Fig 1: FABP4 mediated MMT through Saa1 in vivo and BMDMs of UUO. (A) Heatmap of RNA-seq analysis in kidneys of UUO. (B) RT-PCR confirmed the renal change of Saa1 gene. (C) Immunofluorescence showed the active co-expression of Saa1 and F4/80 in kidneys of UUO. (D) Western blot verified the protein change of Saa1 in kidneys of UUO. (E) ChIP assay demonstrated that UUO enhanced the physical binding of FABP4 protein on the region of the Saa1 gene. (F) Saa1 was knocked down compared with control by siRNA since day 5 and the mRNA levels of Saa1, FABP4, a-SMA, and Col-1 were detected with or without TGF-ß1 by RT-PCR assay. (G) siSaa1 inhibited the MMT cells as shown by two-color immunofluorescence imaging. (H) Western blot verified the in vitro protein change of Saa1, FABP4, a-SMA, and Col-1. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
Fig 2: Hepatic SAA1 suppression in mice via siRNA(A) Experimental design representing suppression of SAA1 in CCl4 and CI injury models and timescale for sample collection.(B and C) Relative expression of liver SAA1 mRNA after 2 days of transfecting five different siRNA(s) in CCl4 and CI injury models, respectively.(D and E) IHC and western blot analyses of selected SAA1-siRNA2 in CCl4 and CI injury models, respectively.Neg-siRNA represents non-specific control. Scale bar represents 100 µm. Where applicable, data represent mean ± SEM *p < 0.05, **p < 0.01 and ***p < 0.001 (n = 3).
Fig 3: SAA1/TLR2 axis induces Rac GTPase-mediated actin reorganization and migration of HSCs(A) Time course of GTPase-Rac1 activation(s) in LX-2 cells at indicated time interval(B and C) Basal and stimulated levels of GTPase-Rac1 in WT and TLR2-/- cells as determined by pull-down assays(D and E) WT and TLR2-/- LX-2 cells were pretreated with NSC 23766 (50 µ?), and activation of GTP-Rac1 was determined by pull-down assay (D), and MLCK and p-MLC at Ser19 activity was determined by western blotting (E).(F and G) Migration assays showing pretreatment of the cells with NSC 23766 (50 µ?) attenuated their migration(s) in agarose spot (F) and Transwell (G) assays. (H) Time course of PI3K activations after treatment of LX-2 cells at indicated time points.(I and J) WT and TLR2-/-cells were pretreated with LY294002 (10 µ?), and the phosphorylation of PI3K at p85 subunits was determined by western blot analysis, and the activations of Rac GTPase were determined by pull-down assay.(K and L) Migration assays showing pretreatment of the cells with LY294002 attenuated their migration(s) in agarose spot (K) and Transwell (L) assays. Photographs are representatives of (n = 6). (Scale bar represents 200 µm).Data represent mean ± SEM *p < 0.01 and **p < 0.001 (n = 3). Photographs are representatives of (n = 6). (Scale bar represents 200 µm). Data represent mean ± SEM *p < 0.01 and **p < 0.001 (n = 3).
Fig 4: SAA1 promotes HSC recruitment in vitro and in vivo(A) Representative IHC immunostaining images showing staining of a-SMA-positive cells in SAA1-siRNA2, Neg-siRNA, and control samples obtained from CCl4 and CI injury models. Bar graph represents quantification of IHC images per 5 fields.(B and C) Representative confocal immunofluorescence images showing co-localization of SAA1 and HSCs in SAA1-siRNA2, Neg-siRNA, and control in CCl4 and CI injury models.(D and E) Transwell migration assay representing co-culture of primary mouse hepatocytes (PMHep) isolated from injured and healthy mouse with JS1 cells (D) and HepG2 cells (SAA1 overexpressing and GFP expressing) with LX-2 (E). The bar graph represents quantification of relative number of migrated cells.(F and G) Agarose spot (F) and Transwell (G) migration assays showing migration of LX-2 cells toward rhSAA1 at indicated time intervals. The bar graph represents quantification of relative number of migrated cells.(Scale bar = A, B, and C/200; G/100 µm: Inset/50 µm). Where applicable, data represent mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001 and (n = 3). iPMHep represents injured primary mouse hepatocytes, hPMHep represents healthy primary mouse hepatocytes, HepG2 (SAA1) represents HepG2 cells overexpressing SAA1, and HepG2 (GFP) represents HepG2 expressing GFP.
Fig 5: Effects of oxysterols on brain pathology and the expression of amyloid precursor protein in the brain. (A) HE staining of the whole brain (n = 3, scale bar: 100 and 20 µm), black arrows: Nuclei pyknosis; (B) number of neurons (/0.05976 mm2) (n = 3, mean ± SEM); (C) density of neurons (cell/mm2) (n = 3, mean ± SEM); (D) APP mRNA (n = 7, median with range); (E) SAA mRNA (n = 7, mean ± SEM); (F) Western blot results of APP and SAA; (G) APP protein (n = 7, mean ± SEM); (H) SAA protein (n = 7, mean ± SEM). *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Supplier Page from Abcam for Anti-SAA1 + SAA2 antibody [EPR19235]