Fig 2: Macrophage polarization is dependent on SOCE channels, and inhibition of these channels disrupts cytokine release(A and B) Cytokine analysis by colorimetric analysis in supernatants of M1 and M2 activated bone marrow-derived macrophage (BMDM) from wild type after treatment with and without SKF96365 (10 µM for 6 h). Data shown are representative of three independent experiments performed in triplicates. Bar graphs depict average ± SD for relative values, ***p = 0.001 (Student's t test).(C and D) Flow cytometer analysis on M1 and M2 activated wild-type mice BMDM with and without SKF96365 (10 µM for 6 h) to show how Ca2+ inhibition interrupts macrophage polarization by blocking surface (CD80 + CD206+) and internal (iNOS and Arginase-1) markers for the different phenotypes. Data shown are representative of three independent experiments.(E) Immunoblot analysis showing the level of expression of wild-type mice M1 activated BMDM pathway genes pNF-?B, NF-?B, pJNK, JNK in media treated for 6 h with SKF96365 (10 µM). Data shown are representative of three independent experiments with similar results.(F) Immunoblot analysis showing the level of expression of wild-type mice M2 activated BMDM pathway genes Arg1, pSTAT6 in media treated for 6 h with SKF96365 (10 µM). Data shown are representative of three independent experiments.(G–I) Primary macrophages derived from bone marrow were pulsed with medium alone (NS), IFN? at 20 ng/mL, IL4 at 20 ng/mL, and cultured for a total of 2, 6, and 24-h period. Cells were harvested and chromatin immunoprecipitation (ChIP) experiments was performed with anti-pNF-?B, anti-pSTAT1, or anti-pSTAT6 at the promoter regions of TRPC1 was assessed by qPCR and expressed as percentage of input. The mean ± SE of NF-?B (G), pSTAT1 (H), pSTAT6 (I) are shown.(J–L) Time-dependent binding to Orai1 promoter region by pSTAT3, pPPARg, and pIRF4 upon IL-4 stimulation; data presented are mean ± SE for each condition. Significant differences were measured by Student's t test (*p < 0.05; NS, non-significant, n = 3).(M–O) Immunoblot analysis showing the level of expression of phosphorylated STAT1 and STAT6 in M1 and M2 cells that were treated for 24 h in 0.5 or 2 mM external calcium. Data shown are representative of three independent experiments. Representative confocal images showing localization of pSTAT1(N) and pSTAT6(O) in cytoplasm and in the nucleus (co-localization with DAPi staining) in cells treated with various calcium concentrations. Scale bar, 10 µm.

Fig 3: TRPC1 siRNA decreases the protein expression levels of TRPC1 in HUVECs. (A) Western blotting demonstrated that TRPC1 siRNA transfection decreased the expression of TRPC1 protein in HUVECs, whereas nonspecific siRNA had no significant effect. (B) Quantification of western blots revealed that TRPC1 siRNA decreased TRPC1 protein expression levels by 74%. Data are expressed as the mean ± standard error of four experiments. ?P<0.05 vs. control; #P<0.05 vs. nonspecific siRNA. siRNA, small interfering RNA, TRPC1, transient receptor potential canonical 1; HUVECs, human umbilical vein endothelial cells.

Fig 4: Cell-type specific TRPC1, TRPC4, and TRPC5 channel upregulation in the hippocampus and cortex after CCI-TBI. (A,B) Representative Western immunoblots using (A) TRPC4 (~120 kDa) and (B) TRPC5 (~110 kDa) antibodies in sham and TBI cortex and hippocampus. Blots were normalized to ß-actin protein (42 kDa) as loading control. (C,D) Summarized data for Western blot quantification of TRPC4 (C, n = 7–13 animals per group) and TRPC5 (D, n = 5–7 animals per group) from microdissected brain regions in mice 7 days after TBI. (E,F) Shown are summarized plots of percent difference in TRPC4 (E) and TRPC5 (F) protein between ipsilateral and contralateral hemispheres of microdissected regions from data in Panels (C,D). All data bars represent the mean ± SEM. *p < 0.05 vs. sham of same subregion. †p < 0.05 vs. contralateral hemisphere of same subregion.

Fig 5: Expression and localization of redox-sensitive TRP channel in the human liver tissue. (A) Expression of mRNAs for TRPV1-4, TRPC1, TRPC4, TRPC5, TRPM2, TRPM7, TRPA1, and GAPDH detected by PCR in the cDNA library from normal human liver. Specific PCR primers used are listed in the Table 1. (B) In situ hybridization analysis of TRPV1, TRPC1, TRPM2, TRPM7, and TRPA1 mRNAs in the paraffin section of the human liver. mRNAs of TRPV1 (a,f), TRPC1 (b,g), TRPM2 (c,h), TRPM7 (d,i) were localized in hepatocytes (*) and Kupffer cells (arrow). TRPA1 (e,j) was localized in the lining of sinusoids and Kupffer cells of the human liver tissue. The images from the analysis of both the antisense (left) and sense probes (right) are shown. Scale bar, 100 µm.