Figure 1 Validation of DEPs using qRT-PCR and Western blotting DEPs, including TSP-1/TGF-β1 signaling pathway and the ECM components, were determined using qRT-PCR. A1: mRNA expression level of TSP-1; A2: mRNA expression level of TGF-β1; A3: mRNA expression level of smad3; A4: mRNA expression level of COL IV; A5: mRNA expression level of FN. The relative gene expression was normalized to that of GAPDH. DEPs: differentially expressed proteins; qRT-PCR: quantitative real-time polymerase chain reaction; TSP-1: thrombospondin-1; TGF-β1: transforming growth factor-β1; ECM: extracellular matrix; COL IV: collagen IV, FN: fibronection; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. Quantitative data are shown as the mean ± standard deviation (n = 6). aP < 0.01, cP < 0.001, compared with the control group. bP < 0.01, dP < 0.05, compared with the model group. B: Western blotting analyses of the TSP-1/TGF-β1 signaling pathway and ECM components. Control: control group; model: model group; PLD: P. linteus decoction intervention group (0.5 g/kg), daily gavage, 8 weeks.

Figure 2 Compound-target network Orange diamonds represent each compound in Phellinus linteus; Blue round nodes represent the putative targets of these compounds. Red square nodes represent the known targets for FSGS obtained from the GeneCards database. FSGS: focal segmental glomeruloscleosis.

Figure 3 PCA inhibits the TSP-1/TGF-β1 signaling pathway in RMCs A: effects of different concentrations of PCA on the cell proliferation of RMCs were determined using the CCK-8 assay. Quantitative data are represented as the mean ± standard deviation (n = 3). PCA: protocatechualdehyde. 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50 μg/mL: cells were treated with PCA for 24 h, aP < 0.01, bP < 0.0001, compared with the control group; B: Western blotting analyses of the TSP-1/TGF-β1 signaling and ECM components. RMCs: rat mesangial cells; CCK-8: cell counting kit-8; TSP-1: thrombospondin-1; TGF-β1: transforming growth factor-β1; ECM: extracellular matrix. Control: control group; 20 mM Glu: high glucose stimulation group; 1.5625, 3.125, 6.25 μg/mL: PCA low-dose, medium-dose, and high-dose group, cells were treated with PCA for 48 h.

Figure 4 Knockdown of TSP-1 protein affects the TGF-β1 signaling pathway A: efficacies of shRNAs were determined in 293T cells by observing the GFP fluorescence intensity under a fluorescence microscope (Olympus IX53). A1: control shRNA; A2: TSP-1 shRNA1; A3: TSP-1 shRNA2; A4: TSP-1 shRNA3. 48 h stimulation, scale bars, 200 μm. B: relative mRNA levels of TSP-1 in 293T cells treated with shRNAs 1-3 for 48 h, respectively. Three independent experiments were analyzed. Quantitative data are represented as the mean ± standard deviation (n = 3). aP < 0.01, compared with the control group, C: protein levels of TSP-1 in 293T cells treated with shRNAs targeting for 48 h. D: efficacy of lentivirus-mediated shRNA interference on TSP-1 was determined in RMCs by observing the GFP fluorescence under a fluorescence microscope (Olympus IX53). D1: control lv, 3 × 108 TU/mL; D2: TSP-1 Lv, 3 × 108 TU/mL. 72 h stimulation, scale bars, 100 μm. E: protein levels of TGF-β1 signaling and ECM components in RMCs treated with the lentivirus targeting TSP-1 for 72 h and the control lentivirus were determined using Western blotting. Control lv: control lentivirus; TSP-1 Lv: Lv carrying TSP-1 shRNA. TSP-1: thrombospondin-1; TGF-β1: transforming growth factor-β 1; GFP: green fluorescent protein; RMCs: rat mesangial cells; ECM: extracellular matrix.