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. 2022 Mar 18;298(5):101847. doi: 10.1016/j.jbc.2022.101847

Figure 4.

Figure 4

Capsaicin blockade of Ca2+entry via TRPV4 channels and the protein expression of TRPV4 in IEC-6 cells and mouse intestinal tissues.A−C, summary tracings of [Ca2+]cyt time course in response to GSK1016790A (GSK, 10–100 nM) and calcium (5 mM) in the absence (0Ca2+, n = 26) or the presence (2Ca2+, n = 23) of calcium and calcium plus HC067047 (HC, 5 μM, n = 19). D, summary data showing the peaks of GSK-increased [Ca2+]cyt signaling as in (A−C). E−G, summary tracings of [Ca2+]cyt time course in response to GSK (10 nM) in the absence or the presence of different dose of capsaicin (Cap, n = 26). H, summary data showing the peaks of GSK-increased [Ca2+]cyt signaling described as in (E−G). I−K, summary tracings of [Ca2+]cyt time course in response to GSK (10 nM) of NC and shTRPV4-1 or shTRPV4-3. L, summary data showing the peaks of GSK-increased [Ca2+]cyt signaling described as in (I−K). The data are presented as mean ± SD. ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001 were performed by Student’s t test. M, immunostaining of TRPV4 proteins to confirm their expression in IEC-6 cells. The upper panels: the specific staining of TRPV4 proteins (in green) and merge with the nuclei of the cells stained with DAPI. The lower panel: the nuclei of the cells stained with DAPI (in blue) without primary antibodies against TRPV4 as a negative control. The scale bar represents 20 μm for each image. N and O, Western blotting analysis of TRPV4 proteins expression in IEC-6 cells (N) and shTRPV4 in IEC-6 cells (O). GAPDH was used as a loading control. Each one is the representative of all images taken from 3 independent experiments with similar results. P, immunohistological analysis on TRPV4 proteins in jejunal and colonic tissues from WT mice (the left panels) and TRPV4 KO mice (the middle panels). The right panels were without primary antibodies against TRPV4 in the intestinal tissues from TRPV4 KO mice as negative controls. The scale bar represents 100 μm for each image. [Ca2+]cyt, cytosolic Ca2+ concentrations; IECs, intestinal epithelial cells; TRPV, transient receptor potential vanilloid.