Journal: The Journal of clinical endocrinology and metabolism

Article Title: Circulating Exosomes Activate Dendritic Cells and Induce Unbalanced CD4+ T Cell Differentiation in Hashimoto Thyroiditis.

doi: 10.1210/jc.2019-00273

Figure Lengend Snippet: Figure 3. HT exosomes bind to and upregulate TLR2/TLR3. DCs were cocultured with HT exosomes or HC exosomes for 24 hours. (a) Immunofluorescence analysis showed that DCs (blue color, marked by DAPI) were colocalized with HT exosomes or HC exosomes (green color, marked by PKH-67) after coculture for 24 hours. TLR2 and TLR3 were marked by Alexa Fluor 594 (red color) (magnification 3400, confocal laser-scanning microscopy). PBMCs were cocultured with HT exosomes or HC exosomes for 24 hours. (b) Representative patterns of CD11c1TLR21, CD11c1TLR31, and CD11c1TLR41 cells as analyzed by flow cytometry. (c) Frequency of CD11c1TLR21, CD11c1TLR31, and CD11c1TLR41 cells in the HT exosomes and HC exosomes groups (n 5 20 per group). The PBS group was used as a negative control (n 5 4). Statistical analyses of multiple groups were performed by Kruskal-Wallis H-test or one-way ANOVA. *P , 0.05; **P , 0.01.

Article Snippet: To perform the inhibition test, according to the literature and the results of our preliminary experiments, DCs were preincubated with the TLR2 antagonist PAb hTLR2 at a concentration of 3 mg/mL (Invitrogen, Carlsbad, CA) or with the TLR3 antagonist CU CPT 4a at a concentration of 30 mg/mL (Tocris Bioscience, Ellisville, MOUK) for 3 hours and then stimulated with 600 mg HT exosomes.

Techniques: Immunofluorescence, Confocal Laser Scanning Microscopy, Flow Cytometry, Negative Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Circulating Exosomes Activate Dendritic Cells and Induce Unbalanced CD4+ T Cell Differentiation in Hashimoto Thyroiditis.

doi: 10.1210/jc.2019-00273

Figure Lengend Snippet: Figure 4. HT exosomes activate the NF-kB signaling pathway in DCs, upregulate the expression of costimulatory molecules, and increase the release of IL-6. DCs were cocultured with HT exosomes or HC exosomes for 24 hours. To perform the inhibition experiments, DCs were pretreated with TLR2/3 inhibitors for 3 hours and then incubated with HT exosomes for 24 hours. (a) Representative stripe images and protein expression of MyD-88, TRIF, and p-P65 in the HC exosomes, HT exosomes (n 5 17 per group), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 8 to 10 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 5). TLR2 inhibitor and TLR3 inhibitor groups were used as positive controls in the inhibition experiments (n 5 8 to 10 per group). The relative protein expression level was corrected to the GAPDH expression level. (b) Expression of IL-6 in the supernatants of the HC exosomes, HT exosomes (n 5 14 per group), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 8 to 13 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 5). TLR2 inhibitor and TLR3 inhibitor groups were used as positive controls in the inhibition experiments (n 5 5). (c) Expression of the costimulatory molecules CD40, CD80, and CD83 on CD11c1DCs in the HC exosomes (n 5 11), HT exosomes (n 5 10), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 6 to 8 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 5). TLR2 inhibitor and TLR3 inhibitor groups were used as positive controls in the inhibition experiments (n 5 6 to 4). Statistical analyses of multiple groups were performed by Kruskal-Wallis H-test or one-way ANOVA. *P , 0.05; **P , 0.01.

Article Snippet: To perform the inhibition test, according to the literature and the results of our preliminary experiments, DCs were preincubated with the TLR2 antagonist PAb hTLR2 at a concentration of 3 mg/mL (Invitrogen, Carlsbad, CA) or with the TLR3 antagonist CU CPT 4a at a concentration of 30 mg/mL (Tocris Bioscience, Ellisville, MOUK) for 3 hours and then stimulated with 600 mg HT exosomes.

Techniques: Expressing, Inhibition, Incubation, Negative Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Circulating Exosomes Activate Dendritic Cells and Induce Unbalanced CD4+ T Cell Differentiation in Hashimoto Thyroiditis.

