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. 2017 Oct 5;2(19):e94920. doi: 10.1172/jci.insight.94920

Figure 4. Activation of calcium signaling–mediated CREB was required for low-potassium-induced VSMC calcification.

Figure 4

(A) Effects of potassium levels on intracellular calcium, determined by Fluo4 NW, in vascular smooth muscle cells (VSMCs) exposed to control (5.4 mM) or low potassium (3.7 mM, Low K+). Results from 3 independent experiments are shown. (B) Effects of low potassium on activation of extracellular signal–regulated kinase (ERK), protein kinase C (PKC), and calcium-activated cAMP response element–binding protein (CREB), determined by Western blot analysis. Representative blots from 3 independent experiments are shown. (C) Effects of pharmacological inhibitors on VSMC calcification. VSMCs were exposed to control or low-potassium media with the indicated inhibitors for 3 weeks. Calcification was determined by Alizarin red staining. (D) Effects of pharmacological inhibitors on activation of CREB. Western blot analysis of phosphorylation of CREB in C. Representative images from 3 independent experiments are shown. (E and F) Effects of CREB knockdown on low-potassium-induced VSMC calcification. VSMCs with CREB knockdown by shRNA (shCREB) or control shRNA (shScr) were exposed to control or low-potassium media for 3 weeks. Calcification was determined by Alizarin red staining. Western blot analysis was performed to determine the expression of CREB, runt-related transcription factor 2 (Runx2), and the autophagic marker, microtubule-associated protein 1 light chain 3 (LC3), in the cytoplasmic form (LC3 I), and conjugated form (LC3 II). Representative blots from 3 independent experiments are shown. (G) Effects of pharmacological inhibitors on autophagy markers. Western blot analysis of LC3 I and II levels in VSMCs exposed to control or low-potassium media for 3 weeks, in the presence or absence of indicated inhibitors. Representative results from 3 independent experiments are shown.