Fig 2: Western blot analysis for Nephrin and TRPC6 in rats injected with shamporter and siRNA.Rats were injected via tail vein with shamporter + control siRNA or shamporter + nephrin siRNA or shamporter + TRPC6 siRNA. Compared with control siRNA injected rats, there was a significant decrease in nephrin protein levels in the rats injected with nephrin siRNA (A). Likewise, there was a significant decrease in TRPC6 protein levels in the rats injected with TRPC6 siRNA (B). Staining for GAPDH or beta actin was used as a protein loading control, and the podocyte specific protein podocin or nephrin were used to ensure that the same amount of podocyte protein was loaded. Densitometry and of the results are shown in the graph. Ratios were compared using t-test (p<0.05); ratios are expressed as median ± min and max values.

Fig 3: Immunohistochemistry for nephrin, podocin and TRPC6.Animals injected with shamporter + control siRNA show a strong linear staining signal for nephrin and podocin, as well as glomerular staining for TRPC6. In animals injected with shamporter + nephrin or TRPC6 specific siRNA, the staining signal was significantly reduced, while the signal for podocin remained unchanged. When nephrin, podocin, or TRPC6 antibodies were omitted, no staining was detected.

Fig 4: Specific POX expression regulated by TRPC6 in human glioma. (A) qPCR analysis of the indicated mRNA levels in LN229 cells. (B) Immunoblotting of total lysates of LN229 cells transfected with GFP and DNC6 (left panel) or control shRNA and shTRPC6 (right panel) with the indicated antibodies. (C) qPCR analysis of POX and p21 mRNA levels in LN229 cells transfected with GFP, DNC6, or WT-TRPC6 (WTC6). (D) Immunoblotting of total lysates from 40 glioma and 39 human normal brain (control) tissues (four samples are shown) probed with the indicated antibodies. GAPDH as loading controls. (E) Immunohistological staining in glioma and control human brain tissues with hematoxylin, GFAP, and POX antibodies. Scale bar, 50 µm. (F) POX mRNA expression in glioma and control tissues detected by in situ hybridization. Antisense probe: POX mRNA; sense probe: negative control. Scale bar, 50 µm (black) or 10 µm (white). Unless stated, data are mean ± SEM of at least three independent experiments in triplicate, t-test, **P < 0.01.

Fig 5: HKC suppresses TRPC6/CnA/NFAT expression in UUO mice. Representative western blots and quantification of the levels of TRPC6 (A), CnA (B) as well as NFAT (C) in sham, and UUO mice with different treatments. GAPDH was used as an internal control. ## P < 0.01, UUO group vs. sham group; * P < 0.05, ** P < 0.01, UUO + HKC group vs. UUO group. Bar graphs represent the mean ± SEM (n = 3).