Journal: Frontiers in Pharmacology

Article Title: HECT, UBA and WWE domain containing 1 represses cholesterol efflux during CD4 + T cell activation in Sjögren’s syndrome

doi: 10.3389/fphar.2023.1191692

Figure Lengend Snippet: ABCA1 could be ubiquitinated by HUWE1 and degraded by proteasomes (A) The cells were treated with cycloheximide for the indicated times, and the expression of ABCA1, ABCG1, and SR-BI was analyzed by Western blotting. The expression of ABCA1 and SR-BI decrease over time. Double asterisks indicate p < 0.01, triple asterisks indicate p < 0.001 vs. control. (B) The expression level of ABCA1 in activated CD4 + T cells was restored by MG132 treatment. The cells were treated with 10 μM of MG132 for the indicated times, and the expression of ABCA1 and SR-BI was analyzed by Western blotting. The expression of ABCA1 increases over time. Double asterisks indicate p < 0.01 vs. control. (C) The interaction of HUWE1 with ABCA1 in CD4 + T cells was detected by co-immunoprecipitation assay. (D) Less ubiquitinated ABCA1 was observed in sh-Huwe1 treated cells than in control cells. The activated CD4 + T cells in the indicated groups were treated with MG132 and were then lysed and immunoprecipitated with anti-ABCA1. The enriched proteins were analyzed by Western blotting with anti-ubiquitin antibodies. (E) The proliferation of cells in the indicated groups was detected by CFSE staining. Bar graph on right shows the proliferation ratio of CD4 + T cells in different groups. Triple asterisks indicate p < 0.001, quadruple asterisks indicate p < 0.0001 vs. control. (F) Cell blasting of CD4 + T cells in the indicated groups was analyzed by size (forward scatter area, FSC) and granularity (side scatter area, SSC) after 24h activation. Asterisk indicates p < 0.05, double asterisks indicate p < 0.01 vs. control. (G) to (H) The CD25 expression in BI8626 (G) - and sh-Huwe1 (H) -treated CD4 + T cells was restored by DIDS. Bar graph on right shows MFI±SD ( n = 3 mice/group from three independent experiments). Data are from one experiment representative of three independent experiments with similar results (A–D) or three independent experiments with biological duplicates in each (E–H) ; ( n = 3).

Article Snippet: Chemical inhibitors included BI8626 (HY-120204, MCE), N-Butyldeoxynojirimycin (NB-DNJ, UGCG inhibitor, HY-17020, MCE), and DIDS (HY-D0086, MCE).

Techniques: Expressing, Western Blot, Control, Co-Immunoprecipitation Assay, Immunoprecipitation, Ubiquitin Proteomics, Staining, Activation Assay

Journal: Nature Communications

Article Title: TRIM21 inhibits irradiation-induced mitochondrial DNA release and impairs antitumour immunity in nasopharyngeal carcinoma tumour models

doi: 10.1038/s41467-023-36523-y

Figure Lengend Snippet: a – d The indicated NPC cells were treated with IR. The relative MTATP8 abundance in the cytosolic fractions was assessed by qPCR ( a ). Cytosolic mtDNA accumulation was evaluated by quantification of cytoplasmic TFAM (indicated by the white arrows) 24 h after IR treatment. Representative images (scale bar, 10 μm) and quantitative results are shown ( n = 30 cells per group) ( b ). The levels of total and phosphorylated STING, TBK1, IRF3 and STAT1 ( c ) and the IFN-β1 level in culture supernatants ( d ) were measured 48 h after IR treatment. e Co-IP with an anti-FLAG antibody revealed the enhanced interaction of two exogenous VDAC2 monomers in lysates of irradiated NPC cells. f Immunoblot analysis showed an increased level of VDAC2 oligomers in NPC cells 24 h after IR treatment. g VDAC2 oligomers in WT and TRIM21-KO NPC cells 24 h after IR treatment. h – j NPC cells were pre-treated with the VDAC2 oligomerization inhibitor DIDS (100 μM) or DMSO for 24 h and were then treated with IR. The relative cytosolic MTATP8 abundance was assessed by qPCR 24 h after IR treatment ( h ). The levels of phosphorylated STING, TBK1, IRF3 and STAT1 ( i ), as well as the expression levels of IFNB1 , CCL5 , and CXCL10 ( j ), were measured 48 h after IR treatment. n = 3 independent experiments. The data are presented as the mean ± SEM ( a , b , d , h and j ). Comparisons were performed using one-way ANOVA with the Tukey’s test for multiple comparisons.

Article Snippet: Cells were treated with the proteasome inhibitor MG132 (10 μM, Sigma) or the autophagy inhibitor CQ (50 μM, Sigma-Aldrich) for 6 h or with the protein synthesis inhibitor CHX (100 μg/ml, Sigma) for 0, 2, 4, 8 or 12 h. For the inhibition of BAX or VDAC2 oligomerization, cells were treated with BAI1 (2 μM, Selleck) or DIDS (100 μM, TargetMol) for 24 h before IR treatment.

Techniques: Co-Immunoprecipitation Assay, Irradiation, Western Blot, Expressing

Journal: Journal of AOAC International

Article Title: Determination of Seven Organic Impurities in FD&C Yellow No. 6 by Ultra-High-Performance Liquid Chromatography.

doi: 10.1093/jaoacint/qsab092

Figure Lengend Snippet: Figure 3. UV-visible absorption spectra of SA, SS, DAADBSA, DONS, SAþRS, SA þ 2N, and SSA.

Article Snippet: The following were used as reference standards without further purification: SA (acid form, 99.9%) from Sigma-Aldrich (Milwaukee, WI), SS (monosodium salt, 95%) from Ark Pharm (Arlington Heights, IL), DAADBSA (97.9%) and SAþRS (trisodium salt, 92.9%) from Wako Pure Chemical Corporation (Osaka, Japan), DONS (disodium salt, 93.3%) from BOC Sciences (Shirley, NY), SSA (monosodium salt, 97.5%) from Combi-Blocks, Inc, and SAþ 2N (monosodium salt, 96.4%) from Sigma-Aldrich.

Techniques: