Downregulation of TRPC4 and TRPC5 Inhibits Smooth Muscle Cell Proliferation without Affecting Endothelial Cell Proliferation

Figures

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  Figure 1. (a) mRNA expression levels of TRPCs (TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6) in VSMCs and VECs measured by RT-qPCR. (b) Western blots and protein expression levels of TRPCs (TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6) in VSMCs and VECs. N = 12. ∗p < 0.05. ∗∗p < 0.01.

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  Figure 2. (a) siTRPC4 (100 nM) and siTRPC5 (100 nM) were transfected into VECs and VSMCs, and cells were examined 48 h after transfection by fluorescence microscopy. a and b are unlabeled Cy3 negative controls for transfection of VECs for 48 h; c and d are TRPC4 siRNA (100 nM) for transfection of VECs for 48 h, e and f are TRPC5 siRNA (100 nM) for transfection of VECs for 48 h; g and h are TRPC4 siRNA (100 nM) and TRPC5 siRNA (100 nM) which were cotransfected into VECs for 48 h. Among them, a, c, e, and g are observations of cell morphology after transfection under brightfield, b, d, f, and h are observations of transfection efficiency under the red fluorescence channel, i and j are unlabeled Cy3 negative controls for transfection of VSMCs for 48 h, k and l are TRPC4 siRNA (100 nM) for transfection of VSMCs for 48 h, m and n are TRPC5 siRNA (100 nM) for transfection of VSMCs for 48 h, and o and p are TRPC4 siRNA (100 nM) and TRPC5 siRNA (100 nM) which were cotransfected into VSMCs for 48 h. Among them, i, k, m, and o are observations of cell morphology after transfection under brightfield, and j, l, n, and p are observations of transfection efficiency under the red fluorescence channel.…

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  Figure 3. (a) The effects of silencing TRPC4 and TRPC5 on VEC and VSMC proliferation assessed by CCK-8 analysis and viable cell counting. ∗p < 0.05 vs. VSMC-CTRL, VSMC siTRPC4, and VSMC siTRPC5. ∗∗p < 0.01. (b) Western blotting analysis of PCNA protein expression. For both VSMCs and VECs, TRPC4, TRPC5, and combined TRPC4 + 5 interference groups were tested, and the results were compared with the NC group. Data are presented as the means +SEM. N = 12. ∗p < 0.05 vs. VSMC-NC.

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  Figure 4. (a) Effects of TRPC knockdown on calcium store depletion in VSMCs. TRPC4, TRPC5, and TRPC4 + TRPC5 were assayed after 7 min. Interfering with TRPC4 and TRPC5 together significantly reduced intracellular calcium levels (N = 6, ∗p < 0.05 vs. scramble siRNA (NC)). (b) Effects of TRPC knockdown on calcium store depletion in VECs. TRPC4, TRPC5, and TRPC4 + TRPC5 interference groups were assayed after 7 min. The increase in intracellular calcium ions in each experimental group was essentially restored to the level of the NC group (N = 6, ∗p < 0.05 vs. NC).