Journal: Science Advances
Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure
doi: 10.1126/sciadv.adn5229
Figure Lengend Snippet: ( A ) Immunoblots and quantification of CCR4 in SVF cells. SVF cells from 10-week-old male C57BL/6 mice were treated with vehicle or AZD2098 (CCR4 antagonist, 100 nM) for 2 days ( n = 3 per group). ( B ) mRNA expression of Ucp1 , Cd137 , and Tmem26 in beige adipocytes. SVF cells from 10-week-old C57BL/6 male mice [divided into four groups: control, rCCL22 (10 ng/ml), CCR4 inhibitor (AZD2098), and AZD2098 + rCCL22] were first treated for 4 days and then induced to beige adipocytes for 5 days at 31°C. ( C and D ) Immunoblots (C) and quantification (D) of UCP1 ( n = 3 per group). ( E ) Immunofluorescence of UCP1 in beige adipocytes. Scale bars, 25 μm. ( F ) Schematic representation of CCR4 KO mouse model generation by the CRISPR strategy. For 8-week-old mice, male Cas9 mice were iWAT-injected with AAV-sgRNAs-CCR4 [2 × 10 13 genome copies/ml] to generate a CCR4 deletion mouse model (CCR4 −/− ). Enhanced green fluorescent protein (EGFP)–labeled AAV-sgRNAs-CCR4 virus was injected into the left iWAT of mice, and EGFP was not detected in the right iWAT and other tissues, verifying the successful injection of the virus. ( G ) Immunoblots and quantification of CCR4 in the iWAT SVF cells ( n = 3 per group). Eight-week-old male Cas9 mice were injected with vehicle or AAV-sgRNA-CCR4 virus for 2 weeks. ( H to K ) Immunofluorescence of UCP1 and H&E staining in iWAT (H); mRNA expression of Ucp1 , Cd137 , and Tmem26 in iWAT (I); and immunoblots and quantification of UCP1(J and K, respectively) ( n = 3 to 6 per group). Eight-week-old male Cas9 mice were injected with vehicle or AAV-sgRNA-CCR4 virus for 2 weeks. Then, wild-type or CCR4 knockout mice were injected with vehicle or rCCL22 (20 μg/kg per day) for 2 weeks at 6°C. Scale bars, 50 μm. Data information: Results are presented as means ± SEM. [(A), (B), (D), (G), (I), and (K)] * P ≤ 0.05 and ** P < 0.01 by nonpaired Student’s t test. ns, not significant.
Article Snippet: Total protein lysates (20 μg) were immunoblotted with rabbit anti–p-FAK (Y397) (1:1000; ABclonal, #AP0302), rabbit anti-FAK (1:1000; ABclonal, A11195), rabbit anti-p65 antibody (1:1000; Abcam, ab32536), rabbit anti-CCR4 antibody (1:1000; Novus Biological, NB56336SS), rabbit anti-UCP1 antibody (1:500; Abcam, ab23841), rabbit anti-tubulin antibody [1:2000; Cell Signaling Technology (CST), 2146S], mouse anti-CCL22 antibody (1:1000; R&D Systems, MAB439-SP), mouse anti-CD206 antibody (Bio-Rad, MAC2235GA), rat anti-F4/80 (1:500; Abcam, ab6640), rat anti-siglecF (1:500; Novus Biological, NBP1-91149), rabbit anti–IL-13 antibody (1:500; ABclonal, A2089), rabbit anti-p-STAT6 antibody (1:500; ABclonal, AP0456), rabbit anti-STAT6 antibody (1:500; ABclonal, A0755), rabbit anti-Histone3 antibody (1:1000; CST, 4499P), followed by goat anti-rat horseradish peroxidase (HRP)–conjugated secondary antibody (1:5000; ABconal, AS028), anti-rabbit HRP-conjugated secondary antibody (1:5000; CST, 7074S), goat anti-mouse HRP-conjugated secondary antibody (1:5000; CST, 96714S).
