Peli1, regulated by m6A modification, suppresses NLRP3 inflammasome activation in atherosclerosis by inhibiting YB-1

Figures

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  Fig. 1. YB-1 knockdown inhibited AS progression in vivo. YB-1 knockdown was induced in ApoE−/− mice combined with HFD treatment. A YB-1 protein level in arterial tissues was examined by western blot. B The atherosclerotic lesions of whole artery were observed using Oil Red O staining, and the atherosclerotic lesions of whole artery were quantified. C HE staining was adopted to analyze lesions in arterial tissues. D, E Lipid accumulation in arterial tissues was measured by Oil red O staining and Sirius red staining. Scale bars represent 50 μm. n = 8 animals. The measurement data were presented as mean ± SD. Comparisons between two groups were performed using the Student’s t-test, while multiple group comparisons were made using one-way ANOVA. ***p < 0.001. AS atherosclerosis, YB-1 Y-box binding protein 1, ApoE−/− apolipoprotein E-deficient, HFD high-fat diet, HE hematoxylin-eosin, SD standard deviation, ANOVA one-way analysis of variance.

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  Fig. 2. YB-1 promoted ox-LDL-induced lipid accumulation in macrophages by regulating NLRP3. A Raw264.7 cells stimulated by ox-LDL were used as AS cellular model, and immunofluorescence was performed to detect nuclear localization in cells. Scale bars represent 25 μm. B, C ChIP and dual-luciferase reporter gene assays were employed to analyze the interaction between YB-1 and NLRP3 promoter. YB-1 knockdown was induced in ox-LDL-treated Raw264.7 cells. D Western blot was adopted to determine YB-1, NLRP3 and caspase-1 levels. E IL-1β and IL-18 levels were analyzed using ELISA. F The protein levels of cleaved IL-1β and pro-IL-1β in cells were examined by western blot. G Lipid accumulation was measured by Oil red O staining. Scale bars represent 25 μm. n = 3 independent experiments. The measurement data were presented as mean ± SD. Comparisons between two groups were performed using the Student’s t-test, while multiple group comparisons were made using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. ox-LDL oxidized low-density lipoprotein, AS atherosclerosis, ChIP chromatin immunoprecipitation, YB-1 Y-box binding protein 1, NLRP3 nucleotide-binding domain (NOD)-like receptor (NLR) family…

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  Fig. 3. Peli1-mediated ubiquitination-dependent degradation of YB-1. A YB-1 ubiquitination level in Raw264.7 cells after ox-LDL treatment was assessed using western blot, and IgG served as the negative control. B CHX chase assay was performed to analyze the protein stability of YB-1 in Raw264.7 cells after ox-LDL treatment. C The interaction between YB-1 and Peli1 was analyzed using CoIP assay. D IP was employed to detect the interaction between YB-1 and Peli1 using a Peli1 overexpression vector with 6X His tag (His-Peli1-pcDNA3.1) and a YB-1 overexpression vector with HA tag (HA-YB-1-pcDNA3.1). E Peli1 overexpression was induced in ox-LDL treated Raw264.7 cells combined with MG132 treatment, and YB-1 protein level in cells was examined using western blot. F CHX chase assay was performed to analyze the protein stability of YB-1 in ox-LDL-treated Raw264.7 cells after Peli1-pcDNA3.1 or pcDNA3.1 transfection. n = 3 independent experiments. The measurement data were presented as mean ± SD. Comparisons between two groups were performed using the Student’s t-test, while multiple group comparisons were made using one-way ANOVA. **p < 0.01, ***p < 0.001. YB-1 Y-box binding protein 1, ox-LDL…

