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Tocris acly inhibitor sb204990
Acly Inhibitor Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/acly inhibitor sb204990/product/Tocris
Average 93 stars, based on 1 article reviews
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acly inhibitor sb204990 - by Bioz Stars, 2023-02
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Tocris sb204990 tocris
Sb204990 Tocris, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb204990 tocris/product/Tocris
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb204990 tocris - by Bioz Stars, 2023-02
93/100 stars

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Structured Review

Tocris sb204990
( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with <t>SB204990</t> (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb204990/product/Tocris
Average 94 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb204990 - by Bioz Stars, 2023-02
94/100 stars

Images

1) Product Images from "ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation"

Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

Journal: eLife

doi: 10.7554/eLife.62394

( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
Figure Legend Snippet: ( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

Techniques Used: Activity Assay, Isolation, Cell Culture, Over Expression, Transfection, Flow Cytometry, Inhibition

( A ) Activated T (Th0) and inducible regulatory T (iTreg) cell polarization in vitro. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain Th0 cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Isolation of cytosolic fraction. Cytosolic fraction without mitochondria and nuclei was isolated from cells prepared as in ( A ). ( C ) Enzymatic activity of ATP-citrate lyase (ACLY). Cells were transfected with GFP-ACLY and cultured as in ( A ) for 24 hr. Enzymatic activity of ACLY was measured and quantified. ( D, E ) Assay for ACLY activity in cells treated with si Acly or SB204990. Cells transfected with small interfering RNA (siRNA) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were induced under Th0- or iTreg (with 0.5 ng/ml transforming growth factor β1 [TGFβ1])-polarization condition for 24 hr as in prior to the measurement of enzymatic activity of ACLY. ( A, B ) One representative experiment out of three independent experiments is represented. ( C–E ) Data represent mean ± SD based on three independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. *p<0.05, **p<0.01.
Figure Legend Snippet: ( A ) Activated T (Th0) and inducible regulatory T (iTreg) cell polarization in vitro. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain Th0 cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Isolation of cytosolic fraction. Cytosolic fraction without mitochondria and nuclei was isolated from cells prepared as in ( A ). ( C ) Enzymatic activity of ATP-citrate lyase (ACLY). Cells were transfected with GFP-ACLY and cultured as in ( A ) for 24 hr. Enzymatic activity of ACLY was measured and quantified. ( D, E ) Assay for ACLY activity in cells treated with si Acly or SB204990. Cells transfected with small interfering RNA (siRNA) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were induced under Th0- or iTreg (with 0.5 ng/ml transforming growth factor β1 [TGFβ1])-polarization condition for 24 hr as in prior to the measurement of enzymatic activity of ACLY. ( A, B ) One representative experiment out of three independent experiments is represented. ( C–E ) Data represent mean ± SD based on three independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. *p<0.05, **p<0.01.

Techniques Used: In Vitro, Isolation, Cell Culture, Flow Cytometry, Activity Assay, Transfection, Small Interfering RNA

( A ) ATP-citrate lyase (ACLY) inhibition reduces de novo fatty acid synthesis (FAS). Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured under inducible regulatory T (iTreg)-polarization condition as in for 24 hr in the presence of [U- 13 C] glucose (11 mM). Cells were collected and subjected to metabolic flux analysis for FAS by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) analysis. n = 4, Student's t-test, mean ± SD. *p<0.05, **p<0.01, ****p<0.0001; ns, nonsignificant. ( B, C ) Functional evaluation of metabolic intermediates from FAS on iTreg differentiation. Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured as in in the presence of different doses of palmitate ( B ) or malonyl-CoA ( C ). CD4 + CD25 + Foxp3 + iTreg cells were assayed by flow cytometry (FCM) (left) and quantified (right). ( D, E ) ACLY inhibition increases carnitine palmitoyltransferase 1 (CPT1) activity and fatty acid oxidation (FAO). In the presence of malonyl-CoA (50 μM) or cerulenin (4 μM), naive CD4 + T cells treated with SB204990 were cultured as in for 24 hr. Cell lysates were used for analyzing CPT1 activity ( D ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and carnitine. Diagram (left) illustrating the OCR at various conditions and associated quantifications (right) are shown ( E ). ( F ) Impact of Cpt1 knockdown on iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNA (siRNA) against Cpt1 were cultured as in . CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). ( B–F ) Data represent mean ± SD of three ( D–F ) or four ( B, C ) independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
Figure Legend Snippet: ( A ) ATP-citrate lyase (ACLY) inhibition reduces de novo fatty acid synthesis (FAS). Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured under inducible regulatory T (iTreg)-polarization condition as in for 24 hr in the presence of [U- 13 C] glucose (11 mM). Cells were collected and subjected to metabolic flux analysis for FAS by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) analysis. n = 4, Student's t-test, mean ± SD. *p<0.05, **p<0.01, ****p<0.0001; ns, nonsignificant. ( B, C ) Functional evaluation of metabolic intermediates from FAS on iTreg differentiation. Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured as in in the presence of different doses of palmitate ( B ) or malonyl-CoA ( C ). CD4 + CD25 + Foxp3 + iTreg cells were assayed by flow cytometry (FCM) (left) and quantified (right). ( D, E ) ACLY inhibition increases carnitine palmitoyltransferase 1 (CPT1) activity and fatty acid oxidation (FAO). In the presence of malonyl-CoA (50 μM) or cerulenin (4 μM), naive CD4 + T cells treated with SB204990 were cultured as in for 24 hr. Cell lysates were used for analyzing CPT1 activity ( D ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and carnitine. Diagram (left) illustrating the OCR at various conditions and associated quantifications (right) are shown ( E ). ( F ) Impact of Cpt1 knockdown on iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNA (siRNA) against Cpt1 were cultured as in . CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). ( B–F ) Data represent mean ± SD of three ( D–F ) or four ( B, C ) independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

Techniques Used: Inhibition, Cell Culture, High Performance Liquid Chromatography, Mass Spectrometry, Functional Assay, Flow Cytometry, Activity Assay, Transfection, Small Interfering RNA

( A–D ) Naive CD4 + T cells treated with SB204990 were cultured under inducible regulatory T (iTreg)-polarization condition as in . Cells were lysed for the detection of HMG-CoA ( A ), mevalonate ( B ), and mevalonate-5-pyrophosphate ( C ) by enzyme-linked immunosorbent assay (ELISA) or stained by Filipin III for evaluating cholesterol by flow cytometry (FCM) ( D ). Data represent mean ± SD of four independent experiments, with significance determined by Student's t-test. ns, nonsignificant.
Figure Legend Snippet: ( A–D ) Naive CD4 + T cells treated with SB204990 were cultured under inducible regulatory T (iTreg)-polarization condition as in . Cells were lysed for the detection of HMG-CoA ( A ), mevalonate ( B ), and mevalonate-5-pyrophosphate ( C ) by enzyme-linked immunosorbent assay (ELISA) or stained by Filipin III for evaluating cholesterol by flow cytometry (FCM) ( D ). Data represent mean ± SD of four independent experiments, with significance determined by Student's t-test. ns, nonsignificant.

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

( A ) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). ( B ) ACLY overexpression affects iTreg cell differentiation. Human naive CD4 + T cells transfected with GFP-ACLY WT were polarized as in ( A ) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). ( C ) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. ( D ) ACLY interaction with CUL3-KLHL25. Cells prepared as in ( A ) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. ( E–G ) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLY WT or -ACLY 3KR were induced under Th0- or iTreg-polarization condition as described in ( A ) for 24 hr ( E, F ) or 72 hr ( G ). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination ( E ), examined by WB ( F ), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) ( G ). ( A–C, F, G ) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test ( A, C ) or one-way analysis of variance (ANOVA) test ( B, F, G ). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in ( A ) and ( D–F ), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.
Figure Legend Snippet: ( A ) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). ( B ) ACLY overexpression affects iTreg cell differentiation. Human naive CD4 + T cells transfected with GFP-ACLY WT were polarized as in ( A ) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). ( C ) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. ( D ) ACLY interaction with CUL3-KLHL25. Cells prepared as in ( A ) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. ( E–G ) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLY WT or -ACLY 3KR were induced under Th0- or iTreg-polarization condition as described in ( A ) for 24 hr ( E, F ) or 72 hr ( G ). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination ( E ), examined by WB ( F ), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) ( G ). ( A–C, F, G ) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test ( A, C ) or one-way analysis of variance (ANOVA) test ( B, F, G ). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in ( A ) and ( D–F ), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.

