Fig 1: Reticulocalbin3 (RCN3) promoted chemoresistance by targeting inositol 1,4,5-trisphosphate receptor 1 (IP3R1)/Ca2+ through inhibiting reactive oxygen species (ROS) production. (A) Western blot showed the expression of chemoresistance proteins MDR1(D-11), ABCG2, and RCN3. (B) After treatment with different concentrations of cisplatin (DDP), CCK-8 showed the cell growth inhibition rate. (C) CCK-8 showed the survival rate of drug-resistant cells after interfering RCN3 expression. (D) Transmission electron microscopy showed the structure of ECA109, ECA109/DDP, and ECA109/DDP-shRCN3 cells. (E) Apoptosis analysis showed apoptosis rates of interfering RCN3 ± calcium chelator (BAPTA AM) resistant cell line, treated with DDP 24 h. (F) Quantitative analysis of mitochondrial membrane potential measurements with JC-1. The change of interfering RCN3 ± calcium chelator (BAPTA AM) resistant cell line, treated with DDP 24 h. (G) ROS analysis showed reactive oxygen of interfering RCN3 ± calcium chelator (BAPTA AM) resistant cell line, treated with DDP 24 h. (H) Apoptosis analysis showed apoptosis rate of overexpression of RCN3 ± siIP3R1, treated with DDP for 24 h. (I) Quantitative analysis of mitochondrial membrane potential measurements with JC-1. The change of overexpression of RCN3 ± siIP3R1 resistant cell line, treated with DDP 24 h. (J) ROS analysis showed overexpression of RCN3 ± si-IP3R1, treated with DDP 24 h. (K) Immunohistochemical staining of RCN3 protein in esophageal squamous cell carcinoma (ESCC) biopsy tissues (left panel). H&E staining of platinum-based chemotherapy ESCC tissues (right panel). *p < 0.05, **p < 0.01, ***p < 0.001, t-test. NC, negative control
Fig 2: Immunofluorescence staining of ZO1 and phalloidin double staining on cross-sectional patellar tendon from wild-type mice and Scx-Cre; Rcn3fl/fl (tendon-specific Rcn3 loss-of-function model) littermates during postnatal tendon maturation (A). H&E staining of the cross-sectional patellar tendon (B). Cell area (C) and protrusion number (D) of the patellar tendon. (Scale bar indicates 5 µm (A) and 20 µm (B), * indicates P < 0.05, and ** indicates P < 0.01 between genotypes, n = 3).
Fig 3: Quantitative real-time PCR of Achilles tendons from wild-type mice and Scx-Cre; Rcn3fl/fl (tendon-specific Rcn3 loss-of-function model) littermates at P28 (C). (* indicates P < 0.05, ** indicates P < 0.01 and # indicates P < 0.001 between genotypes, wild-type mice n = 6, Scx-Cre; Rcn3fl/fl mice n = 5).
Fig 4: The weight of wild-type mice and Scx-Cre; Rcn3fl/fl mice at P30 (A). Immunohistochemical analysis of Rcn3 on patellar and Achilles tendons from wild-type mice and Scx-Cre; Rcn3fl/fl littermates during postnatal tendon maturation (B). (Brown color indicates Rcn3, Scale bar indicates 20 µm (B) and n = 5 (A)).
Fig 5: 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
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