Extended Data Fig. 7: SMC-specific Trpm7 deletion suppresses CRTC2/CREB signaling.
From: The TRPM7 chanzyme in smooth muscle cells drives abdominal aortic aneurysm in mice
(a-b) Immunoblots of phosphorylated CREB in WT or MKO cells pretreated for 30 mins with TRPM7 blocker (NS8593, 10 μM), Zn2+ chelator (TPEN, 10 μM), Ca2+ chelator (BAPTA-AM, 10 μM), adenylyl cyclase inhibitor (SQ22536, 10 μM), PKA inhibitor (H89, 10 μM), CaMKKβ blocker (STO-609, 1 μM), CaMKII blocker (KN93, 5 μM), Pyk2 blocker (PF431396, 10 μM) or calcineurin inhibitor (CsA, 1 μM), followed by 30-min naltriben stimulation (20 μM). n = 3. (c-d) Statistics of Fig. 5c. n = 6-9 for c, n = 3-5 for d. (e-f) Statistics of Fig. 5d. n = 6-8 for e, n = 3-4 for f. (g-h) Statistics of Fig. 5e. n = 4 for g, n = 6-7 for h. (i) Immunoblots of the denoted protein in M7KO cells rescued with empty vector, M7Ch-WT or M7Ch-PD upon saline or Ang II stimulation. n = 6-8. (j-k) Statistics of Extended Data Fig. 7i. n = 6-8 for j, n = 8 for k. (l) Immunofluorescence of CRTC2 in InfraAo from saline or PPE-perfused Ai9sm-tdT/0 mice. Ai9sm-tdT/0 mice were generated by breeding Ai9(RCL-tdT) mice (JAX 007909) with another SMC Cre driver (Tagln-Cre, JAX 017491). SMC in these mice is labeled by tdTomato for fluorescent visualization. CRTC2 was shown in green, tdTomato autofluorescence in red and DAPI in blue. CRTC2 nuclear localization (white arrows) in SMC was analyzed based on nuclear staining (cyan) in tdTomato-positive cell (red). Scale bar=50μm. n = 3. (m) Immunofluorescence of CRTC2 in InfraAo from saline or PPE-perfused mice. Analysis is identical to Fig. 5f. n = 3. Scale bar=50μm. (n-o) Detection of CRTC2 in extracted cytosolic or nuclear protein from WT or M7KO cells treated with saline or Ang II. n = 3. (p) Immunoblots of CRTC2 in extracted cytosolic or nuclear protein from HEK293 cells transfected with empty vector, M7Ch-WT or M7Ch-PD upon naltriben stimulation. n = 3. (q-r) Statistics of Extended Data Fig. 7p. The differences were determined by two-way ANOVA (Figs. 7c–h,j,kn,o) or one-way ANOVA (Figs. 7b,q,r), followed by Tukey’s post-hoc test.