Techniques: Western Blot, Expressing, Control, Immunofluorescence, CRISPR, Injection, Labeling, Virus, Staining, Knock-Out
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![( A ) Immunoblots and quantification of <t>CCR4</t> in SVF cells. SVF cells from 10-week-old male C57BL/6 mice were treated with vehicle or AZD2098 (CCR4 antagonist, 100 nM) for 2 days ( n = 3 per group). ( B ) mRNA expression of Ucp1 , Cd137 , and Tmem26 in beige adipocytes. SVF cells from 10-week-old C57BL/6 male mice [divided into four groups: control, rCCL22 (10 ng/ml), CCR4 inhibitor (AZD2098), and AZD2098 + rCCL22] were first treated for 4 days and then induced to beige adipocytes for 5 days at 31°C. ( C and D ) Immunoblots (C) and quantification (D) of UCP1 ( n = 3 per group). ( E ) Immunofluorescence of UCP1 in beige adipocytes. Scale bars, 25 μm. ( F ) Schematic representation of CCR4 KO mouse model generation by the CRISPR strategy. For 8-week-old mice, male Cas9 mice were iWAT-injected with AAV-sgRNAs-CCR4 [2 × 10 13 genome copies/ml] to generate a CCR4 deletion mouse model (CCR4 −/− ). Enhanced green fluorescent protein (EGFP)–labeled AAV-sgRNAs-CCR4 virus was injected into the left iWAT of mice, and EGFP was not detected in the right iWAT and other tissues, verifying the successful injection of the virus. ( G ) Immunoblots and quantification of CCR4 in the iWAT SVF cells ( n = 3 per group). Eight-week-old male Cas9 mice were injected with vehicle or AAV-sgRNA-CCR4 virus for 2 weeks. ( H to K ) Immunofluorescence of UCP1 and H&E staining in iWAT (H); mRNA expression of Ucp1 , Cd137 , and Tmem26 in iWAT (I); and immunoblots and quantification of UCP1(J and K, respectively) ( n = 3 to 6 per group). Eight-week-old male Cas9 mice were injected with vehicle or AAV-sgRNA-CCR4 virus for 2 weeks. Then, wild-type or CCR4 knockout mice were injected with vehicle or rCCL22 (20 μg/kg per day) for 2 weeks at 6°C. Scale bars, 50 μm. Data information: Results are presented as means ± SEM. [(A), (B), (D), (G), (I), and (K)] * P ≤ 0.05 and ** P < 0.01 by nonpaired Student’s t test. ns, not significant.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4298/pmc11204298/pmc11204298__sciadv.adn5229-f4.jpg)
![( A and B ) Immunoblots and quantification of CCR4 in T reg cells (CD4 + CD25 + CD127 + FoxP3 + ), eosinophils (EO; CD45 + F4/80 + CD11b + SiglecF + ), M2 macrophages (M2; CD45 + CD64 + F4/80 + CD206 + ), dendritic cells (DC; CD11c + I-A/I-E + CD103 + ), SVF cells, and MAs, isolated from five 10-week-old male C57BL/6 mice iWAT by flow cytometry sorting, respectively. ( C and D ) Flow cytometry analysis for eosinophils in iWAT from 10-week-old C57BL/6 male mice receiving sham or LNR for 7 days at 6°C ( n = 4 per group). ( E and F ) Flow cytometry analysis of eosinophils in iWAT from 10-week-old male mice ( n = 4 per group). Littermate control or CCR4 knockout mice were injected with vehicle or rCCL22 (20 μg/kg per day) into bilateral iWAT for 14 days at 6 ° C. ( G ) Immunofluorescence of CCR4 in eosinophils sorted from (E) and (F). Scale bars, 10 μm. ( H to J ) Immunofluorescence (H), immunoblots (I), and quantification (J) of UCP1 in beige adipocytes ( n = 3 per group). Eosinophils from the iWAT SVF cells were sorted, treated with vehicle or rCCL22 (10 ng/ml) for 4 days at 31°C, and subsequently induced to differentiate into beige adipocytes for 5 days. Scale bar, 25 μm. ( K ) Schematic representation picture. Eosinophils (from BMDEs, transferred with vehicle or AAV-sgRNA-CCR4 virus for 3 days) and iWAT SVF cells (sorted out eosinophils) were cocultured for 2 days, treated with vehicle or rCCL22 for 4 days at 31°C and induced to form beige adipocytes for 5 days. SVF cells were obtained from 10-week-old Cas9 male iWAT depots. Eosinophils were obtained from the bone marrow of 10-week-old Cas9 male mice. ( L to N ) Immunofluorescence (L) of UCP1, immunoblots (M), and quantification (N) of CCR4 and UCP1 in eosinophils or beige adipocytes ( n = 3 per group). Scale bars, 25 μm. Data information: Results are presented as means ± SEM. [(D), (F), and (N)] * P ≤ 0.05 by nonpaired Student’s t test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4298/pmc11204298/pmc11204298__sciadv.adn5229-f5.jpg)
![( A and B ) Immunoblots (A) and quantification (B) of CCR4, p-FAK, and p65 protein in eosinophils cultured with a vehicle, AZD2098 (CCR4 inhibitor, 100 nM), rCCL22 (10 ng/ml), or AZD2098 + rCCL22 (10 ng/ml) for 48 hours ( n = 3 per group). Eosinophils were isolated and induced from BMDEs of 10-week-old male C57BL/6 mice receiving 6°C stimulation for 14 days. ( C ) p65 translocation in eosinophil cells cultured with vehicle, AZD2098 (100 nM), rCCL22 (10 ng/ml), or AZD2098 + rCCL22 (10 ng/ml) for 48 hours ( n = 3 per group). Scale bars, 10 μm. ( D and E ) Immunoblots (D) and quantification (E) of p-FAK and p65 protein in eosinophils cultured with vehicle, PF5733228 (FAK inhibitor, 5 μM), rCCL22 (10 ng/ml), or PF5733228 + rCCL22 (10 ng/ml) for 48 hours. ( F to I ) Immunofluorescence (F) of UCP1, mRNA expression of Ucp1 (G), immunoblots (H), and quantification of UCP1 (I) in beige adipocytes ( n = 3 per group). Eosinophils (treated with vehicle or PF573228 for 2 days) and iWAT SVF cells (without eosinophils) were cocultured for 4 days, and beige adipocytes were then induced for 5 days at 31°C. SVF cells were obtained from 10-week-old C57BL/6 male mouse iWAT. Eosinophils were obtained from the BMDMs of 10-week-old C57BL/6 male mice. Scale bar, 25 μm. Data information: Results are presented as means ± SEM. [(B), (E), (G), and (I)] * P ≤ 0.05 and ** P < 0.01 by nonpaired Student’s t test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4298/pmc11204298/pmc11204298__sciadv.adn5229-f6.jpg)