Fig. 4: B7-1 mediates podocyte injury through activating β-catenin signaling in vitro and glomerular mini-organ culture.
A Heatmap plot of transcriptomic analysis showing the changes in the Wnt/β-catenin signaling, cell differentiation, actin cytoskeleton reorganization and glomerular development at different groups. n = 3. MPC5 cells were transfected with B7-1 siRNA(siB7-1) or negative control(si-NC) for 24 h, following 0.25 μg/ml of ADR treatment for another 24 hours. B GSEA analysis of different comparisons among groups show ADR induced genes enriched in Wnt/β-catenin pathway, and blocked by B7-1 interference. Ctl: siNC group; ADR: ADR + siNC group; SiB7-1: ADR + siB7-1 group. n = 3. C, D Western blot and quantification of B7-1, active β-catenin, Nephrin and Desmin in different groups as indicated. *P < 0.05, **P < 0.01 versus siNC group. #P < 0.05, ##P < 0.01 versus ADR + siNC group. n = 3. E, F Representative images showing the expression of active β-catenin, F-actin, Zo-1 and Fibronectin in MPC5 cells. Cells were transfected with B7-1 expressing plasmid (pFlag-B7-1) or empty vector (pcDNA) for 24 hours. Bar = 10 or 20 μm. G Western blot showing the protein expression of B7-1, active β-catenin, Zo-1, Synaptopodin, Nephrin, Fibronectin and Desmin in indicated groups. n = 3. H Western blot showing the expression of active β-catenin, Zo-1, Nephrin, Fibronectin, Desmin and WT-1. MPC5 cells were pre-treated with ICG-001 (5 μmol/L) for one hour, following transfection with pFlag-B7-1 plasmid (or pcDNA) for another 24 hours. n = 3. I Representative image showing the isolated rat glomeruli under light microscopy. J Representative images showing staining of Podocin and Synaptopodin in isolated rat glomeruli treated with B7-1 lentivirus (Lenti-B7-1) or negative control lentivirus (Lenti-NC). Bar = 20 μm. K Western blot showing the expression of Flag tag, active β-catenin, Zo-1, Podocalyxin, WT1, and Fibronectin in isolated rat glomeruli treated with indicated treatment. The glomeruli were first pre-treated with ICG-001 (5 μmol/L) or DMSO, following infection with B7-1 expressing lentivirus or negative control for another 72 hours. n = 3. L Experimental scheme showing the isolated glomeruli from B7-1flox/flox mice treated with adenovirus expressing NPHS2-cre recombinase to knockdown B7-1 in podocytes and co-treated with ADR. The mouse glomeruli were isolated by magnetic beads and plated on the six-well plates, treated with the indicated adenovirus for 48 hours and followed by ADR (0.125 μg/ml) treatment for another 24 hours. M Graph showing the mRNA level of B7-1. **P < 0.01 versus negative control group (AdV-NC). ##P < 0.01 versus ADR group (AdV-NC + ADR). n = 3. N Western blot showing the expression of Nephrin and active β-catenin in isolated B7-1flox/flox mouse glomeruli. O Graphs showing the mRNA levels of Desmin and Fibronectin in isolated B7-1flox/flox mouse glomeruli. *P < 0.05, **P < 0.01 versus negative AdV-NC group. #P < 0.05 versus AdV-NC + ADR group. n = 3.ADR Adriamycin, DMSO Dimethyl sulfoxide. The full length original western blots for all results are provided in Supplementary File 1.
