Fig 1: Downregulation of Cx43 and STIP1 in the H/R cellular model. The H9C2 cellular model of H/R was constructed (A). Compared with the NC group, SLDT assay showed decreased intercellular gap junctions in the H/R group (B); CCK‐8 assay revealed that H9C2 cell viability was decreased in the H/R group (C); flow cytometry revealed increased H9C2 cell apoptosis in the H/R group (D); the elevated level of ROS was detected by DCFH‐DA in the H/R group (E); the declined level of SOD, and increased levels of MDA and LDH were tested by ELISA in the H/R group (F–H); qRT‐PCR (I) and western blotting (J) showed that the expression of Cx43 and STIP1 was downregulated in cardiomyocytes in the H/R group. Three independent experiments were performed, *p < .05 compared with the NC group. CCK‐8, cell counting kit 8; Cx43, connexin 43; DCFH‐DA, 2′,7′‐dichlorodihydrofluorescein; ELISA, enzyme‐linked immunosorbent assay; H/R, hypoxia/reoxygenation; LDH, lactic dehydrogenase; MDA, malondialdehyde; NC, negative control; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; ROS, reactive oxygen species; SLDT, scrape‐loading and dye transfer; SOD, superoxide dismutase; STIP1, stress‐induced phosphoprotein 1.
Fig 2: Effects of STIP1 on intercellular communication and oxidative stress in H/R‐treated H9C2 cells. Compared with the H/R + OE‐NC group, qRT‐PCR showed the successful transfection of OE‐STIP1 (A). After overexpression of STIP1, western blotting showed that the protein expression of STIP1 and Cx43 was elevated (B); SLDT assay showed enhanced intercellular gap junctions (C); CCK‐8 assay showed increased H9C2 cell viability (D); flow cytometry revealed decreased H9C2 cell apoptosis (E); DCFH‐DA assay exhibited the reduced level of ROS (F); ELISA displayed the upregulated level of SOD and downregulated levels of MDA and LDH (G–I). Three independent experiments were performed. *p < .05 compared with the H/R + OE‐NC group. CCK‐8, cell counting kit 8; Cx43, connexin 43; DCFH‐DA, 2′,7′‐dichlorodihydrofluorescein; ELISA, enzyme‐linked immunosorbent assay; H/R, hypoxia/reoxygenation; LDH, lactic dehydrogenase; MDA, malondialdehyde; NC, negative control; oe, overexpression; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; ROS, reactive oxygen species; SLDT, scrape‐loading and dye transfer; SOD, superoxide dismutase; STIP1, stress‐induced phosphoprotein 1.
Fig 3: Low expression of Cx43 and STIP1 in the isolated Langendorff‐perfused rat heart model. Compared with the control group, qRT‐PCR (A) and western blotting (B) showed that the expression of Cx43 and STIP1 was reduced in myocardial tissues in the I/R group. n = 8, *p < .05 compared with the control group. Cx43, connexin 43; I/R, ischemia/reperfusion; qRT‐PCR, quantitative reverse transcription‐polymerase chain reaction; STIP1, stress‐induced phosphoprotein 1.
Fig 4: Inhibitory effect of HSP90 on Cx43 ubiquitination. Western blotting showed that sh‐HSP90 transfection inhibited the expression of HSP90 (A). Co‐IP assay illustrated that the ubiquitination level of Cx43 in the H/R group was higher than that in the NC group and that sh‐HSP90 transfection further enhanced Cx43 ubiquitination (B). Each assay was run in triplicate, *p < .05 compared with the NC + sh‐NC, H/R + sh‐NC, NC, H/R, or NC + sh‐HSP90 group. Co‐IP, co‐immunoprecipitation; H/R, hypoxia reoxygenation; NC, negative control; sh‐HSP90, HSP90 knockdown vector.
Fig 5: Binding of Cx43 to HSP70 and HSP90. The string database predicted a potential interaction between STIP1 and HSP90 (A). Co‐IP assay showed that STIP1 could directly bind to HSP90 and HSP70 in myocardial tissues of the I/R group (B) and H9C2 cells of the H/R group (C). Fluorescence co‐localization assay revealed that Cx43‐HSP70 and Cx43‐HSP90 existed in complex forms (D). Co‐IP assay displayed that Cx43 bound to HSP90 and HSP70 in rat myocardial tissues, where compared with the control group, HSP70 expression was increased and HSP90 expression was decreased in the immune complex pulled down by the Cx43 antibody in the I/R group (E). Co‐IP assay displayed that Cx43 bound to HSP90 and HSP70 in H9C2 cells, where compared with the NC group, HSP70 expression was increased and HSP90 expression was decreased in the immune complex pulled down by the Cx43 antibody in the H/R group (F). Each experiment was independently repeated three times. Co‐IP, co‐immunoprecipitation; Cx43, connexin 43; H/R, hypoxia reoxygenation; I/R, ischemia reperfusion; NC, negative control; STIP1, stress‐induced phosphoprotein 1.
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