Journal: Cell Death & Disease
Article Title: Knockout of integrin αvβ6 protects against renal inflammation in chronic kidney disease by reduction of pro-inflammatory macrophages
doi: 10.1038/s41419-024-06785-5
Figure Lengend Snippet: A TKPTS cells transfected with si-NC or si- Itgb6 were pre-stimulated with hypoxia for 24 h or not, and then the relative mRNA levels of macrophage activators in TKPTS cells were detected with qPCR and shown with heatmap ( n = 3 per group). B Western blot of IL-34 in si-NC- or si- Itgb6 -transfected hypoxic TKPTS cells. The quantification of the relative levels of IL-34/β-actin is shown ( n = 4 per group). C TKPTS cells transfected with si-NC or si- Itgb6 were pre-stimulated with hypoxia for 24 h or not, and then IL-34 in supernatant was detected by ELISA ( n = 3 per group). D , E Western blot and immunostaining of IL-34 in uIRI-7d or AAN kidneys from WT and Itgb6 -/- mice (scale bar, 50 μm). The quantification of the relative levels of IL-34/α-Tubulin and quantification of IL-34 positive area proportion are shown ( n = 3–5 per group). F Immunofluorescence staining for IL-34 (red), KIM-1 (green), and DAPI (blue) in WT-uIRI-7d kidney sections (scale bar, 50 μm). G Schematic of an in vitro rescue experiment. TKPTS cells transfected with si-NC or si- Itgb6 were pre-stimulated with hypoxia for 24 h or not, and then co-cultured with RAW264.7 cells for 12 h. RmIL-34 or vehicle was supplemented into the co-culture system as indicated. H , I Migration and pro-inflammatory differentiation of RAW264.7 cells in the co-culture system (scale bar, 25 μm) ( n = 3-4 per group). J Schematic of another in vitro reverse rescue experiment. TKPTS cells transfected with oe-NC or oe- Itgb6 plasmids were pre-stimulated with hypoxia for 24 h or not, and then co-cultured with RAW264.7 cells for 12 h. Anti-IL-34 antibody (αIL-34) or isotype control (10 μg/mL) was added into the co-culture system as indicated. K , L Migration and pro-inflammatory differentiation of RAW264.7 cells in the co-culture system (scale bar, 25 μm) ( n = 3 per group). M Schematic of an in vivo rescue experiment. Itgb6 -/- mice were intraperitoneally supplemented with recombinant mouse IL-34 (rmIL-34) or vehicle (1 μg/dose) immediately and 3 days after uIRI, and WT-IRI-7d mice received vehicle served as controls. N Relative mRNA levels of renal inflammatory factors ( Tnα , Il1b , Ccl2 , and Il6 ) in WT and Itgb6 -/- mice supplemented with rmIL-34 or vehicle were detected by qPCR ( n = 4-5 per group). O Comparison of pro-inflammatory (Ly6c + F4/80 int ) macrophages in WT and Itgb6 -/- mice supplemented with rmIL-34 or vehicle ( n = 4-5 per group). P Western blot of α-SMA and fibronectin in kidneys of WT and Itgb6 -/- mice supplemented with rmIL-34 or vehicle. The quantifications of the relative levels of α-SMA/α-Tubulin and fibronectin/α-Tubulin are shown ( n = 3 per group). Q Representative images and quantifications of Sirius Red staining in kidney sections from WT and Itgb6 -/- mice supplemented with rmIL-34 or vehicle (scale bar, 100 μm) ( n = 4-5 per group). Data are presented as mean ± SEM of three biological replicates. Student’s t -test ( B , D , E , K , L ) and one-way ANOVA ( C , H – Q ) were performed. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. # p < 0.05; ## p < 0.01; ### p < 0.001; #### p < 0.0001; compared with si- Itgb6 + rmIL-34 group (0 ng/mL) ( H , I ); # p < 0.05; ## p < 0.01; up-regulated degree by oe- Itgb6 was compared between isotype (light blue background) and αIL-34 group (light orange background) ( K , L ); ns, not significant.
Article Snippet: The antibodies used in western blot were as follows: goat anti-mouse integrin β6 antibody (RD, AF2389), mouse anti-mouse α-SMA Ab (Sigma-Aldrich, A5228), rabbit anti-mouse Fibronectin Ab (BOSTER, BA1772), sheep anti-mouse IL-34 Ab (RD, AF5195), rabbit anti-mouse YAP Ab (CST, 14074 S), rabbit anti-mouse p-YAP (S127) Ab (CST, 4911 S), mouse anti-mouse GAPDH Ab (Abcam, ab8245), mouse anti-mouse α-Tubulin Ab (CST, 12351 S), and mouse anti-mouse β-actin Ab (Abcam, ab8226).
Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Immunostaining, Immunofluorescence, Staining, In Vitro, Cell Culture, Co-Culture Assay, Migration, Control, In Vivo, Recombinant, Comparison
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