Techniques Used: Expressing, Isolation, Cell Culture, Western Blot, Over Expression, Cell Differentiation, Transfection, Flow Cytometry, Inhibition, Immunoprecipitation, Negative Control

( A, B ) Mice were supplied with dextran sodium sulfate (DSS) (2%, w/v) in drinking water. Meanwhile, ATP-citrate lyase (ACLY) inhibitor (SB204990, 37.5 mg/kg/d) was administrated once every 2 days by oral gavage. 10 days later, regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) ( A ) and colonic lamina propria (cLP) ( B ) were examined by flow cytometry (FCM). ( C–F ) Systematic evaluation of colitis in mice administrated with ACLY inhibitor. Body weight ( C ) and disease activity index (DAI) score ( D ), based on body weight loss, stool consistency, and blood in the stool, were recorded daily. 10 days later, entire colons from mice treated in different ways were removed for length assessment ( E ) and hematoxylin and eosin (H and E) staining for histopathological changes ( F ). Scale bar, 200 μm. In ( F ), one representative experiment out of three biological replicas is represented. ( A–E ) Data represent mean ± SD (n = 3), with significance determined by one-way analysis of variance (ANOVA) test ( A, B, E ) and two-way ANOVA test ( C, D ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure Legend Snippet: ( A, B ) Mice were supplied with dextran sodium sulfate (DSS) (2%, w/v) in drinking water. Meanwhile, ATP-citrate lyase (ACLY) inhibitor (SB204990, 37.5 mg/kg/d) was administrated once every 2 days by oral gavage. 10 days later, regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) ( A ) and colonic lamina propria (cLP) ( B ) were examined by flow cytometry (FCM). ( C–F ) Systematic evaluation of colitis in mice administrated with ACLY inhibitor. Body weight ( C ) and disease activity index (DAI) score ( D ), based on body weight loss, stool consistency, and blood in the stool, were recorded daily. 10 days later, entire colons from mice treated in different ways were removed for length assessment ( E ) and hematoxylin and eosin (H and E) staining for histopathological changes ( F ). Scale bar, 200 μm. In ( F ), one representative experiment out of three biological replicas is represented. ( A–E ) Data represent mean ± SD (n = 3), with significance determined by one-way analysis of variance (ANOVA) test ( A, B, E ) and two-way ANOVA test ( C, D ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Techniques Used: Flow Cytometry, Activity Assay, Staining

( A ) Inhibition of ATP-citrate lyase (ACLY) by SB204990 facilitates inducible regulatory T (iTreg) differentiation. Naive CD4 + T cells treated with SB204990 were induced as in . CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Assay for iTreg inhibitory function in vitro. Cells prepared as in ( A ) were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained naive CD4 + T cells in the presence of Dynabeads Mouse T-Activator CD3/CD28 for 72 hr. CD4 + T-cell proliferation was analyzed by FCM (left) and quantified (right) based on three independent experiments. One-way analysis of variance (ANOVA) test, mean ± SD. **p<0.01. ( C–E ) Systematic evaluation of colitis in mice adoptively transferred with different cells. ( C ) Body weight (left) and disease activity index (DAI) score (right), based on body weight loss, stool consistency, and blood in the stool, were recorded weekly. 7 weeks later, entire colons from mice treated in different ways were removed for length assessment ( D ) and hematoxylin and eosin (H and E) staining for histopathological changes ( E ). Scale bar, 200 μm. ( C, D ) Data represent mean ± SD (n = 4), with significance determined by two-way ANOVA test ( C ) and one-way ANOVA test ( D ). *p<0.05, ***p<0.001, and ****p<0.0001. ( A, E ) One representative experiment out of four biological replicas is represented.
Figure Legend Snippet: ( A ) Inhibition of ATP-citrate lyase (ACLY) by SB204990 facilitates inducible regulatory T (iTreg) differentiation. Naive CD4 + T cells treated with SB204990 were induced as in . CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Assay for iTreg inhibitory function in vitro. Cells prepared as in ( A ) were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained naive CD4 + T cells in the presence of Dynabeads Mouse T-Activator CD3/CD28 for 72 hr. CD4 + T-cell proliferation was analyzed by FCM (left) and quantified (right) based on three independent experiments. One-way analysis of variance (ANOVA) test, mean ± SD. **p<0.01. ( C–E ) Systematic evaluation of colitis in mice adoptively transferred with different cells. ( C ) Body weight (left) and disease activity index (DAI) score (right), based on body weight loss, stool consistency, and blood in the stool, were recorded weekly. 7 weeks later, entire colons from mice treated in different ways were removed for length assessment ( D ) and hematoxylin and eosin (H and E) staining for histopathological changes ( E ). Scale bar, 200 μm. ( C, D ) Data represent mean ± SD (n = 4), with significance determined by two-way ANOVA test ( C ) and one-way ANOVA test ( D ). *p<0.05, ***p<0.001, and ****p<0.0001. ( A, E ) One representative experiment out of four biological replicas is represented.

Techniques Used: Inhibition, Flow Cytometry, In Vitro, Staining, Activity Assay


Figure Legend Snippet:

Techniques Used: Activation Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Isolation, Activity Assay, Recombinant, Plasmid Preparation, Software


Structured Review

Tocris sb204990
( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with <t>SB204990</t> (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb204990/product/Tocris
Average 96 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb204990 - by Bioz Stars, 2023-02
96/100 stars

Images

1) Product Images from "ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation"

Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

Journal: eLife

doi: 10.7554/eLife.62394

( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
Figure Legend Snippet: ( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

Techniques Used: Activity Assay, Isolation, Cell Culture, Over Expression, Transfection, Flow Cytometry, Inhibition

( A ) Activated T (Th0) and inducible regulatory T (iTreg) cell polarization in vitro. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain Th0 cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Isolation of cytosolic fraction. Cytosolic fraction without mitochondria and nuclei was isolated from cells prepared as in ( A ). ( C ) Enzymatic activity of ATP-citrate lyase (ACLY). Cells were transfected with GFP-ACLY and cultured as in ( A ) for 24 hr. Enzymatic activity of ACLY was measured and quantified. ( D, E ) Assay for ACLY activity in cells treated with si Acly or SB204990. Cells transfected with small interfering RNA (siRNA) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were induced under Th0- or iTreg (with 0.5 ng/ml transforming growth factor β1 [TGFβ1])-polarization condition for 24 hr as in prior to the measurement of enzymatic activity of ACLY. ( A, B ) One representative experiment out of three independent experiments is represented. ( C–E ) Data represent mean ± SD based on three independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. *p<0.05, **p<0.01.
Figure Legend Snippet: ( A ) Activated T (Th0) and inducible regulatory T (iTreg) cell polarization in vitro. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain Th0 cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Isolation of cytosolic fraction. Cytosolic fraction without mitochondria and nuclei was isolated from cells prepared as in ( A ). ( C ) Enzymatic activity of ATP-citrate lyase (ACLY). Cells were transfected with GFP-ACLY and cultured as in ( A ) for 24 hr. Enzymatic activity of ACLY was measured and quantified. ( D, E ) Assay for ACLY activity in cells treated with si Acly or SB204990. Cells transfected with small interfering RNA (siRNA) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were induced under Th0- or iTreg (with 0.5 ng/ml transforming growth factor β1 [TGFβ1])-polarization condition for 24 hr as in prior to the measurement of enzymatic activity of ACLY. ( A, B ) One representative experiment out of three independent experiments is represented. ( C–E ) Data represent mean ± SD based on three independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. *p<0.05, **p<0.01.

Techniques Used: In Vitro, Isolation, Cell Culture, Flow Cytometry, Activity Assay, Transfection, Small Interfering RNA

( A ) ATP-citrate lyase (ACLY) inhibition reduces de novo fatty acid synthesis (FAS). Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured under inducible regulatory T (iTreg)-polarization condition as in for 24 hr in the presence of [U- 13 C] glucose (11 mM). Cells were collected and subjected to metabolic flux analysis for FAS by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) analysis. n = 4, Student's t-test, mean ± SD. *p<0.05, **p<0.01, ****p<0.0001; ns, nonsignificant. ( B, C ) Functional evaluation of metabolic intermediates from FAS on iTreg differentiation. Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured as in in the presence of different doses of palmitate ( B ) or malonyl-CoA ( C ). CD4 + CD25 + Foxp3 + iTreg cells were assayed by flow cytometry (FCM) (left) and quantified (right). ( D, E ) ACLY inhibition increases carnitine palmitoyltransferase 1 (CPT1) activity and fatty acid oxidation (FAO). In the presence of malonyl-CoA (50 μM) or cerulenin (4 μM), naive CD4 + T cells treated with SB204990 were cultured as in for 24 hr. Cell lysates were used for analyzing CPT1 activity ( D ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and carnitine. Diagram (left) illustrating the OCR at various conditions and associated quantifications (right) are shown ( E ). ( F ) Impact of Cpt1 knockdown on iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNA (siRNA) against Cpt1 were cultured as in . CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). ( B–F ) Data represent mean ± SD of three ( D–F ) or four ( B, C ) independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
Figure Legend Snippet: ( A ) ATP-citrate lyase (ACLY) inhibition reduces de novo fatty acid synthesis (FAS). Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured under inducible regulatory T (iTreg)-polarization condition as in for 24 hr in the presence of [U- 13 C] glucose (11 mM). Cells were collected and subjected to metabolic flux analysis for FAS by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) analysis. n = 4, Student's t-test, mean ± SD. *p<0.05, **p<0.01, ****p<0.0001; ns, nonsignificant. ( B, C ) Functional evaluation of metabolic intermediates from FAS on iTreg differentiation. Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured as in in the presence of different doses of palmitate ( B ) or malonyl-CoA ( C ). CD4 + CD25 + Foxp3 + iTreg cells were assayed by flow cytometry (FCM) (left) and quantified (right). ( D, E ) ACLY inhibition increases carnitine palmitoyltransferase 1 (CPT1) activity and fatty acid oxidation (FAO). In the presence of malonyl-CoA (50 μM) or cerulenin (4 μM), naive CD4 + T cells treated with SB204990 were cultured as in for 24 hr. Cell lysates were used for analyzing CPT1 activity ( D ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and carnitine. Diagram (left) illustrating the OCR at various conditions and associated quantifications (right) are shown ( E ). ( F ) Impact of Cpt1 knockdown on iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNA (siRNA) against Cpt1 were cultured as in . CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). ( B–F ) Data represent mean ± SD of three ( D–F ) or four ( B, C ) independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

Techniques Used: Inhibition, Cell Culture, High Performance Liquid Chromatography, Mass Spectrometry, Functional Assay, Flow Cytometry, Activity Assay, Transfection, Small Interfering RNA

( A–D ) Naive CD4 + T cells treated with SB204990 were cultured under inducible regulatory T (iTreg)-polarization condition as in . Cells were lysed for the detection of HMG-CoA ( A ), mevalonate ( B ), and mevalonate-5-pyrophosphate ( C ) by enzyme-linked immunosorbent assay (ELISA) or stained by Filipin III for evaluating cholesterol by flow cytometry (FCM) ( D ). Data represent mean ± SD of four independent experiments, with significance determined by Student's t-test. ns, nonsignificant.
Figure Legend Snippet: ( A–D ) Naive CD4 + T cells treated with SB204990 were cultured under inducible regulatory T (iTreg)-polarization condition as in . Cells were lysed for the detection of HMG-CoA ( A ), mevalonate ( B ), and mevalonate-5-pyrophosphate ( C ) by enzyme-linked immunosorbent assay (ELISA) or stained by Filipin III for evaluating cholesterol by flow cytometry (FCM) ( D ). Data represent mean ± SD of four independent experiments, with significance determined by Student's t-test. ns, nonsignificant.

