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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