Fig 2: UCHL1 deletion suppresses lipid uptake.Macrophages were exposed to DMSO, oxLDL (50 µg/ml), LDN57444 (20 µM), and LDN57444 + oxLDL for 24 h. UCHL1 siRNA (50 nM) retreated for 24 h, then adding oxLDL (50 µg/ml) for additional 24 h in macrophages. Before cells were harvest for 6 h, Dil-oxLDL were used to incubate cells. The images were captured by confocal microscopy (a, b) and by flow cytometry (e, f). The showed images were from three experiments. c, d, g The quantitative analysis was presented. *p < 0.05, **p < 0.01, ***p < 0.001 versus oxLDL treatment group.

Fig 3: Elevation of CHST3 expression and 6S-CS isomer abundance at the site of cortical injury and ipsilateral thalamus at 7 days post-CCI. Representative images of RNAscope analysis paired with immunofluorescent analysis showed low expression of the 6S-CS-specific sulfotransferase, CHST3, and GFAP in the contralateral cortex (A) relative to the ipsilateral cortex (B) at 7 days post-CCI, and quantified in (C) and (D). LC-MS/MS analysis found an increase in the relative abundance of ipsilateral 6S-CS compared to the contralateral cortex (E). Low expression of CHST3 and GFAP was observed in the contralateral thalamus (F) compared to the ipsilateral thalamus (G) 7 days post-CCI, and quantified in (H) and (I). A significant increase in the relative abundance of 6S-CS was measured in the ipsilateral thalamus (J), compared to the contralateral thalamus 7 days post-CCI. Low expression of CHST3, yet normal expression of UCHL1, was observed in the contralateral thalamus (K) compared to the ipsilateral thalamus (L) 7 days post-CCI, and quantified in (M) and (N). (C–E,H–J,M,N) Student’s t-test. Values represent means ± SD (N = 4, *P < 0.05; ***P < 0.001). Scale bars = 20 µm. CHST3, carbohydrate sulfotransferase 3; CCI, controlled cortical impact; UCHL1, ubiquitin C-terminal hydrolase L1; GFAP, Glial fibrillary acidic protein.

Fig 4: Post-MI myocardial upregulation of Uchl1 occurred primarily in the cardiomyocytes of the border zone in mice. Shown are representative micrographs of immunofluorescence staining for Uchl1 (red) and sarcomeric a-actin (green) in mouse hearts 7 days after LAD ligation or sham surgery. The sections were counter-stained with DAPI to visualize nuclear DNA (blue). The heart scale bar, 500 µm.

Fig 5: UCH-L1 regulates the transcription of ERa gene via EGFR pathway. (A) MCF-7 or T47D cells were transfected with a control plasmid or a myc-his-UCH-L1 plasmid. (B and C) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or UCH-L1 siRNAs for 72h (B), or were treated with 10 µM LDN for 24h (C). The ERa mRNA level was analyzed by real-time PCR. (D) MCF-7 cells were transfected with a control plasmid or a Flag-EGFR plasmid. The mRNA level of ERa was measured by real-time PCR. The expressions of EGFR and ERa were measured by western blot. ß-actin was used as a loading control. Results shown are Mean ± s.d., n=3. *, p <0.05; **, p <0.01. (E) MCF-7/AdrR cells were transfected with a non-targeting siRNA or an UCH-L1 siRNA, followed by transfection with a Flag-EGFR expression plasmid. (F) MCF-7 cells overexpressing UCH-L1 were transfected with a non-targeting siRNA or an EGFR siRNA. The mRNA level of ERa was measured by real-time PCR. The expressions of UCH-L1, ERa and EGFR were measured by western blot. ß-actin was used as a loading control.