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

( A ) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). ( B ) ACLY overexpression affects iTreg cell differentiation. Human naive CD4 + T cells transfected with GFP-ACLY WT were polarized as in ( A ) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). ( C ) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. ( D ) ACLY interaction with CUL3-KLHL25. Cells prepared as in ( A ) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. ( E–G ) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLY WT or -ACLY 3KR were induced under Th0- or iTreg-polarization condition as described in ( A ) for 24 hr ( E, F ) or 72 hr ( G ). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination ( E ), examined by WB ( F ), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) ( G ). ( A–C, F, G ) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test ( A, C ) or one-way analysis of variance (ANOVA) test ( B, F, G ). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in ( A ) and ( D–F ), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.
Figure Legend Snippet: ( A ) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). ( B ) ACLY overexpression affects iTreg cell differentiation. Human naive CD4 + T cells transfected with GFP-ACLY WT were polarized as in ( A ) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). ( C ) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. ( D ) ACLY interaction with CUL3-KLHL25. Cells prepared as in ( A ) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. ( E–G ) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLY WT or -ACLY 3KR were induced under Th0- or iTreg-polarization condition as described in ( A ) for 24 hr ( E, F ) or 72 hr ( G ). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination ( E ), examined by WB ( F ), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) ( G ). ( A–C, F, G ) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test ( A, C ) or one-way analysis of variance (ANOVA) test ( B, F, G ). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in ( A ) and ( D–F ), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.

Techniques Used: Expressing, Isolation, Cell Culture, Western Blot, Over Expression, Cell Differentiation, Transfection, Flow Cytometry, Inhibition, Immunoprecipitation, Negative Control

( A, B ) Mice were supplied with dextran sodium sulfate (DSS) (2%, w/v) in drinking water. Meanwhile, ATP-citrate lyase (ACLY) inhibitor (SB204990, 37.5 mg/kg/d) was administrated once every 2 days by oral gavage. 10 days later, regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) ( A ) and colonic lamina propria (cLP) ( B ) were examined by flow cytometry (FCM). ( C–F ) Systematic evaluation of colitis in mice administrated with ACLY inhibitor. Body weight ( C ) and disease activity index (DAI) score ( D ), based on body weight loss, stool consistency, and blood in the stool, were recorded daily. 10 days later, entire colons from mice treated in different ways were removed for length assessment ( E ) and hematoxylin and eosin (H and E) staining for histopathological changes ( F ). Scale bar, 200 μm. In ( F ), one representative experiment out of three biological replicas is represented. ( A–E ) Data represent mean ± SD (n = 3), with significance determined by one-way analysis of variance (ANOVA) test ( A, B, E ) and two-way ANOVA test ( C, D ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure Legend Snippet: ( A, B ) Mice were supplied with dextran sodium sulfate (DSS) (2%, w/v) in drinking water. Meanwhile, ATP-citrate lyase (ACLY) inhibitor (SB204990, 37.5 mg/kg/d) was administrated once every 2 days by oral gavage. 10 days later, regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) ( A ) and colonic lamina propria (cLP) ( B ) were examined by flow cytometry (FCM). ( C–F ) Systematic evaluation of colitis in mice administrated with ACLY inhibitor. Body weight ( C ) and disease activity index (DAI) score ( D ), based on body weight loss, stool consistency, and blood in the stool, were recorded daily. 10 days later, entire colons from mice treated in different ways were removed for length assessment ( E ) and hematoxylin and eosin (H and E) staining for histopathological changes ( F ). Scale bar, 200 μm. In ( F ), one representative experiment out of three biological replicas is represented. ( A–E ) Data represent mean ± SD (n = 3), with significance determined by one-way analysis of variance (ANOVA) test ( A, B, E ) and two-way ANOVA test ( C, D ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Techniques Used: Flow Cytometry, Activity Assay, Staining

( A ) Inhibition of ATP-citrate lyase (ACLY) by SB204990 facilitates inducible regulatory T (iTreg) differentiation. Naive CD4 + T cells treated with SB204990 were induced as in . CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Assay for iTreg inhibitory function in vitro. Cells prepared as in ( A ) were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained naive CD4 + T cells in the presence of Dynabeads Mouse T-Activator CD3/CD28 for 72 hr. CD4 + T-cell proliferation was analyzed by FCM (left) and quantified (right) based on three independent experiments. One-way analysis of variance (ANOVA) test, mean ± SD. **p<0.01. ( C–E ) Systematic evaluation of colitis in mice adoptively transferred with different cells. ( C ) Body weight (left) and disease activity index (DAI) score (right), based on body weight loss, stool consistency, and blood in the stool, were recorded weekly. 7 weeks later, entire colons from mice treated in different ways were removed for length assessment ( D ) and hematoxylin and eosin (H and E) staining for histopathological changes ( E ). Scale bar, 200 μm. ( C, D ) Data represent mean ± SD (n = 4), with significance determined by two-way ANOVA test ( C ) and one-way ANOVA test ( D ). *p<0.05, ***p<0.001, and ****p<0.0001. ( A, E ) One representative experiment out of four biological replicas is represented.
Figure Legend Snippet: ( A ) Inhibition of ATP-citrate lyase (ACLY) by SB204990 facilitates inducible regulatory T (iTreg) differentiation. Naive CD4 + T cells treated with SB204990 were induced as in . CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Assay for iTreg inhibitory function in vitro. Cells prepared as in ( A ) were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained naive CD4 + T cells in the presence of Dynabeads Mouse T-Activator CD3/CD28 for 72 hr. CD4 + T-cell proliferation was analyzed by FCM (left) and quantified (right) based on three independent experiments. One-way analysis of variance (ANOVA) test, mean ± SD. **p<0.01. ( C–E ) Systematic evaluation of colitis in mice adoptively transferred with different cells. ( C ) Body weight (left) and disease activity index (DAI) score (right), based on body weight loss, stool consistency, and blood in the stool, were recorded weekly. 7 weeks later, entire colons from mice treated in different ways were removed for length assessment ( D ) and hematoxylin and eosin (H and E) staining for histopathological changes ( E ). Scale bar, 200 μm. ( C, D ) Data represent mean ± SD (n = 4), with significance determined by two-way ANOVA test ( C ) and one-way ANOVA test ( D ). *p<0.05, ***p<0.001, and ****p<0.0001. ( A, E ) One representative experiment out of four biological replicas is represented.

Techniques Used: Inhibition, Flow Cytometry, In Vitro, Staining, Activity Assay


Figure Legend Snippet:

Techniques Used: Activation Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Isolation, Activity Assay, Recombinant, Plasmid Preparation, Software


Structured Review

Tocris sb204990
( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with <t>SB204990</t> (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
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1) Product Images from "ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation"

Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

Journal: eLife

doi: 10.7554/eLife.62394

( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
Figure Legend Snippet: ( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

Techniques Used: Activity Assay, Isolation, Cell Culture, Over Expression, Transfection, Flow Cytometry, Inhibition

( A ) Activated T (Th0) and inducible regulatory T (iTreg) cell polarization in vitro. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain Th0 cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Isolation of cytosolic fraction. Cytosolic fraction without mitochondria and nuclei was isolated from cells prepared as in ( A ). ( C ) Enzymatic activity of ATP-citrate lyase (ACLY). Cells were transfected with GFP-ACLY and cultured as in ( A ) for 24 hr. Enzymatic activity of ACLY was measured and quantified. ( D, E ) Assay for ACLY activity in cells treated with si Acly or SB204990. Cells transfected with small interfering RNA (siRNA) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were induced under Th0- or iTreg (with 0.5 ng/ml transforming growth factor β1 [TGFβ1])-polarization condition for 24 hr as in prior to the measurement of enzymatic activity of ACLY. ( A, B ) One representative experiment out of three independent experiments is represented. ( C–E ) Data represent mean ± SD based on three independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. *p<0.05, **p<0.01.
Figure Legend Snippet: ( A ) Activated T (Th0) and inducible regulatory T (iTreg) cell polarization in vitro. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain Th0 cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Isolation of cytosolic fraction. Cytosolic fraction without mitochondria and nuclei was isolated from cells prepared as in ( A ). ( C ) Enzymatic activity of ATP-citrate lyase (ACLY). Cells were transfected with GFP-ACLY and cultured as in ( A ) for 24 hr. Enzymatic activity of ACLY was measured and quantified. ( D, E ) Assay for ACLY activity in cells treated with si Acly or SB204990. Cells transfected with small interfering RNA (siRNA) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were induced under Th0- or iTreg (with 0.5 ng/ml transforming growth factor β1 [TGFβ1])-polarization condition for 24 hr as in prior to the measurement of enzymatic activity of ACLY. ( A, B ) One representative experiment out of three independent experiments is represented. ( C–E ) Data represent mean ± SD based on three independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. *p<0.05, **p<0.01.

Techniques Used: In Vitro, Isolation, Cell Culture, Flow Cytometry, Activity Assay, Transfection, Small Interfering RNA

( A ) ATP-citrate lyase (ACLY) inhibition reduces de novo fatty acid synthesis (FAS). Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured under inducible regulatory T (iTreg)-polarization condition as in for 24 hr in the presence of [U- 13 C] glucose (11 mM). Cells were collected and subjected to metabolic flux analysis for FAS by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) analysis. n = 4, Student's t-test, mean ± SD. *p<0.05, **p<0.01, ****p<0.0001; ns, nonsignificant. ( B, C ) Functional evaluation of metabolic intermediates from FAS on iTreg differentiation. Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured as in in the presence of different doses of palmitate ( B ) or malonyl-CoA ( C ). CD4 + CD25 + Foxp3 + iTreg cells were assayed by flow cytometry (FCM) (left) and quantified (right). ( D, E ) ACLY inhibition increases carnitine palmitoyltransferase 1 (CPT1) activity and fatty acid oxidation (FAO). In the presence of malonyl-CoA (50 μM) or cerulenin (4 μM), naive CD4 + T cells treated with SB204990 were cultured as in for 24 hr. Cell lysates were used for analyzing CPT1 activity ( D ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and carnitine. Diagram (left) illustrating the OCR at various conditions and associated quantifications (right) are shown ( E ). ( F ) Impact of Cpt1 knockdown on iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNA (siRNA) against Cpt1 were cultured as in . CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). ( B–F ) Data represent mean ± SD of three ( D–F ) or four ( B, C ) independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
Figure Legend Snippet: ( A ) ATP-citrate lyase (ACLY) inhibition reduces de novo fatty acid synthesis (FAS). Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured under inducible regulatory T (iTreg)-polarization condition as in for 24 hr in the presence of [U- 13 C] glucose (11 mM). Cells were collected and subjected to metabolic flux analysis for FAS by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) analysis. n = 4, Student's t-test, mean ± SD. *p<0.05, **p<0.01, ****p<0.0001; ns, nonsignificant. ( B, C ) Functional evaluation of metabolic intermediates from FAS on iTreg differentiation. Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured as in in the presence of different doses of palmitate ( B ) or malonyl-CoA ( C ). CD4 + CD25 + Foxp3 + iTreg cells were assayed by flow cytometry (FCM) (left) and quantified (right). ( D, E ) ACLY inhibition increases carnitine palmitoyltransferase 1 (CPT1) activity and fatty acid oxidation (FAO). In the presence of malonyl-CoA (50 μM) or cerulenin (4 μM), naive CD4 + T cells treated with SB204990 were cultured as in for 24 hr. Cell lysates were used for analyzing CPT1 activity ( D ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and carnitine. Diagram (left) illustrating the OCR at various conditions and associated quantifications (right) are shown ( E ). ( F ) Impact of Cpt1 knockdown on iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNA (siRNA) against Cpt1 were cultured as in . CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). ( B–F ) Data represent mean ± SD of three ( D–F ) or four ( B, C ) independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

Techniques Used: Inhibition, Cell Culture, High Performance Liquid Chromatography, Mass Spectrometry, Functional Assay, Flow Cytometry, Activity Assay, Transfection, Small Interfering RNA

( A–D ) Naive CD4 + T cells treated with SB204990 were cultured under inducible regulatory T (iTreg)-polarization condition as in . Cells were lysed for the detection of HMG-CoA ( A ), mevalonate ( B ), and mevalonate-5-pyrophosphate ( C ) by enzyme-linked immunosorbent assay (ELISA) or stained by Filipin III for evaluating cholesterol by flow cytometry (FCM) ( D ). Data represent mean ± SD of four independent experiments, with significance determined by Student's t-test. ns, nonsignificant.
Figure Legend Snippet: ( A–D ) Naive CD4 + T cells treated with SB204990 were cultured under inducible regulatory T (iTreg)-polarization condition as in . Cells were lysed for the detection of HMG-CoA ( A ), mevalonate ( B ), and mevalonate-5-pyrophosphate ( C ) by enzyme-linked immunosorbent assay (ELISA) or stained by Filipin III for evaluating cholesterol by flow cytometry (FCM) ( D ). Data represent mean ± SD of four independent experiments, with significance determined by Student's t-test. ns, nonsignificant.

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

( A ) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). ( B ) ACLY overexpression affects iTreg cell differentiation. Human naive CD4 + T cells transfected with GFP-ACLY WT were polarized as in ( A ) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). ( C ) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. ( D ) ACLY interaction with CUL3-KLHL25. Cells prepared as in ( A ) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. ( E–G ) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLY WT or -ACLY 3KR were induced under Th0- or iTreg-polarization condition as described in ( A ) for 24 hr ( E, F ) or 72 hr ( G ). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination ( E ), examined by WB ( F ), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) ( G ). ( A–C, F, G ) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test ( A, C ) or one-way analysis of variance (ANOVA) test ( B, F, G ). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in ( A ) and ( D–F ), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.
Figure Legend Snippet: ( A ) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). ( B ) ACLY overexpression affects iTreg cell differentiation. Human naive CD4 + T cells transfected with GFP-ACLY WT were polarized as in ( A ) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). ( C ) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. ( D ) ACLY interaction with CUL3-KLHL25. Cells prepared as in ( A ) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. ( E–G ) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLY WT or -ACLY 3KR were induced under Th0- or iTreg-polarization condition as described in ( A ) for 24 hr ( E, F ) or 72 hr ( G ). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination ( E ), examined by WB ( F ), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) ( G ). ( A–C, F, G ) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test ( A, C ) or one-way analysis of variance (ANOVA) test ( B, F, G ). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in ( A ) and ( D–F ), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.

Techniques Used: Expressing, Isolation, Cell Culture, Western Blot, Over Expression, Cell Differentiation, Transfection, Flow Cytometry, Inhibition, Immunoprecipitation, Negative Control

( A, B ) Mice were supplied with dextran sodium sulfate (DSS) (2%, w/v) in drinking water. Meanwhile, ATP-citrate lyase (ACLY) inhibitor (SB204990, 37.5 mg/kg/d) was administrated once every 2 days by oral gavage. 10 days later, regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) ( A ) and colonic lamina propria (cLP) ( B ) were examined by flow cytometry (FCM). ( C–F ) Systematic evaluation of colitis in mice administrated with ACLY inhibitor. Body weight ( C ) and disease activity index (DAI) score ( D ), based on body weight loss, stool consistency, and blood in the stool, were recorded daily. 10 days later, entire colons from mice treated in different ways were removed for length assessment ( E ) and hematoxylin and eosin (H and E) staining for histopathological changes ( F ). Scale bar, 200 μm. In ( F ), one representative experiment out of three biological replicas is represented. ( A–E ) Data represent mean ± SD (n = 3), with significance determined by one-way analysis of variance (ANOVA) test ( A, B, E ) and two-way ANOVA test ( C, D ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.
Figure Legend Snippet: ( A, B ) Mice were supplied with dextran sodium sulfate (DSS) (2%, w/v) in drinking water. Meanwhile, ATP-citrate lyase (ACLY) inhibitor (SB204990, 37.5 mg/kg/d) was administrated once every 2 days by oral gavage. 10 days later, regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) ( A ) and colonic lamina propria (cLP) ( B ) were examined by flow cytometry (FCM). ( C–F ) Systematic evaluation of colitis in mice administrated with ACLY inhibitor. Body weight ( C ) and disease activity index (DAI) score ( D ), based on body weight loss, stool consistency, and blood in the stool, were recorded daily. 10 days later, entire colons from mice treated in different ways were removed for length assessment ( E ) and hematoxylin and eosin (H and E) staining for histopathological changes ( F ). Scale bar, 200 μm. In ( F ), one representative experiment out of three biological replicas is represented. ( A–E ) Data represent mean ± SD (n = 3), with significance determined by one-way analysis of variance (ANOVA) test ( A, B, E ) and two-way ANOVA test ( C, D ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Techniques Used: Flow Cytometry, Activity Assay, Staining

( A ) Inhibition of ATP-citrate lyase (ACLY) by SB204990 facilitates inducible regulatory T (iTreg) differentiation. Naive CD4 + T cells treated with SB204990 were induced as in . CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Assay for iTreg inhibitory function in vitro. Cells prepared as in ( A ) were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained naive CD4 + T cells in the presence of Dynabeads Mouse T-Activator CD3/CD28 for 72 hr. CD4 + T-cell proliferation was analyzed by FCM (left) and quantified (right) based on three independent experiments. One-way analysis of variance (ANOVA) test, mean ± SD. **p<0.01. ( C–E ) Systematic evaluation of colitis in mice adoptively transferred with different cells. ( C ) Body weight (left) and disease activity index (DAI) score (right), based on body weight loss, stool consistency, and blood in the stool, were recorded weekly. 7 weeks later, entire colons from mice treated in different ways were removed for length assessment ( D ) and hematoxylin and eosin (H and E) staining for histopathological changes ( E ). Scale bar, 200 μm. ( C, D ) Data represent mean ± SD (n = 4), with significance determined by two-way ANOVA test ( C ) and one-way ANOVA test ( D ). *p<0.05, ***p<0.001, and ****p<0.0001. ( A, E ) One representative experiment out of four biological replicas is represented.
Figure Legend Snippet: ( A ) Inhibition of ATP-citrate lyase (ACLY) by SB204990 facilitates inducible regulatory T (iTreg) differentiation. Naive CD4 + T cells treated with SB204990 were induced as in . CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Assay for iTreg inhibitory function in vitro. Cells prepared as in ( A ) were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained naive CD4 + T cells in the presence of Dynabeads Mouse T-Activator CD3/CD28 for 72 hr. CD4 + T-cell proliferation was analyzed by FCM (left) and quantified (right) based on three independent experiments. One-way analysis of variance (ANOVA) test, mean ± SD. **p<0.01. ( C–E ) Systematic evaluation of colitis in mice adoptively transferred with different cells. ( C ) Body weight (left) and disease activity index (DAI) score (right), based on body weight loss, stool consistency, and blood in the stool, were recorded weekly. 7 weeks later, entire colons from mice treated in different ways were removed for length assessment ( D ) and hematoxylin and eosin (H and E) staining for histopathological changes ( E ). Scale bar, 200 μm. ( C, D ) Data represent mean ± SD (n = 4), with significance determined by two-way ANOVA test ( C ) and one-way ANOVA test ( D ). *p<0.05, ***p<0.001, and ****p<0.0001. ( A, E ) One representative experiment out of four biological replicas is represented.

Techniques Used: Inhibition, Flow Cytometry, In Vitro, Staining, Activity Assay


Figure Legend Snippet:

Techniques Used: Activation Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Isolation, Activity Assay, Recombinant, Plasmid Preparation, Software


Structured Review

Tocris sb204990
Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb204990/product/Tocris
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb204990 - by Bioz Stars, 2023-02
93/100 stars

Images


Structured Review

Tocris sb204990
A, ACL specific inhibitors BMS303141 and <t>SB204990</t> decreased transdifferentiation yield. (n=3) B, ACL siRNA impaired transdifferentiation efficiency. (n=3) C, ACL knockdown reduced the effect of PolyI:C to induce glycolysis as reflected by ECAR measured by Seahorse assay. (n=3) D, LDHA inhibitor reduces the nuclear level of ACL induced by PolyI:C. (n=3) E. Schematic model of the control of glycolytic switch in transdifferentiation. Innate immune signaling induces a glycolytic switch and glucose is used to generate lactate or citrate, the latter is transported to the nucleus. There, it will be converted into acetyl-coA by ACL, and to support histone acetyl-coA and transdifferentiation. (*, 0.01 <p< 0.05; **, 0.001<p<0.01)
Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb204990/product/Tocris
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb204990 - by Bioz Stars, 2023-02
86/100 stars

Images

1) Product Images from "A Glycolytic Switch is Required for Transdifferentiation to Endothelial Lineage"

Article Title: A Glycolytic Switch is Required for Transdifferentiation to Endothelial Lineage

Journal: Circulation

doi: 10.1161/CIRCULATIONAHA.118.035741

A, ACL specific inhibitors BMS303141 and SB204990 decreased transdifferentiation yield. (n=3) B, ACL siRNA impaired transdifferentiation efficiency. (n=3) C, ACL knockdown reduced the effect of PolyI:C to induce glycolysis as reflected by ECAR measured by Seahorse assay. (n=3) D, LDHA inhibitor reduces the nuclear level of ACL induced by PolyI:C. (n=3) E. Schematic model of the control of glycolytic switch in transdifferentiation. Innate immune signaling induces a glycolytic switch and glucose is used to generate lactate or citrate, the latter is transported to the nucleus. There, it will be converted into acetyl-coA by ACL, and to support histone acetyl-coA and transdifferentiation. (*, 0.01 <p< 0.05; **, 0.001<p<0.01)
Figure Legend Snippet: A, ACL specific inhibitors BMS303141 and SB204990 decreased transdifferentiation yield. (n=3) B, ACL siRNA impaired transdifferentiation efficiency. (n=3) C, ACL knockdown reduced the effect of PolyI:C to induce glycolysis as reflected by ECAR measured by Seahorse assay. (n=3) D, LDHA inhibitor reduces the nuclear level of ACL induced by PolyI:C. (n=3) E. Schematic model of the control of glycolytic switch in transdifferentiation. Innate immune signaling induces a glycolytic switch and glucose is used to generate lactate or citrate, the latter is transported to the nucleus. There, it will be converted into acetyl-coA by ACL, and to support histone acetyl-coA and transdifferentiation. (*, 0.01

Techniques Used:


Structured Review

Tocris sb204990
A, ACL specific inhibitors BMS303141 and <t>SB204990</t> decreased transdifferentiation yield. (n=3) B, ACL siRNA impaired transdifferentiation efficiency. (n=3) C, ACL knockdown reduced the effect of PolyI:C to induce glycolysis as reflected by ECAR measured by Seahorse assay. (n=3) D, LDHA inhibitor reduces the nuclear level of ACL induced by PolyI:C. (n=3) E. Schematic model of the control of glycolytic switch in transdifferentiation. Innate immune signaling induces a glycolytic switch and glucose is used to generate lactate or citrate, the latter is transported to the nucleus. There, it will be converted into acetyl-coA by ACL, and to support histone acetyl-coA and transdifferentiation. (*, 0.01 <p< 0.05; **, 0.001<p<0.01)
Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb204990/product/Tocris
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb204990 - by Bioz Stars, 2023-02
86/100 stars

Images

1) Product Images from "A Glycolytic Switch is Required for Transdifferentiation to Endothelial Lineage"

Article Title: A Glycolytic Switch is Required for Transdifferentiation to Endothelial Lineage

Journal: Circulation

doi: 10.1161/CIRCULATIONAHA.118.035741

A, ACL specific inhibitors BMS303141 and SB204990 decreased transdifferentiation yield. (n=3) B, ACL siRNA impaired transdifferentiation efficiency. (n=3) C, ACL knockdown reduced the effect of PolyI:C to induce glycolysis as reflected by ECAR measured by Seahorse assay. (n=3) D, LDHA inhibitor reduces the nuclear level of ACL induced by PolyI:C. (n=3) E. Schematic model of the control of glycolytic switch in transdifferentiation. Innate immune signaling induces a glycolytic switch and glucose is used to generate lactate or citrate, the latter is transported to the nucleus. There, it will be converted into acetyl-coA by ACL, and to support histone acetyl-coA and transdifferentiation. (*, 0.01 <p< 0.05; **, 0.001<p<0.01)
Figure Legend Snippet: A, ACL specific inhibitors BMS303141 and SB204990 decreased transdifferentiation yield. (n=3) B, ACL siRNA impaired transdifferentiation efficiency. (n=3) C, ACL knockdown reduced the effect of PolyI:C to induce glycolysis as reflected by ECAR measured by Seahorse assay. (n=3) D, LDHA inhibitor reduces the nuclear level of ACL induced by PolyI:C. (n=3) E. Schematic model of the control of glycolytic switch in transdifferentiation. Innate immune signaling induces a glycolytic switch and glucose is used to generate lactate or citrate, the latter is transported to the nucleus. There, it will be converted into acetyl-coA by ACL, and to support histone acetyl-coA and transdifferentiation. (*, 0.01

Techniques Used:


Structured Review

Tocris sb204990
FASN deletion leads to ATP citrate lyase (ACLY) inhibition, blocking 3D growth. a Representative pictures of the spheroids recovered from the different genotypes growing under ultralow attachment conditions for 72 h (left). Spheroid diameter ( n = 30 spheroids, FASN lox/lox -PyMT; n = 24 FASN ∆/∆ -PyMT) and number of cells over 72 h (right). Scale bars, 500 μM. Presented data are the mean values ± SD. *** P < 0.001; Student’s t test. b OCR assay performed with cells recovered from the previous experiment and monolayer cultures. Oligomycin, FCCP, and antimycin/rotenone were added at the indicated time point. Data are represented as the means ± SEM. c Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in monolayer compared with 3D conditions in PyMT MEFs loaded with MitoSOX Red (FASN lox/lox -PyMT, n = 4; FASN ∆/∆ -PyMT, n = 5). The quantification of MitoSOX Red fluorescence intensity is shown on the left. Presented data are the mean values ± SD. *** P < 0.001, Student’s t test. d Quantification of MitoSOX Red fluorescence intensity by flow cytometry analysis in FASN lox/lox -PyMT, FASN ∆/∆ -PyMT, and DMNQ-treated (10 μM; 20 min) FASN lox/lox -PyMT MEFs ( n = 3). e Effect of 10 μM DMNQ treatment in the ability of FASN lox/lox -PyMT MEFs to form spheroids. Scale bars, 500 μM. Representative pictures for each condition are shown. f Representative pictures showing the effects of sodium citrate (5 mM) or <t>SB204990</t> (30 μM) in the formation of tumor spheroids by FASN lox/lox -PyMT MEFs. Scale bars, 500 μM. The lower charts show an increase in ROS production by sodium citrate and SB204990. Representative flow cytometry analysis was shown ( n = 4). g Effects in OCR suppression caused by sodium citrate or SB204990 treatment in FASN lox/lox -PyMT MEFs. Presented data are the mean values ± SEM. Three independent experiments ( h ) ACLY activity in FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT MEFs. i , j Cell viability was determined in FASN lox/lox -PyMT MEFs at 24, 48, and 72 h in the presence of various concentrations of SB204990 ( n = 8) ( i ) or sodium citrate ( n = 7) ( j ) under 2D culture conditions. In h – j represented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; Student’s t test
Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb204990/product/Tocris
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb204990 - by Bioz Stars, 2023-02
86/100 stars

Images

1) Product Images from "Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells"

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

Journal: Nature Communications

doi: 10.1038/s41467-019-13028-1

FASN deletion leads to ATP citrate lyase (ACLY) inhibition, blocking 3D growth. a Representative pictures of the spheroids recovered from the different genotypes growing under ultralow attachment conditions for 72 h (left). Spheroid diameter ( n = 30 spheroids, FASN lox/lox -PyMT; n = 24 FASN ∆/∆ -PyMT) and number of cells over 72 h (right). Scale bars, 500 μM. Presented data are the mean values ± SD. *** P < 0.001; Student’s t test. b OCR assay performed with cells recovered from the previous experiment and monolayer cultures. Oligomycin, FCCP, and antimycin/rotenone were added at the indicated time point. Data are represented as the means ± SEM. c Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in monolayer compared with 3D conditions in PyMT MEFs loaded with MitoSOX Red (FASN lox/lox -PyMT, n = 4; FASN ∆/∆ -PyMT, n = 5). The quantification of MitoSOX Red fluorescence intensity is shown on the left. Presented data are the mean values ± SD. *** P < 0.001, Student’s t test. d Quantification of MitoSOX Red fluorescence intensity by flow cytometry analysis in FASN lox/lox -PyMT, FASN ∆/∆ -PyMT, and DMNQ-treated (10 μM; 20 min) FASN lox/lox -PyMT MEFs ( n = 3). e Effect of 10 μM DMNQ treatment in the ability of FASN lox/lox -PyMT MEFs to form spheroids. Scale bars, 500 μM. Representative pictures for each condition are shown. f Representative pictures showing the effects of sodium citrate (5 mM) or SB204990 (30 μM) in the formation of tumor spheroids by FASN lox/lox -PyMT MEFs. Scale bars, 500 μM. The lower charts show an increase in ROS production by sodium citrate and SB204990. Representative flow cytometry analysis was shown ( n = 4). g Effects in OCR suppression caused by sodium citrate or SB204990 treatment in FASN lox/lox -PyMT MEFs. Presented data are the mean values ± SEM. Three independent experiments ( h ) ACLY activity in FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT MEFs. i , j Cell viability was determined in FASN lox/lox -PyMT MEFs at 24, 48, and 72 h in the presence of various concentrations of SB204990 ( n = 8) ( i ) or sodium citrate ( n = 7) ( j ) under 2D culture conditions. In h – j represented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; Student’s t test
Figure Legend Snippet: FASN deletion leads to ATP citrate lyase (ACLY) inhibition, blocking 3D growth. a Representative pictures of the spheroids recovered from the different genotypes growing under ultralow attachment conditions for 72 h (left). Spheroid diameter ( n = 30 spheroids, FASN lox/lox -PyMT; n = 24 FASN ∆/∆ -PyMT) and number of cells over 72 h (right). Scale bars, 500 μM. Presented data are the mean values ± SD. *** P < 0.001; Student’s t test. b OCR assay performed with cells recovered from the previous experiment and monolayer cultures. Oligomycin, FCCP, and antimycin/rotenone were added at the indicated time point. Data are represented as the means ± SEM. c Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in monolayer compared with 3D conditions in PyMT MEFs loaded with MitoSOX Red (FASN lox/lox -PyMT, n = 4; FASN ∆/∆ -PyMT, n = 5). The quantification of MitoSOX Red fluorescence intensity is shown on the left. Presented data are the mean values ± SD. *** P < 0.001, Student’s t test. d Quantification of MitoSOX Red fluorescence intensity by flow cytometry analysis in FASN lox/lox -PyMT, FASN ∆/∆ -PyMT, and DMNQ-treated (10 μM; 20 min) FASN lox/lox -PyMT MEFs ( n = 3). e Effect of 10 μM DMNQ treatment in the ability of FASN lox/lox -PyMT MEFs to form spheroids. Scale bars, 500 μM. Representative pictures for each condition are shown. f Representative pictures showing the effects of sodium citrate (5 mM) or SB204990 (30 μM) in the formation of tumor spheroids by FASN lox/lox -PyMT MEFs. Scale bars, 500 μM. The lower charts show an increase in ROS production by sodium citrate and SB204990. Representative flow cytometry analysis was shown ( n = 4). g Effects in OCR suppression caused by sodium citrate or SB204990 treatment in FASN lox/lox -PyMT MEFs. Presented data are the mean values ± SEM. Three independent experiments ( h ) ACLY activity in FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT MEFs. i , j Cell viability was determined in FASN lox/lox -PyMT MEFs at 24, 48, and 72 h in the presence of various concentrations of SB204990 ( n = 8) ( i ) or sodium citrate ( n = 7) ( j ) under 2D culture conditions. In h – j represented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; Student’s t test

Techniques Used: Inhibition, Blocking Assay, Flow Cytometry, Fluorescence, Activity Assay


Structured Review

Tocris sb204990
FASN deletion leads to ATP citrate lyase (ACLY) inhibition, blocking 3D growth. a Representative pictures of the spheroids recovered from the different genotypes growing under ultralow attachment conditions for 72 h (left). Spheroid diameter ( n = 30 spheroids, FASN lox/lox -PyMT; n = 24 FASN ∆/∆ -PyMT) and number of cells over 72 h (right). Scale bars, 500 μM. Presented data are the mean values ± SD. *** P < 0.001; Student’s t test. b OCR assay performed with cells recovered from the previous experiment and monolayer cultures. Oligomycin, FCCP, and antimycin/rotenone were added at the indicated time point. Data are represented as the means ± SEM. c Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in monolayer compared with 3D conditions in PyMT MEFs loaded with MitoSOX Red (FASN lox/lox -PyMT, n = 4; FASN ∆/∆ -PyMT, n = 5). The quantification of MitoSOX Red fluorescence intensity is shown on the left. Presented data are the mean values ± SD. *** P < 0.001, Student’s t test. d Quantification of MitoSOX Red fluorescence intensity by flow cytometry analysis in FASN lox/lox -PyMT, FASN ∆/∆ -PyMT, and DMNQ-treated (10 μM; 20 min) FASN lox/lox -PyMT MEFs ( n = 3). e Effect of 10 μM DMNQ treatment in the ability of FASN lox/lox -PyMT MEFs to form spheroids. Scale bars, 500 μM. Representative pictures for each condition are shown. f Representative pictures showing the effects of sodium citrate (5 mM) or <t>SB204990</t> (30 μM) in the formation of tumor spheroids by FASN lox/lox -PyMT MEFs. Scale bars, 500 μM. The lower charts show an increase in ROS production by sodium citrate and SB204990. Representative flow cytometry analysis was shown ( n = 4). g Effects in OCR suppression caused by sodium citrate or SB204990 treatment in FASN lox/lox -PyMT MEFs. Presented data are the mean values ± SEM. Three independent experiments ( h ) ACLY activity in FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT MEFs. i , j Cell viability was determined in FASN lox/lox -PyMT MEFs at 24, 48, and 72 h in the presence of various concentrations of SB204990 ( n = 8) ( i ) or sodium citrate ( n = 7) ( j ) under 2D culture conditions. In h – j represented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; Student’s t test
Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb204990/product/Tocris
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb204990 - by Bioz Stars, 2023-02
86/100 stars

Images

1) Product Images from "Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells"

Article Title: Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

Journal: Nature Communications

doi: 10.1038/s41467-019-13028-1

FASN deletion leads to ATP citrate lyase (ACLY) inhibition, blocking 3D growth. a Representative pictures of the spheroids recovered from the different genotypes growing under ultralow attachment conditions for 72 h (left). Spheroid diameter ( n = 30 spheroids, FASN lox/lox -PyMT; n = 24 FASN ∆/∆ -PyMT) and number of cells over 72 h (right). Scale bars, 500 μM. Presented data are the mean values ± SD. *** P < 0.001; Student’s t test. b OCR assay performed with cells recovered from the previous experiment and monolayer cultures. Oligomycin, FCCP, and antimycin/rotenone were added at the indicated time point. Data are represented as the means ± SEM. c Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in monolayer compared with 3D conditions in PyMT MEFs loaded with MitoSOX Red (FASN lox/lox -PyMT, n = 4; FASN ∆/∆ -PyMT, n = 5). The quantification of MitoSOX Red fluorescence intensity is shown on the left. Presented data are the mean values ± SD. *** P < 0.001, Student’s t test. d Quantification of MitoSOX Red fluorescence intensity by flow cytometry analysis in FASN lox/lox -PyMT, FASN ∆/∆ -PyMT, and DMNQ-treated (10 μM; 20 min) FASN lox/lox -PyMT MEFs ( n = 3). e Effect of 10 μM DMNQ treatment in the ability of FASN lox/lox -PyMT MEFs to form spheroids. Scale bars, 500 μM. Representative pictures for each condition are shown. f Representative pictures showing the effects of sodium citrate (5 mM) or SB204990 (30 μM) in the formation of tumor spheroids by FASN lox/lox -PyMT MEFs. Scale bars, 500 μM. The lower charts show an increase in ROS production by sodium citrate and SB204990. Representative flow cytometry analysis was shown ( n = 4). g Effects in OCR suppression caused by sodium citrate or SB204990 treatment in FASN lox/lox -PyMT MEFs. Presented data are the mean values ± SEM. Three independent experiments ( h ) ACLY activity in FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT MEFs. i , j Cell viability was determined in FASN lox/lox -PyMT MEFs at 24, 48, and 72 h in the presence of various concentrations of SB204990 ( n = 8) ( i ) or sodium citrate ( n = 7) ( j ) under 2D culture conditions. In h – j represented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; Student’s t test
Figure Legend Snippet: FASN deletion leads to ATP citrate lyase (ACLY) inhibition, blocking 3D growth. a Representative pictures of the spheroids recovered from the different genotypes growing under ultralow attachment conditions for 72 h (left). Spheroid diameter ( n = 30 spheroids, FASN lox/lox -PyMT; n = 24 FASN ∆/∆ -PyMT) and number of cells over 72 h (right). Scale bars, 500 μM. Presented data are the mean values ± SD. *** P < 0.001; Student’s t test. b OCR assay performed with cells recovered from the previous experiment and monolayer cultures. Oligomycin, FCCP, and antimycin/rotenone were added at the indicated time point. Data are represented as the means ± SEM. c Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in monolayer compared with 3D conditions in PyMT MEFs loaded with MitoSOX Red (FASN lox/lox -PyMT, n = 4; FASN ∆/∆ -PyMT, n = 5). The quantification of MitoSOX Red fluorescence intensity is shown on the left. Presented data are the mean values ± SD. *** P < 0.001, Student’s t test. d Quantification of MitoSOX Red fluorescence intensity by flow cytometry analysis in FASN lox/lox -PyMT, FASN ∆/∆ -PyMT, and DMNQ-treated (10 μM; 20 min) FASN lox/lox -PyMT MEFs ( n = 3). e Effect of 10 μM DMNQ treatment in the ability of FASN lox/lox -PyMT MEFs to form spheroids. Scale bars, 500 μM. Representative pictures for each condition are shown. f Representative pictures showing the effects of sodium citrate (5 mM) or SB204990 (30 μM) in the formation of tumor spheroids by FASN lox/lox -PyMT MEFs. Scale bars, 500 μM. The lower charts show an increase in ROS production by sodium citrate and SB204990. Representative flow cytometry analysis was shown ( n = 4). g Effects in OCR suppression caused by sodium citrate or SB204990 treatment in FASN lox/lox -PyMT MEFs. Presented data are the mean values ± SEM. Three independent experiments ( h ) ACLY activity in FASN lox/lox -PyMT versus FASN ∆/∆ -PyMT MEFs. i , j Cell viability was determined in FASN lox/lox -PyMT MEFs at 24, 48, and 72 h in the presence of various concentrations of SB204990 ( n = 8) ( i ) or sodium citrate ( n = 7) ( j ) under 2D culture conditions. In h – j represented data are the mean values ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001; Student’s t test

Techniques Used: Inhibition, Blocking Assay, Flow Cytometry, Fluorescence, Activity Assay

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    Tocris acly inhibitor sb204990
    Acly Inhibitor Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris sb204990 tocris
    Sb204990 Tocris, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris sb204990
    ( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with <t>SB204990</t> (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.
    Sb204990, supplied by Tocris, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb204990/product/Tocris
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sb204990 - by Bioz Stars, 2023-02
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    ( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

    Journal: eLife

    Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

    doi: 10.7554/eLife.62394

    Figure Lengend Snippet: ( A, B ) Detection of ATP-citrate lyase (ACLY) activity in inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. ( A ) Assessment of enzymatic activity for ACLY, acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) from Th0 or iTreg cells. ( B ) Assay for cytosol acetyl-CoA and oxaloacetic acid (OAA) from Th0 or iTreg cells. ( C ) ACLY overexpression affects iTreg differentiation. Naive CD4 + T cells were transfected with GFP-ACLY and cultured under Th0- or iTreg-polarization condition as in ( A ). Cells were analyzed by flow cytometry (FCM) to evaluate GFP + CD25 + Foxp3 + iTreg cells. ( D, E ) Inhibition of ACLY influences iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNAs (siRNAs) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr. CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). Data represent mean ± SD of three independent experiments, with significance determined by Student's t-test ( A, B ) or one-way analysis of variance (ANOVA) test ( C–E ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

    Article Snippet: Chemical compound, drug , SB204990 , Tocris Bioscience , Cat. #: 154566-12-8 , 100 μM.

    Techniques: Activity Assay, Isolation, Cell Culture, Over Expression, Transfection, Flow Cytometry, Inhibition

    ( A ) Activated T (Th0) and inducible regulatory T (iTreg) cell polarization in vitro. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain Th0 cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Isolation of cytosolic fraction. Cytosolic fraction without mitochondria and nuclei was isolated from cells prepared as in ( A ). ( C ) Enzymatic activity of ATP-citrate lyase (ACLY). Cells were transfected with GFP-ACLY and cultured as in ( A ) for 24 hr. Enzymatic activity of ACLY was measured and quantified. ( D, E ) Assay for ACLY activity in cells treated with si Acly or SB204990. Cells transfected with small interfering RNA (siRNA) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were induced under Th0- or iTreg (with 0.5 ng/ml transforming growth factor β1 [TGFβ1])-polarization condition for 24 hr as in prior to the measurement of enzymatic activity of ACLY. ( A, B ) One representative experiment out of three independent experiments is represented. ( C–E ) Data represent mean ± SD based on three independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. *p<0.05, **p<0.01.

    Journal: eLife

    Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

    doi: 10.7554/eLife.62394

    Figure Lengend Snippet: ( A ) Activated T (Th0) and inducible regulatory T (iTreg) cell polarization in vitro. Naive CD4 + T cells isolated from mice were cultured with Dynabeads Mouse T-Activator CD3/CD28 and rmIL-2 for 72 hr to obtain Th0 cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to generate iTreg cells. CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Isolation of cytosolic fraction. Cytosolic fraction without mitochondria and nuclei was isolated from cells prepared as in ( A ). ( C ) Enzymatic activity of ATP-citrate lyase (ACLY). Cells were transfected with GFP-ACLY and cultured as in ( A ) for 24 hr. Enzymatic activity of ACLY was measured and quantified. ( D, E ) Assay for ACLY activity in cells treated with si Acly or SB204990. Cells transfected with small interfering RNA (siRNA) against Acly ( D ) or treated with SB204990 (100 μM) ( E ) were induced under Th0- or iTreg (with 0.5 ng/ml transforming growth factor β1 [TGFβ1])-polarization condition for 24 hr as in prior to the measurement of enzymatic activity of ACLY. ( A, B ) One representative experiment out of three independent experiments is represented. ( C–E ) Data represent mean ± SD based on three independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. *p<0.05, **p<0.01.

    Article Snippet: Chemical compound, drug , SB204990 , Tocris Bioscience , Cat. #: 154566-12-8 , 100 μM.

    Techniques: In Vitro, Isolation, Cell Culture, Flow Cytometry, Activity Assay, Transfection, Small Interfering RNA

    ( A ) ATP-citrate lyase (ACLY) inhibition reduces de novo fatty acid synthesis (FAS). Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured under inducible regulatory T (iTreg)-polarization condition as in for 24 hr in the presence of [U- 13 C] glucose (11 mM). Cells were collected and subjected to metabolic flux analysis for FAS by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) analysis. n = 4, Student's t-test, mean ± SD. *p<0.05, **p<0.01, ****p<0.0001; ns, nonsignificant. ( B, C ) Functional evaluation of metabolic intermediates from FAS on iTreg differentiation. Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured as in in the presence of different doses of palmitate ( B ) or malonyl-CoA ( C ). CD4 + CD25 + Foxp3 + iTreg cells were assayed by flow cytometry (FCM) (left) and quantified (right). ( D, E ) ACLY inhibition increases carnitine palmitoyltransferase 1 (CPT1) activity and fatty acid oxidation (FAO). In the presence of malonyl-CoA (50 μM) or cerulenin (4 μM), naive CD4 + T cells treated with SB204990 were cultured as in for 24 hr. Cell lysates were used for analyzing CPT1 activity ( D ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and carnitine. Diagram (left) illustrating the OCR at various conditions and associated quantifications (right) are shown ( E ). ( F ) Impact of Cpt1 knockdown on iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNA (siRNA) against Cpt1 were cultured as in . CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). ( B–F ) Data represent mean ± SD of three ( D–F ) or four ( B, C ) independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

    Journal: eLife

    Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

    doi: 10.7554/eLife.62394

    Figure Lengend Snippet: ( A ) ATP-citrate lyase (ACLY) inhibition reduces de novo fatty acid synthesis (FAS). Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured under inducible regulatory T (iTreg)-polarization condition as in for 24 hr in the presence of [U- 13 C] glucose (11 mM). Cells were collected and subjected to metabolic flux analysis for FAS by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) analysis. n = 4, Student's t-test, mean ± SD. *p<0.05, **p<0.01, ****p<0.0001; ns, nonsignificant. ( B, C ) Functional evaluation of metabolic intermediates from FAS on iTreg differentiation. Naive CD4 + T cells were treated with SB204990 (100 μM) and cultured as in in the presence of different doses of palmitate ( B ) or malonyl-CoA ( C ). CD4 + CD25 + Foxp3 + iTreg cells were assayed by flow cytometry (FCM) (left) and quantified (right). ( D, E ) ACLY inhibition increases carnitine palmitoyltransferase 1 (CPT1) activity and fatty acid oxidation (FAO). In the presence of malonyl-CoA (50 μM) or cerulenin (4 μM), naive CD4 + T cells treated with SB204990 were cultured as in for 24 hr. Cell lysates were used for analyzing CPT1 activity ( D ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and carnitine. Diagram (left) illustrating the OCR at various conditions and associated quantifications (right) are shown ( E ). ( F ) Impact of Cpt1 knockdown on iTreg differentiation. Naive CD4 + T cells transfected with small interfering RNA (siRNA) against Cpt1 were cultured as in . CD4 + CD25 + Foxp3 + iTreg cells were assayed by FCM (left) and quantified (right). ( B–F ) Data represent mean ± SD of three ( D–F ) or four ( B, C ) independent experiments, with significance determined by one-way analysis of variance (ANOVA) test. **p<0.01, ***p<0.001, and ****p<0.0001; ns, nonsignificant.

