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. 2021 Mar 28;296:100606. doi: 10.1016/j.jbc.2021.100606

Figure 2.

Figure 2

HASMC PDE1C activation in response to ER (Ca2+) store depletion or SOCE activation.A and B, cAMP PDE activity in homogenates generated from control siRNA (A) or PDE1C siRNA (B), transfected HASMCs under control conditions (Krebs), following ER(Ca2+) store depletion [Ca2+ free + CPA (10 μM)] or following stepwise ER(Ca2+) depletion and SOCE activation [Krebs + CPA (10 μM)]. n = 4 experiments where each experiment was carried out in triplicate. One-way ANOVA, F = 3.76, p = 0.0343, Dunnett’s multiple comparison’s test was conducted, ∗p = 0.0485 control cells (Krebs) versus either ER(Ca2+) depletion and ∗p = 0.0415 control cells (Krebs) versus SOCE activation. C, HASMC cAMP PDE activity in homogenates without (black) or with Ca2+/CaM (red) in the presence of a combination of cilostamide (5 μM) + Ro 20 to 1724 (10 μM), a combination of C33 (1 μM) and cilostamide (5 μM) + Ro 20 to 1724 (10 μM), or a combination of PF-04827736 (1 μM) and cilostamide (5 μM) + Ro-20 to 1724 (10 μM). n = 3 experiments where each experiment was carried out in triplicate. Tukey’s multiple comparisons test was conducted, ∗p = 0.011 compared with control, ∗∗∗∗p < 0.0001 compared with control, ####p < 0.0001 compared with Ca2+/CaM. Two-way ANOVA, F (3, 16) = 8.207, p = 0.0016 interaction between control versus Ca2+/CaM conditions, F (3, 16) = 157.9 within each individual drug condition (control versus Ca2+/CaM treatment), p < 0.0001, F (1, 16) = 19.96, p = 0.0004 between the different drug treatments. Tukey’s multiple comparisons test was conducted, ∗p = 0.011 compared with control, ∗∗∗∗p < 0.0001 compared with control, ####p < 0.0001 compared with Ca2+/CaM. D, representative anti-PDE1C immunoblot of lysates generated from control siRNA-transfected HASMCs (siCtrl) or PDE1C-silenced (siPDE1C) HASMCs and quantitation of the efficiency of PDE1C-silencing in these cells (plot). ∗∗∗p < 0.01 versus siCtrl, n = 3, ∗∗∗∗p < 0.001 versus siCtrl, n = 3, Student’s unpaired t test. CaM, calmodulin; cAMP, cyclic AMP; CPA, cyclopiazonic acid; HASMC, human arterial smooth muscle cell; PDE, phosphodiesterase; PDE1C, phosphodiesterase 1C; SOCE, store-operated calcium entry.