Fig 2: TRIM16 perturbs the interactions of the p62-KEAP1-NRF2 complex A, BCo-IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16.CCo-IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16.DCo-IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16.EGraphical representation of data presented in Figs 2 and 3. Source data are available online for this figure.

Fig 3: MC1R disruption amplifies microglia activation and alters Nrf2 response to aSyn overexpression in the nigrostriatal pathway. MC1Re/e and WT mice were injected unilaterally with human WT aSyn AAV into the SN and sacrificed 8 weeks later: A Iba1 staining and B morphological classification and quantification of iba1-positive cells in the SN. n = 4 mice/group. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 30 µm. C IL-1a, IL-6, TNFa, and ICAM1 mRNA levels in ventral midbrain. Measurements were normalized by dividing values by the mean of the WT contralateral side. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. D Representative oxyblots for protein carbonyls and the corresponding Ponceau S staining in the ipsilateral ventral midbrain and E quantification of band density. Measurements were normalized by dividing values by the mean of WT control and multiplying by 100. Two-tail Student’s t-test. n = 3 mice/group. F Immunoblot for Nrf2 using ventral midbrain tissue and G quantification of Nrf2 band density in original values or H normalized to contralateral side by dividing values by the mean of the contralateral side and multiplying by 100. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group.I SN sections double-stained for Nrf2 and TH and J quantification of nuclear and cytoplasmic Nrf2. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. Scale bar, 20 µm. K mRNA levels of Nrf2 target genes HO-1, NQO1, GCLM, and GCLC in the ipsilateral ventral midbrain. Measurements were normalized by dividing values by the mean of WT control. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: (I) The protein levels of phosphorylated Nrf-2 and cleaved caspases 3 were determined by Western blotting. ß-actin was used as an internal control. (II) The protein expression of cleaved caspase 3 and Nrf-2 were detected by immunohistochemical assays (the positive reaction of anti-cleaved caspase 3 and anti-Nrf-2 antibody were brown); (a) the kidney from the control group, (b–d) the kidney from M25, M55, and M110 groups, respectively, showed a positive reaction in the glomerulus and medulla kidney.

Fig 5: Human telomerase reverse transcriptase (hTERT) increases NRF2 expression by recruiting YBX1 to bind to the NRF2 promoter. (A) Flow chart for screening of potential hTERT-recruited NRF2 transcription factors. (B) The YBX1 and NRF2 mRNA expression level was identified by qRT-PCR after downregulation of YBX1. (C) Luciferase activity of the NRF2 promoter was detected after downregulation of YBX1. (D) NRF2 promoter with a 5' biotin label was used to pull-down YBX1. Mock beads were used as a negative control. (E) Diagrammatic drawing of NRF2 P2 fragment. The P2 fragment was divided into 5 fragments. (F) YBX1 antibody was used to immunoprecipitate binding fragments of the NRF2 promoter under the condition of hTERT overexpression and fragments were identified by ChIP-qPCR with six primers (Left). Statistical analysis of ChIP-qPCR. (G) Luciferase activity of P2 fragment containing different mutant sites was detected after YBX1 overexpression. (H) HCT116 cell lysates were prepared for separate IP with hTERT and YBX1 antibody and then evaluated via western blotting. (I) The subcellular localization and the colocalization of hTERT and YBX1 were examined in SW620 cells via dual immunofluorescence using confocal microscopy. (J) YBX1 in cell nuclei and cytoplasm was identified by western blotting after overexpression of Flag-hTERT. (K) The NRF2 mRNA expression level was identified by qRT-PCR after overexpression of hTERT and simultaneous knockdown of YBX1. (L) Luciferase activity of the NRF2 promoter was detected after overexpression of hTERT and simultaneous knockdown of YBX1. (M) The NRF2 protein level was identified by western blotting after overexpression of hTERT and simultaneous knockdown of YBX1 (Left). Statistical analysis of western blotting (Right). *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance.