acly inhibitor sb204990  (Tocris)

Journal: Communications Biology

Article Title: KLHL25-ACLY module functions as a switch in the fate determination of the differentiation of iTreg/Th17

doi: 10.1038/s42003-025-07917-z

Figure Lengend Snippet: a , b JNK inhibition reduces the transcription and protein level of Klhl25 . Naïve CD4 + T cells cultured under iTreg polarization condition were treated with specific inhibitors against Smad3, ERK, JNK, and p38 at the onset for 48 h. a Klhl25 mRNA level was assessed by qPCR. b KLHL25 protein level was analyzed by WB. c , d JNK inhibition attenuates the binding of CUL3-KLHL25 with ACLY and ACLY ubiquitination. Naïve CD4 + T cells transfected with pMIG- Klhl25 overexpression virus were cultured under iTreg polarization condition in the presence of JNK inhibitor (10 μM) for 48 h. Cells were treated with MG132 (10 μM) for 6 h before immunoprecipitated with antibodies against ACLY, and then the binding of ACLY with CUL3-KLHL25 was assessed by WB ( c ), the ubiquitination of ACLY was determined by WB ( d ). IgG serves as a negative control. e JNK inhibition suppresses TGFβ1-induced ACLY degradation. Naïve CD4 + T cells were transfected and cultured as described in the legend to ( c , d ) before the analysis of ACLY protein level by WB. ( f , g ) Impact of KLHL25 overexpression on CPT1 activity and iTreg differentiation upon JNK inhibition. Cells were transfected and cultured as described in the legend to ( c , d ) before analysis for CPT1 enzymatic activity assay by kit ( f ). Cells transfected and cultured as described in the legend to ( c , d ) for 72 h were stained for CD4 + Foxp3 + iTreg analyses by flow cytometry (FCM) (left) and quantified (right) ( g ). h Impact of Acly knockdown on iTreg differentiation upon JNK inhibition. Naïve CD4 + T cells transfected with Acly targeting siRNAs were cultured as described in the legend to ( g ) and then stained for CD4 + Foxp3 + iTreg analyses by FCM (left) and quantified (right). For ( a – h ), data are representative as mean ± SD (n = 3 biologically independent samples) with p values determined by one-way ANOVA test. For WB in ( b – h ), one representative experiment out of three is represented. The values indicate mean intensities based on three biological replicas.

Article Snippet: ACLY inhibitor: SB204990 (154566-12-8, Tocris Bioscience); ACC inhibitor: PF05175157 (TOCRIS, 5709); Proteasome inhibitor: MG132 (S2619, Selleck); ERK inhibitor: U0126 (tlrl-u0126, InvivoGen); JNK inhibitor: SP600125 (Selleck, S1460); p38 inhibitor: SB203580 (tlrl-sb20, InvivoGen); Smad3 inhibitor: SIS3 HCl (S7959, Selleck); STAT3 inhibitor: S31-201(S1155, Selleck).

Techniques: Inhibition, Cell Culture, Binding Assay, Ubiquitin Proteomics, Transfection, Over Expression, Virus, Immunoprecipitation, Negative Control, Activity Assay, Enzyme Activity Assay, Staining, Flow Cytometry, Knockdown

Journal: Communications Biology

Article Title: KLHL25-ACLY module functions as a switch in the fate determination of the differentiation of iTreg/Th17

doi: 10.1038/s42003-025-07917-z

Figure Lengend Snippet: a IL-6-triggered-ERK reduces the transcriptional activity of Klhl25 promoter. HEK293T cells were transfected with pGL 4.0- Klhl25 and treated with IL-6 and (or) TGFβ1 in the presence (or absence) of ERK inhibitor (15 μM) before dual luciferase reporter assay. b , c ERK but not STAT3 inhibition rescues Klhl25 mRNA and protein level. Naïve CD4 + T cells were cultured under iTreg or Th17 polarization conditions for 48 h in the presence (or absence) of ERK inhibitor and STAT3 inhibitor (10 μM). The mRNA level of Klhl25 was assayed by qPCR ( b ) and the protein level was detected by WB ( c ). d IL-6-triggered-ERK inhibits the association of ACLY with CUL3-KLHL25 during human Th17 differentiation. Naive CD4 + T cells isolated from human peripheral blood were cultured under iTreg or Th17 polarization conditions for 48 h in the presence (or absence) of ERK inhibitor. Cells were immunoprecipitated with antibodies against ACLY for WB analysis. IgG serves as a negative control. e , f Knockdown of Nfya abolishes the effect of ERK inhibition on the transcription and expression of Klhl25 . Naive CD4 + T cells transfected with Nfya targeting siRNAs were cultured under iTreg or Th17 polarization conditions in the presence (or absence) of ERK inhibitor and subsequently subjected to qPCR ( e ) or WB ( f ) analysis. g Knockdown of Nfya abolishes the effect of ERK inhibition on ACLY ubiquitination. Cells transfected and cultured as described in the legend to ( e , f ) were treated with MG132 before immunoprecipitated with antibodies against ACLY ( g ) or Ub ( h ) for WB analysis. IgG serves as a negative control. h , i Knockdown of Nfya abolishes the effect of ERK inhibition on FAO and Th17 differentiation. h Cells were cultured as described in the legend to ( e , f ). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and L-carnitine. Diagram (up) illustrating the OCR at various conditions and associated quantifications (down) are shown. i Cells cultured as described in the legend to ( e , f ) for 72 h were analyzed by flow cytometry (FCM) for the differentiation of CD4 + IL-17A + Th17 (left) and quantified (right). j Knockdown of Klhl25 abolishes the effect of ERK inhibition on Th17 differentiation. Naive CD4 + T cells transfected with Klhl25 targeting siRNAs were cultured under iTreg or Th17 polarization conditions in the presence (or absence) of ERK inhibitor and subsequently analyzed by FCM for the differentiation of CD4 + IL-17A + Th17 (left) and quantified (right). For ( a – j ), data are representative as mean ± SD (n = 3 biologically independent samples) with p values determined by one-way ANOVA test. For WB in ( c , d , f , g ), one representative experiment out of three is represented. The values indicate mean intensities based on three biological replicas.

Article Snippet: ACLY inhibitor: SB204990 (154566-12-8, Tocris Bioscience); ACC inhibitor: PF05175157 (TOCRIS, 5709); Proteasome inhibitor: MG132 (S2619, Selleck); ERK inhibitor: U0126 (tlrl-u0126, InvivoGen); JNK inhibitor: SP600125 (Selleck, S1460); p38 inhibitor: SB203580 (tlrl-sb20, InvivoGen); Smad3 inhibitor: SIS3 HCl (S7959, Selleck); STAT3 inhibitor: S31-201(S1155, Selleck).

Techniques: Activity Assay, Transfection, Luciferase, Reporter Assay, Inhibition, Cell Culture, Isolation, Immunoprecipitation, Negative Control, Knockdown, Expressing, Ubiquitin Proteomics, Flow Cytometry