Fig 1: Fatty acid metabolism disorders play a central role in renal fibrosis and are closely related to β-catenin.a Representative micrographs showing renal expression of CPT1A (upper), PPARα (middle), as well as Masson staining (bottom) in healthy subjects and different patients with CKD. Human renal biopsy sections were obtained from healthy subjects (paracancerous tissue) and patients with IgA nephropathy (IgAN), membranous nephropathy (MN), lupus nephritis (LN), and diabetic nephropathy (DN). Arrows indicate positive staining (scale bar: 50 µm). b Quantitative analysis of immunohistochemical staining of CPT1A in patients with CKD and healthy subjects. n = 5 (healthy subjects); n = 20 (CKD patients). **P < 0.01 versus healthy subjects. c Quantitative analysis of immunohistochemical staining of PPARα in patients with CKD and healthy subjects. n = 5 (healthy subjects); n = 20 (CKD patients). **P < 0.01 versus healthy subjects. d Linear regression showing the Pearson correlation coefficient (r) and P value between CPT1A and fibrosis score from CKD patients, respectively. n = 20. e Linear regression showing the Pearson correlation coefficient (r) and P value between PPARα and fibrosis score from CKD patients, respectively. n = 20. f Representative transmission electron microscopy (TEM) images showing the lipid droplets in renal tubular epithelial cells in a patient with diabetic nephropathy. Arrows indicate positive staining; scale bar: 2 µm. g Representative micrographs showing the staggered localization of CPT1A and β-catenin in a patient with IgA nephropathy. Sequential kidney paraffin sections were stained with antibodies against β-catenin or CPT1A. Arrows indicate positive staining; scale bar: 50 µm. h Representative micrographs showing co-localization of β-catenin and ADRP in patients with IgAN and diabetic nephropathy (DN). Kidney frozen sections were stained for β-catenin (red) and ADRP (green) by immunofluorescence. Arrows indicate positive staining; scale bar: 50 µm. The data were analyzed by using Student’s t-test or Pearson correlation analysis.
Fig 2: Treatment with GalNAc-siPlin2 ameliorates MASLD induced by obesity. A; Schematic representation of obesity-induced MASLD in leptin-deficient (ob/ob) mice subcutaneously injected with 4 mg/kg GalNAc-siPlin2, GalNAc-siNC, or PBS. B: RT-qPCR analysis of relative mRNA levels of hepatic Plin2 in ob/ob mice subcutaneously injected with 4 mg/kg GalNAc-siPlin2, GalNAc-siNC, or PBS. C: Immunoblotting analysis of hepatic PLIN2 protein levels in ob/ob mice subcutaneously injected with 4 mg/kg GalNAc-siPlin2, GalNAc-siNC, or PBS. D,E: Body weight (D) and liver-to-body weight ratio (E) for ob/ob mice subcutaneously injected with 4 mg/kg GalNAc-siPlin2, GalNAc-siNC, or PBS. F: Histological analysis of liver sections from ob/ob mice subcutaneously injected with 4 mg/kg GalNAc-siPlin2, GalNAc-siNC, or PBS. Quantification of Oil red O staining–positive areas and TUNEL-positive cells is shown. Scale bar, 50 μm. G-J: The levels of hepatic TG (G), hepatic TC (H), serum TG (I), and serum TC (J) from ob/ob mice subcutaneously injected with 4 mg/kg GalNAc-siPlin2, GalNAc-siNC, or PBS. K,L: The levels of serum ALT (K) and serum AST (L) in ob/ob mice subcutaneously injected with 4 mg/kg GalNAc-siPlin2, GalNAc-siNC, or PBS. Data are shown as mean ± SEM. For all the panels, n = 6 mice per group. P values were calculated using one-way ANOVA followed by Tukey's multiple comparison tests. “ns” denotes no significant difference. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GalNAc, N-acetylgalactosamine; MASLD, metabolic dysfunction–associated steatotic liver disease; RT-qPCR, quantitative real-time PCR; TC, total cholesterol; TG, triglyceride.
Fig 3: Cardiac macrophage infiltration, interstitial fibrosis, and apoptosis. A–C: Representative light micrographs of cardiac ventricles derived from Wt, AKO, and AKO+cHSL mice. Macrophages were immunostained with a specific antibody against F4/80. The scale bars represent 100 μm. D–F: Macrophage localization in cardiac ventricles. Tissue sections were subjected to immunostaining procedures with a specific antibody against F4/80 and PLIN2, and they were analyzed via confocal microscopy. Representative images are presented showing macrophages (indicated in green, with arrows), the LD surface (indicated in red), and the nucleus (indicated in blue). The scale bars represent 10 μm. G–I: Representative light micrographs of cardiac ventricles derived from Wt, AKO, and AKO+cHSL mice subjected to staining procedures with Masson’s trichrome staining. The scale bars represent 100 μm. J–L: Apoptotic cells in cardiac ventricles of Wt, AKO, and AKO+cHSL mice. DNA fragmentation was detected using the TUNEL assay, and representative images have been presented. TUNEL-positive nuclei are shown in green (with arrows). The scale bars represent 20 μm. Five mice per group were selected, two sections per mouse were analyzed, and representative images have been presented.
Fig 4: IMCL-PLIN2 intensity correlation analysis in twin pairs. (A) representative image showing differences between groups. Gray level indicates marker signal, cyan indicates segmented sarcolemma. Note active twin type II fiber with high intensity IMCL significantly colocalized by PLIN2 (magenta arrows), unlike the inactive twin (orange arrows). Bar = 10 μm. Scatter plot is relative to the arrowed type II fiber; (B) fiber type as main effect, with LTPA combined; (C) LTPA as main effect, with fiber type combined, main effect differences denoted with # (p <0.01); combined group differences denoted with ** (p <0.001). Dots in (B,C) represent individual cells.
Fig 5: Amino acid binding activates Ubr1 by relieving its auto‐inhibition. In S2 cells, the Ubr1‐C deletion mutant Ubr1ΔC (amino acids 1–1531) bound to Plin2 in an amino acid‐independent manner (A). Interaction between Ubr1‐N (amino acids 1–1031) and Ubr1‐C (amino acids 1532–1824) was observed during amino acid deficiency (B and C), while refeeding cells with Schneider's medium (B) or adding amino acids to the lysate (C) suppressed this interaction. In fed S2 cells (D, upper panel), auto‐inhibitory interactions were observed in double‐site (Ubr1G157D, D244N‐N, Ubr1D160E, H247Y‐N), but not single‐site Ubr1‐N mutants (type 1, Ubr1G157D‐N and Ubr1D160E‐N; type 2, Ubr1D244N‐N and Ubr1H247Y‐N). In amino acid‐starved cells (D, lower panel), exogenous Lys (type 1) prevented type 2, but not type 1 Ubr1‐N mutants recognizing Ubr1‐C. Similarly, addition of Leu (type 2) to cell lysate released the auto‐inhibitory interaction of type 1, but not type 2 amino acid‐binding defective Ubr1‐N mutants. Neither Lys nor Leu affected auto‐inhibition in double‐site Ubr1‐N mutants.
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