doi: 10.1210/jc.2019-00273

Figure Lengend Snippet: Figure 5. HT exosomes modulate the differentiation of Th1, Th17, and Treg cells and the secretion of cytokines. PBMCs were incubated with HT exosomes or HC exosomes for 1 to 4 days. To perform the inhibition experiments, PBMCs were pretreated for 3 hours with TLR2 or TLR3 inhibitors and then incubated with HT exosomes for 1 to 4 days. (a) Representative pictures of CD41IFN-g1Th1, CD41IL-17A1Th17A, and CD41CD251Foxp31 Treg cells as analyzed by flow cytometry in the inhibition experiments. (b) Frequency of CD41IFN-g1Th1, CD41IL- 17A1Th17A, and CD41CD251Foxp31 Treg cells in the HC exosomes, HT exosomes (n 5 30 per group), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 10 to 12 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 3). TLR2 inhibitor and TLR3 inhibitor groups were used as a positive control in the inhibition experiments (n 5 5). (c) Levels of IFN-g, IL-17A, and IL-10 in the cell supernatants as determined by ELISA in the HC exosomes, HT exosomes (n 5 18 to 23 per group), HT exosomes 1 TLR2 inhibitor, and HT exosomes 1 TLR3 inhibitor groups (n 5 12 to 20 per group). PBS was added to the medium of a group without exosomes and inhibitors as a negative control (n 5 3). TLR2 inhibitor and TLR3 inhibitor groups were used as a positive control in the inhibition experiments (n 5 5). HT-exo 1 TLR2 inhibitor: HT exosomes 1 TLR2 inhibitor; HT-exo 1 TLR3 inhibitor: HT exosomes 1 TLR3 inhibitor. Statistical analyses of multiple groups were performed by Kruskal-Wallis H-test or one-way ANOVA. *P , 0.05; **P , 0.01.

Article Snippet: To perform the inhibition test, according to the literature and the results of our preliminary experiments, DCs were preincubated with the TLR2 antagonist PAb hTLR2 at a concentration of 3 mg/mL (Invitrogen, Carlsbad, CA) or with the TLR3 antagonist CU CPT 4a at a concentration of 30 mg/mL (Tocris Bioscience, Ellisville, MOUK) for 3 hours and then stimulated with 600 mg HT exosomes.

Techniques: Incubation, Inhibition, Flow Cytometry, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Indoleamine 2,3-dioxygenase-regulated macrophages metabolic reprogramming rescues tacrolimus-induced nephrotoxicity

doi: 10.3389/fphar.2026.1784153

Figure Lengend Snippet: KYN supplementation alleviates TAC-induced energy metabolism disorder by restoring FAO. (A) Relative abundance of TCA cycle–related metabolites in the renal cortex (n = 5). (B) Relative abundance of glycolysis-related metabolites in the renal cortex (n = 5). (C) Schematic diagram of energy metabolism pathways based on metabolomic data, including the TCA cycle, glycolysis, and β-oxidation; red arrows indicate metabolites upregulated by TAC treatment, and blue arrows indicate metabolites downregulated by TAC treatment. (D) Heatmap of fatty acid metabolism–related compounds in the renal cortex (n = 5). (E,F) Seahorse analysis showing mitochondrial oxygen consumption rate (OCR) in renal cortical cells using glucose (E) or palmitic acid (F) as substrates (n = 4). (G) Quantification of basal and maximal OCR in renal cortical cells under glucose or palmitic acid substrate conditions (n = 4). (H) RT-qPCR analysis of FAO-related gene expression (MCAD, LCAD, CPT1, CPT2) in the renal cortex (n = 3). (I) Schematic diagram of mitochondrial fatty acid transport and β-oxidation pathways. (J) Western blot analysis of CPT1, CPT2, and CACT protein expression in the renal cortex, with β-tubulin as a loading control. (K) Densitometric quantification of CPT1, CPT2, and CACT protein bands from Western blots (n = 3). Data are presented as mean ± SD. Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: CIT, citrate; αKG, alpha-ketoglutarate; SUC, succinate; OAA, oxaloacetate; FUM, fumarate; MAL, malate; PYR, pyruvate; LAC, lactate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; F1,6BP, fructose-1,6-bisphosphate; MCAD, medium-chain acyl-CoA dehydrogenase; LCAD, long-chain acyl-CoA dehydrogenase; CPT1/2, carnitine palmitoyltransferase 1/2; CACT, carnitine-acylcarnitine translocase; ACSL, acyl-CoA synthetase long-chain family member; OCR, oxygen consumption rate.

Article Snippet: Primary antibodies used in this study included: IDO1 (Proteintech, Cat# 13268-1-AP, 1:1000), CPT1 (ABclonal, Cat# A5307, 1:2000), CPT2 (Proteintech, Cat# 26555-1-AP, 1:8000), CACT (ABclonal, Cat# A13956, 1:400), β-tubulin (Proteintech, Cat# 80713-1-RR, 1:10,000), HRP-conjugated Goat Anti-Rabbit IgG (H + L) (ABclonal, Cat# SA00001-2, 1:10,000).