    Article Snippet: Chemical compound, drug , SB204990 , Tocris Bioscience , Cat. #: 154566-12-8 , 100 μM.

    Techniques: Inhibition, Cell Culture, High Performance Liquid Chromatography, Mass Spectrometry, Functional Assay, Flow Cytometry, Activity Assay, Transfection, Small Interfering RNA

    ( A–D ) Naive CD4 + T cells treated with SB204990 were cultured under inducible regulatory T (iTreg)-polarization condition as in . Cells were lysed for the detection of HMG-CoA ( A ), mevalonate ( B ), and mevalonate-5-pyrophosphate ( C ) by enzyme-linked immunosorbent assay (ELISA) or stained by Filipin III for evaluating cholesterol by flow cytometry (FCM) ( D ). Data represent mean ± SD of four independent experiments, with significance determined by Student's t-test. ns, nonsignificant.

    Journal: eLife

    Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

    doi: 10.7554/eLife.62394

    Figure Lengend Snippet: ( A–D ) Naive CD4 + T cells treated with SB204990 were cultured under inducible regulatory T (iTreg)-polarization condition as in . Cells were lysed for the detection of HMG-CoA ( A ), mevalonate ( B ), and mevalonate-5-pyrophosphate ( C ) by enzyme-linked immunosorbent assay (ELISA) or stained by Filipin III for evaluating cholesterol by flow cytometry (FCM) ( D ). Data represent mean ± SD of four independent experiments, with significance determined by Student's t-test. ns, nonsignificant.

    Article Snippet: Chemical compound, drug , SB204990 , Tocris Bioscience , Cat. #: 154566-12-8 , 100 μM.

    Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

    ( A ) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). ( B ) ACLY overexpression affects iTreg cell differentiation. Human naive CD4 + T cells transfected with GFP-ACLY WT were polarized as in ( A ) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). ( C ) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. ( D ) ACLY interaction with CUL3-KLHL25. Cells prepared as in ( A ) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. ( E–G ) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLY WT or -ACLY 3KR were induced under Th0- or iTreg-polarization condition as described in ( A ) for 24 hr ( E, F ) or 72 hr ( G ). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination ( E ), examined by WB ( F ), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) ( G ). ( A–C, F, G ) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test ( A, C ) or one-way analysis of variance (ANOVA) test ( B, F, G ). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in ( A ) and ( D–F ), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.

    Journal: eLife

    Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

    doi: 10.7554/eLife.62394

    Figure Lengend Snippet: ( A ) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4 + T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFβ1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). ( B ) ACLY overexpression affects iTreg cell differentiation. Human naive CD4 + T cells transfected with GFP-ACLY WT were polarized as in ( A ) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). ( C ) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFβ1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. ( D ) ACLY interaction with CUL3-KLHL25. Cells prepared as in ( A ) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. ( E–G ) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLY WT or -ACLY 3KR were induced under Th0- or iTreg-polarization condition as described in ( A ) for 24 hr ( E, F ) or 72 hr ( G ). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination ( E ), examined by WB ( F ), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) ( G ). ( A–C, F, G ) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test ( A, C ) or one-way analysis of variance (ANOVA) test ( B, F, G ). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in ( A ) and ( D–F ), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.

    Article Snippet: Chemical compound, drug , SB204990 , Tocris Bioscience , Cat. #: 154566-12-8 , 100 μM.

    Techniques: Expressing, Isolation, Cell Culture, Western Blot, Over Expression, Cell Differentiation, Transfection, Flow Cytometry, Inhibition, Immunoprecipitation, Negative Control

    ( A, B ) Mice were supplied with dextran sodium sulfate (DSS) (2%, w/v) in drinking water. Meanwhile, ATP-citrate lyase (ACLY) inhibitor (SB204990, 37.5 mg/kg/d) was administrated once every 2 days by oral gavage. 10 days later, regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) ( A ) and colonic lamina propria (cLP) ( B ) were examined by flow cytometry (FCM). ( C–F ) Systematic evaluation of colitis in mice administrated with ACLY inhibitor. Body weight ( C ) and disease activity index (DAI) score ( D ), based on body weight loss, stool consistency, and blood in the stool, were recorded daily. 10 days later, entire colons from mice treated in different ways were removed for length assessment ( E ) and hematoxylin and eosin (H and E) staining for histopathological changes ( F ). Scale bar, 200 μm. In ( F ), one representative experiment out of three biological replicas is represented. ( A–E ) Data represent mean ± SD (n = 3), with significance determined by one-way analysis of variance (ANOVA) test ( A, B, E ) and two-way ANOVA test ( C, D ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

    Journal: eLife

    Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

    doi: 10.7554/eLife.62394

    Figure Lengend Snippet: ( A, B ) Mice were supplied with dextran sodium sulfate (DSS) (2%, w/v) in drinking water. Meanwhile, ATP-citrate lyase (ACLY) inhibitor (SB204990, 37.5 mg/kg/d) was administrated once every 2 days by oral gavage. 10 days later, regulatory T (Treg) cells in mesenteric lymph nodes (MLNs) ( A ) and colonic lamina propria (cLP) ( B ) were examined by flow cytometry (FCM). ( C–F ) Systematic evaluation of colitis in mice administrated with ACLY inhibitor. Body weight ( C ) and disease activity index (DAI) score ( D ), based on body weight loss, stool consistency, and blood in the stool, were recorded daily. 10 days later, entire colons from mice treated in different ways were removed for length assessment ( E ) and hematoxylin and eosin (H and E) staining for histopathological changes ( F ). Scale bar, 200 μm. In ( F ), one representative experiment out of three biological replicas is represented. ( A–E ) Data represent mean ± SD (n = 3), with significance determined by one-way analysis of variance (ANOVA) test ( A, B, E ) and two-way ANOVA test ( C, D ). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

    Article Snippet: Chemical compound, drug , SB204990 , Tocris Bioscience , Cat. #: 154566-12-8 , 100 μM.

    Techniques: Flow Cytometry, Activity Assay, Staining

    ( A ) Inhibition of ATP-citrate lyase (ACLY) by SB204990 facilitates inducible regulatory T (iTreg) differentiation. Naive CD4 + T cells treated with SB204990 were induced as in . CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Assay for iTreg inhibitory function in vitro. Cells prepared as in ( A ) were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained naive CD4 + T cells in the presence of Dynabeads Mouse T-Activator CD3/CD28 for 72 hr. CD4 + T-cell proliferation was analyzed by FCM (left) and quantified (right) based on three independent experiments. One-way analysis of variance (ANOVA) test, mean ± SD. **p<0.01. ( C–E ) Systematic evaluation of colitis in mice adoptively transferred with different cells. ( C ) Body weight (left) and disease activity index (DAI) score (right), based on body weight loss, stool consistency, and blood in the stool, were recorded weekly. 7 weeks later, entire colons from mice treated in different ways were removed for length assessment ( D ) and hematoxylin and eosin (H and E) staining for histopathological changes ( E ). Scale bar, 200 μm. ( C, D ) Data represent mean ± SD (n = 4), with significance determined by two-way ANOVA test ( C ) and one-way ANOVA test ( D ). *p<0.05, ***p<0.001, and ****p<0.0001. ( A, E ) One representative experiment out of four biological replicas is represented.

    Journal: eLife

    Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

    doi: 10.7554/eLife.62394

    Figure Lengend Snippet: ( A ) Inhibition of ATP-citrate lyase (ACLY) by SB204990 facilitates inducible regulatory T (iTreg) differentiation. Naive CD4 + T cells treated with SB204990 were induced as in . CD4 + CD25 + Foxp3 + iTreg cells were evaluated by flow cytometry (FCM). ( B ) Assay for iTreg inhibitory function in vitro. Cells prepared as in ( A ) were cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-stained naive CD4 + T cells in the presence of Dynabeads Mouse T-Activator CD3/CD28 for 72 hr. CD4 + T-cell proliferation was analyzed by FCM (left) and quantified (right) based on three independent experiments. One-way analysis of variance (ANOVA) test, mean ± SD. **p<0.01. ( C–E ) Systematic evaluation of colitis in mice adoptively transferred with different cells. ( C ) Body weight (left) and disease activity index (DAI) score (right), based on body weight loss, stool consistency, and blood in the stool, were recorded weekly. 7 weeks later, entire colons from mice treated in different ways were removed for length assessment ( D ) and hematoxylin and eosin (H and E) staining for histopathological changes ( E ). Scale bar, 200 μm. ( C, D ) Data represent mean ± SD (n = 4), with significance determined by two-way ANOVA test ( C ) and one-way ANOVA test ( D ). *p<0.05, ***p<0.001, and ****p<0.0001. ( A, E ) One representative experiment out of four biological replicas is represented.

    Article Snippet: Chemical compound, drug , SB204990 , Tocris Bioscience , Cat. #: 154566-12-8 , 100 μM.

    Techniques: Inhibition, Flow Cytometry, In Vitro, Staining, Activity Assay

    Journal: eLife

    Article Title: ACLY ubiquitination by CUL3-KLHL25 induces the reprogramming of fatty acid metabolism to facilitate iTreg differentiation

    doi: 10.7554/eLife.62394

    Figure Lengend Snippet:

    Article Snippet: Chemical compound, drug , SB204990 , Tocris Bioscience , Cat. #: 154566-12-8 , 100 μM.

    Techniques: Activation Assay, Cell Isolation, Enzyme-linked Immunosorbent Assay, Isolation, Activity Assay, Recombinant, Plasmid Preparation, Software