Fig 2: The phosphorylation of p62 at S349 is involved in TFEB-mediated Nrf2 activation. (a) The levels of phosphorylated p62 at S349 and total p62 in H4 cells (Control) and H4 cells stably expressing TFEB (TFEB) were analyzed by immunoblotting using anti-phospho-specific p62 (S349) and anti-p62 antibodies, respectively. (b) HEK 293 cells with the wild type or haplotype (+/−) p62 gene were transiently transfected with the Myc-Nrf2 expression plasmid and together with or without the pHM6-TFEB expression plasmid. The protein levels of Nrf2, phosphorylated p62 at S349, p62 and TFEB were analyzed by immunoblotting using anti-Myc, phospho p62 (S349), p62 and HA antibodies. The asterisk on the Nrf2 panel indicates a non-specific band. (c) Scatter plot represents the relative ratio of Nrf2 and phosphorylated p62 at S349 normalized with that of actin. (d) H4 cells stably expressing TFEB were transiently transfected with the ARE-Luc reporter plasmid along with the wild-type or mutant p62 (S349A) plasmid. The number under graph indicates the amount of plasmids transfected in each well. The luciferase activity in the cells was measured following 24 h incubation. Full blots are provided in Supplementary Fig. S10. Data shown are mean ± S.E. of four independent experiments and were analyzed using Student’s t test. (***p < 0.001).

Fig 3: Schematic diagram showing the mechanism of Nrf2 activation by TFEB. TFEB first stabilizes Nrf2 through the repression of DCAF11, its E3 ubiquitin ligase, and activates Nrf2, resulting in the increased expression and phosphorylation of p62 at S349. Then, increased phosphorylated p62 disrupts the binding of Keap1 and Nrf2, thus leading to sustained Nrf2 activation through a positive feedback loop.

Fig 4: TFEB increases the expression of Nrf2-response genes. (a) The protein levels of Nrf2-response genes in H4 cells (Control) and H4 cells stably expressing TFEB (TFEB) were analyzed by immunoblotting using anti-TFEB, Nrf2, HO-1, GSTM1, p62, and NDP52 antibodies, respectively. Full blots are provided in Supplementary Fig. S10. (b) The mRNA levels of Nrf2-response genes were analyzed by qRT-PCR as described in Methods. Bar graph represents the relative mRNA level of genes in TFEB cells compared to H4 cells. Data shown are mean ± S.E. of three independent experiments and were analyzed using Student’s t test. (*p < 0.05; ***p < 0.001).

Fig 5: Wogonin activates Nrf2 signalling in liver tissue of LPS‐induced sepsis mice. A‐E, Representative IHC images and IHC score analysis of Nrf2, HO‐1, SOD1, SOD2 and p‐p65 (Ser536) in liver tissue from LPS‐induced septic mice (n = 5). The number of scored fields = 10. Magnification, 400×. Scale bar = 50 μm. The concentration of wogonin was 50.0 mg/kg. The results are presented as mean ± SD through at least 3 independent experiments and were analysed by one‐way analysis of variance (ANOVA). ns, no statistical significance, *P < .05, **P < .01, ***P < .001