Techniques: Metabolomic, Quantitative RT-PCR, Gene Expression, Western Blot, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Levocarnitine improves cardiac energy metabolic remodeling in myocarditis mice

doi: 10.3389/fphar.2025.1706936

Figure Lengend Snippet: LC improves myocardial mitochondrial substrate utilization in EAM mice. (A) Serum H-FABP levels in each group of mice, n = 3. (B–D) Western blot analysis of OCTN-2 and CPT-1B proteins, n = 3. (E) FFA and (F) LAC levels in myocardial tissue from each group of mice, n = 3. Data are expressed as Mean ± SEM with individual data points shown. FABP, heart-type fatty acid-binding protein; OCTN2, organic carnitine transporter novel type 2; CPT-1B, carnitine palmitoyltransferase-1B; FFA, free fatty acid; LAC, lactic acid.

Article Snippet: The PVDF membrane was soaked in 5% skim milk and incubated at room temperature for 2 h. Primary antibodies—GAPDH (60004-1-Ig, Proteintech, Wuhan, China), CPT1B (DF3904, Affinity, USA), OCTN2 (16331-1-AP, Proteintech, Wuhan, China), Akt (60203-2-Ig, Proteintech, Wuhan, China), p-Akt (80642-1-rr, Proteintech, Wuhan, China), and PGC-1α (Ab 313559, Abcam, Shanghai, China)—were incubated at 4 °C overnight.

Techniques: Western Blot, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: Lysosomal exocytosis of HSP70 stimulates monocytic BMP6 expression in Sjögren’s syndrome

doi: 10.1172/JCI152780

Figure Lengend Snippet: ( A ) Venn diagram showing increased expression of potential ligands for TLR4 (listed inside the largest oval) in minor salivary glands (yellow oval), saliva (blue oval), and/or serum/plasma (shown in red) from patients with SS. ( B ) Serum HSP70 and BMP6 levels in patients with SS ( n = 50) and HVs ( n = 30). Boxes represent first, second, and third quartiles. †† P < 0.01, by Wilcoxon test. RFU, relative fluorescence units. ( C and D ) THP1 cells were treated with the indicated recombinant proteins at the indicated concentration for 4 hours. C, control. ( E ) THP1 cells were treated with boiled HSP70 (1 μg/mL) or LPS (100 ng/mL) for 4 hours. ( F ) THP1 cells were pretreated with TLR1/2 antagonist (CUCPT22, 20 μM) or TAK242 (40 μM) 1 hour prior to HSP70 simulation. ( G ) WT, MYD88 –/– , or IRF3 –/– THP1 cells were stimulated with HSP70 for 4 hours. TNFA and BMP6 transcript levels in the cells were quantified after each treatment using the ΔΔCt method relative to ACTB expression. Values shown are mean ± SEM of 3 independent experiments. ** P < 0.01, by Student’s t test with multiple testing correction using Dunnett’s method ( C – G ).

Article Snippet: The following chemical reagents and recombinant proteins were purchased: LPS, ZDEVD, YVAD, and vacuolin-1 (MilliporeSigma); loxoribine, 3M002, and ODN2216 (InvivoGen); ZVAD and HSP60, -70, and -90 (Enzo Life Sciences); CUCPT22 and TAK242 (Tocris Bioscience); IFN-γ and HMGB1 (R&D Systems); and mouse HSP70-neutralizing antibody (Thermo Fisher Scientific, MA3-009).

Techniques: Expressing, Clinical Proteomics, Fluorescence, Recombinant, Concentration Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Lysosomal exocytosis of HSP70 stimulates monocytic BMP6 expression in Sjögren’s syndrome

doi: 10.1172/JCI152780

Figure Lengend Snippet: ( A ) Scatter plot showing enriched gene ontology in patients with SS with high BMP6 expression compared with those with normal BMP6 expression. ( B ) Correlation between LAMP3 and BMP6 transcript expression in minor salivary glands of patients with SS ( n = 43). ( C ) Schematic showing the methods used in the in vitro assays. ( D ) Representative Western blot with the indicated antibodies using lysates of salivary acinar or ductal cells 72 hours after transfection with empty and/or LAMP3 expression plasmids. ( E ) HSP70 concentration in culture supernatant collected 96 hours after transfection. ( F ) THP1 cells were treated with the culture supernatant of acinar or ductal cells with or without CUCPT22 (20 μM) or TAK242 (40 μM). BMP6 transcript levels in THP1 cells were evaluated 20 hours after stimulation using the ΔΔCt method relative to ACTB . Values shown are the mean ± SEM of 3 independent experiments. ** P < 0.01, by Student’s t test with multiple testing correction using Tukey’s method ( E and F ).

Article Snippet: The following chemical reagents and recombinant proteins were purchased: LPS, ZDEVD, YVAD, and vacuolin-1 (MilliporeSigma); loxoribine, 3M002, and ODN2216 (InvivoGen); ZVAD and HSP60, -70, and -90 (Enzo Life Sciences); CUCPT22 and TAK242 (Tocris Bioscience); IFN-γ and HMGB1 (R&D Systems); and mouse HSP70-neutralizing antibody (Thermo Fisher Scientific, MA3-009).

Techniques: Expressing, In Vitro, Western Blot, Transfection, Concentration Assay