Fig 1: The impact of IRI surgery on renal physiological function and the expression levels of renal fibrosis-related factors in kidney tissue(A and B) The effect of IRI surgery on renal function, where A shows the creatinine levels in the blood of mice in the Sham, IRI_2d, and IRI_1w groups; B shows the blood urea nitrogen levels in the blood of mice in the Sham, IRI_2d, and IRI_1w groups.(C–H) Expression of Spp1, TGF-β, Smad2/3, P-Smad2/3, Col1a1, and α-SMA in kidney tissues using ELISA.(I and J) Expression of Spp1, TGF-β, Smad2/3, P-Smad2/3, Col1a1, and α-SMA in kidney tissues using immunohistochemistry. In all the images, compared to the Sham group, ∗ represents p < 0.05, ∗∗ represents p < 0.01, and ∗∗∗ represents p < 0.001; the bar in the image equals 750 μm.
Fig 2: Investigation into the impact of Spp1 on fibroblast behavior(A and B) Scratch experiment to evaluate the effect of Spp1 on fibroblast migration.(C and D) Transwell chamber experiment to assess the effect of Spp1 on fibroblast migration.(E) CCK8 experiment to measure the effect of Spp1 on fibroblast proliferation.(F) qRT-PCR to detect the expression levels of Col1a1 and α-SMA in fibroblasts. In all the images, compared to the sh-NC or oe-NC groups, ∗ represents p < 0.05, ∗∗ represents p < 0.01, and ∗∗∗ represents p < 0.001; bar in the image equals 750 μm or 500 μm; each experiment was conducted three times with six replicates each time.
Fig 3: The effect of adding A83-01 inhibitor under TGF-β-induced conditions on fibroblast behaviorNote: (A) Western blot analysis of the effects of overexpressing Spp1 under TGF-β-induced conditions with or without adding A83-01 inhibitor on the expression of Smad2/3, P-Smad2/3, Col1a1, and α-SMA.(B–D) Protein expression grayscale analysis to compare the expression and phosphorylation differences of the proteins. In all the images, ∗ represents p < 0.05, ∗∗ represents p < 0.01, and ∗∗∗ represents p < 0.001; each experiment was conducted three times with six replicates each time.
Fig 4: The impact of Spp1−/− mice on alleviating the effects of IRI surgery on renal physiological function and the expression levels of kidney fibrosis-related factors in their tissues(A and B) The impact of IRI surgery on renal function. Panel A shows the creatinine levels in the blood of mice in each group. Panel B shows the urea nitrogen levels in the blood of mice in each group.(C–H) Expression levels of Spp1, TGF-β, Smad2/3, P-Smad2/3, Col1a1, and α-SMA in kidney tissues using ELISA technique.(I and J) Expression levels of Spp1, TGF-β, Smad2/3, P-Smad2/3, Col1a1, and α-SMA in kidney tissues using immunohistochemistry technique. In all the panels above, ∗ represents p < 0.05 when comparing the two groups, ∗∗ represents p < 0.01, and ∗∗∗ represents p < 0.001; the bar in the figure equals 50 μm.
Fig 5: Molecular mechanism of Spp1 in regulating fibroblast behavior(A) Western blot analysis of the effects of knocking down or overexpressing Spp1 on the expression of Smad2/3, Col1a1, and α-SMA under TGF-β induced conditions.(B–D) Protein expression grayscale analysis to compare the expression and phosphorylation differences of the proteins.(E–H) Double immunofluorescence staining of P-Smad2/3 (red) and Col1a1 (green) or α-SMA (green) to validate whether Spp1 regulates Col1a1 and α-SMA expression through the phosphorylation of Smad2/3. Blue represents cell nuclei stained with DAPI. In all the images, compared to the sh-NC or oe-NC groups, ∗ represents p < 0.05, ∗∗ represents p < 0.01, and ∗∗∗ represents p < 0.001; bar equals 20 μm; each experiment was conducted three times with six replicates each time.
from Cell Signaling Technology for FastScan ™ COL1A1 ELISA Kit