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  Fig. 4. Peli1 was regulated by m6A modification. A m6A dot blot assay was conducted to analyze total m6A level in Raw264.7 cells after ox-LDL treatment. B The m6A level of Peli1 in Raw264.7 cells after ox-LDL treatment was examined using MeRIP assay. C The m6A level of Peli1 in arterial tissues was examined using MeRIP assay. D The interaction between METTL3 and Peli1 was analyzed using RNA pull-down assay. E The m6A level of Peli1 in ox-LDL treated Raw264.7 cells after METTL3 knockdown was examined using MeRIP assay. F, G Peli1 expression level in ox-LDL treated Raw264.7 cells after METTL3 knockdown was examined using qRT-PCR and western blot. n = 3 independent experiments. The measurement data were presented as mean ± SD. Comparisons between two groups were performed using the Student’s t-test. ***p < 0.001. m6A N6-methyladenosine, Peli1 pellino 1, MeRIP methylated RNA immunoprecipitation, METTL3 methyltransferase-like 3, ox-LDL oxidized low-density lipoprotein, qRT-PCR quantitative real-time polymerase chain reaction, SD standard deviation.

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  Fig. 5. YTHDC2 recognized m6A modified Peli1 and mediated its mRNA degradation. A, B The interaction between YTHDC2 and Peli1 was analyzed using RIP and RNA pull-down assays. C, D YTHDC2 overexpression and METTL3 knockdown were induced in ox-LDL-treated Raw264.7 cells. C, D Peli1 expression level was measured by qRT-PCR and western blot. E RNA degradation assay was employed to analyze Peli1 mRNA stability in cells. n = 3 independent experiments. The measurement data were presented as mean ± SD. Comparisons between two groups were performed using the Student’s t-test, while multiple group comparisons were made using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. YTHDC2 YTH domain-containing protein 2, Peli1 pellino 1, RIP RNA immunoprecipitation, METTL3 methyltransferase-like 3, ox-LDL oxidized low-density lipoprotein, SD standard deviation, ANOVA one-way analysis of variance.

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  Fig. 6. Peli1 inhibited the activation of NLRP3 inflammasome in macrophages through ubiquitination of YB-1. Peli1 overexpression and YB-1 overexpression were induced in Raw264.7 cells and ox-LDL treatment was subsequently performed. A YB-1, NLRP3 and caspase-1 levels were measured using western blot. B IL-1β and IL-18 levels were analyzed using ELISA. C The protein levels of cleaved IL-1β and pro-IL-1β in cells were detected by western blot. D Lipid accumulation was measured by Oil red O staining. Scale bars represent 25 μm. n = 3 independent experiments. The measurement data were presented as mean ± SD. Comparisons between two groups were performed using the Student’s t-test, while multiple group comparisons were made using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Peli1 pellino 1, YB-1 Y-box binding protein 1, ox-LDL oxidized low-density lipoprotein, NLRP3 nucleotide-binding domain (NOD)-like receptor (NLR) family pyrin domain-containing protein 3, IL interleukin, ELISA enzyme-linked immunosorbent assay, SD standard deviation, ANOVA one-way analysis of variance.

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  Fig. 7. Peli1 inhibited AS progression in vivo through ubiquitination of YB-1. Peli1 overexpression and YB-1 overexpression were induced in ApoE−/− mice combined with HFD treatment. A YB-1, NLRP3 and caspase-1 protein levels in arterial tissues were measured using western blot. B Ubiquitination assay was performed to detect ubiquitination level of YB-1 in arterial tissues. C The atherosclerotic lesions of whole artery were observed using Oil Red O staining, and the atherosclerotic lesions of whole artery were quantified. D HE staining was employed to analyze lesions in arterial tissues. Scale bars represent 50 μm. E, F Lipid accumulation in arterial tissues was measured by Oil red O staining and Sirius red staining. Scale bars represent 50 μm. G Serum IL-1β and IL-18 levels were analyzed using ELISA. n = 8 animals. The measurement data were presented as mean ± SD. Comparisons between two groups were performed using the Student’s t-test, while multiple group comparisons were made using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. Peli1 pellino 1, YB-1 Y-box binding protein 1, ApoE−/− apolipoprotein E-deficient, HFD high-fat diet, HE hematoxylin-eosin, IL interleukin, ELISA…