Fig 2: Loss of functional SELENOK in melanoma cells leads to decreased proliferation(A) Western blot analysis showed similar SELENOK levels in primary human melanocytes and three human melanoma cell lines. GAPDH was used as a loading control. (B) A diagram illustrates how CRISPR/Cas9 was used to edit the genome of SK-MEL-28 cells, generating a truncated version of SELENOK with its functional domain deleted. (C) Western blot confirmed presence of full-length SELENOK in w.t. cells, both full-length and truncated SELENOK in Clone 3, but only truncated SELENOK in Clone 7 cells. Only Clone 7 exhibited reduced IP3R levels. GAPDH was used as a loading control. (D) Equal numbers of cells were plated in replicate wells (N = 5 per cell line) and proliferation was measured over a 4-day period. Clone 7 showed reduced growth on days 1–3. Results are expressed as mean + SEM and a one-way ANOVA with Tukey post-test was used to analyze groups. Means at each time point without a common letter differ, p < 0.05. (E) Scratch assays were performed in triplicate comparing w.t. SK-MEL-28 cells to Clone 3 and 7 cells. Results showed less enclosure of the scratched region for Clone 7 cells. For D–E, results represent two independent experiments and a one-way ANOVA was used to analyze groups with Tukey post-test used to compare means of each group. Results are expressed as mean + SEM and means without a common letter differ, p < 0.05.

Fig 3: Reticulocalbin3 (RCN3) regulated MMP-2 and MMP-9 expression through inositol 1,4,5-trisphosphate receptor 1 (IP3R1)–Ca2+–calcium/calmodulin-dependent protein kinase (CaMKII). (A) In esophageal squamous cell carcinoma (ESCC) cell lines, RCN3 is positively correlated with IP3R1 expression. *p < 0.05, Pearson's correlation coefficient. (B) Co-IP analysis showed that RCN3 interacted with IP3R1. (C) Immunofluorescence staining showed there the expression and localization of RCN3 and IP3R1 in ECA109 cells. (D–G) Fluo-4 AM showed intracellular Ca2+ concentration. (H) Western blot analysis the expression of related proteins of the IP3R1–Ca2+–CaMKII–c-Jun signaling pathway. (I) Expression of MMP-2 and MMP-9 inhibited by IP3R–Ca2+–CaMKII blockers in OV-RCN3 ESCC cells. (J) Invasion ability of OV-RCN3 ESCC cell inhibited by IP3R–Ca2+–CaMKII blockers. *p < 0.05, **p < 0.01, ***p < 0.001, t-test. NC, negative control

Fig 4: IP3R1 remodeling in aortic tissues from patients with HF.(A) Representative immunoblots showing total IP3R1 expression, phosphorylation of serine/tyrosine kinases, and AKAP9 binding to the channels, as assessed by immunoprecipitation (IP: IP3R1); RyR2 phosphorylation levels of PKA-induced RyR2 phosphorylation on serine 2808 (pSer-2808); and p-MLC20 levels in aortic tissues from patients with HF (n = 5) and controls (n = 4). (B–G) Quantification of the immunoblots shown in A. Individual values with the mean ± SEM are shown. *P < 0.05 versus control, by 2-tailed Student’s t test.

Fig 5: Functional role of VSMC IP3R1 in HF.(A) LVEF evaluated by serial echocardiography following surgical ligation of the LAD. (B–D) Neurohormonal activation in HF assessed by measuring blood levels of catecholamines and aldosterone. (E) Representative Masson’s trichrome–stained images showing interstitial cardiac fibrosis. Scale bars: 50 µm. (F) Quantification of interstitial cardiac fibrosis. (G) Measurement of cardiac afterload (ratio of end-systolic pressure and stroke volume); other hemodynamic parameters are reported in Supplemental Table 2. (H) Coronary flow reserve determined in vivo in IP3R1fl/fl and IP3R1VSMC–/– mice. (I) Representative immunoblots of denuded mesenteric arteries (n =8 mice/group) showing MLC protein phosphorylation levels in sham-operated and MI IP3R1fl/fl and IP3R1VSMC–/– mice. (J) Quantification of results in I. Data are shown as individual values with the mean ± SEM. *P < 0.05 versus sham, by 2-tailed Student’s t test; #P < 0.05 versus MI IP3R1fl/fl mice, by repeated-measures ANOVA.