNA was determined by qPCR and normalized against β-actin. Error bars represent ± S.D. of triplicate experiments. The two-tailed Student’s t-test was used. NS denotes no significance (d). Scatterplots revealed that A20 and PFKL were not correlated at the mRNA level. Clinical data of non-cancerous liver samples and hepatocellular carcinoma samples were based on GSE364 (e). Data from the GEPIA website showed that A20 has no correlation with PFKL at the mRNA level (r = 0.16) (f). (The two-tailed Student’s t-test was used. The symbol * shows statistically significant differences with *p < 0.05, **p < 0.01, and ***p < 0.001).