Fig 2: Redistribution of astrocytic Cx43 after OGD/R injury and the effects of SalB and CBX. We cultured primary astrocytes and performed cytoimmunofluorescent staining for Cx43. a1 In the normal group, Cx43 was mainly expressed discontinuously in the plasma membrane. b1 At high magnification, Cx43 mainly expressed at the gap junction and some were punctate distributed. a2, b2 In the OGD/R group, Cx43 was mainly expressed in the cytoplasm, which existed in the shape of block and grain. a3, b3 Compared to the OGD/R group, the OGD/R-SalB group exhibited weaker cytoplasmic Cx43 staining but enhanced plasma membrane Cx43 staining. The Cx43 expressed at gap junctions was morphologically similar to that in the normal group. a4, b4 The OGD/R-CBX group exhibited staining results similar to those of the OGD/R-SalB group, though the Cx43 at gap junctions covered a larger area than in the control and OGD/R-SalB groups. Scale bar = 50 µm

Fig 3: Evaluating the purity of primary cultured astrocytes and Cx43 expression in astrocytic subcellular components. Primary astrocytes were prepared from newborn mice. a Western blotting analyses of Cx43 levels were conducted with samples prepared from astrocytes from the control, OGD/R, OGD/R-SalB, and OGD/R-CBX groups. Representative protein bands are displayed for total protein extractions and the plasma membrane and cytoplasm compartments. b After OGD/R injury, Cx43 levels were significantly upregulated in the cytoplasm but downregulated in the plasma membrane. SalB treatment reversed these effects. The four groups did not significantly differ in total Cx43 levels. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 50 μm

Fig 4: Number of branches in different groups following tube formation assay (the resulting data are presented as the mean ± SD; *P<0.01 vs. NC; n=30 in each group). NC, normal control group; Cx43, connexin 43; siRNA, small-interfering RNA.

Fig 5: OGD/R injury with SalB or CBX application influenced astrocytic phosphorylated Cx43 and corresponding kinases. a1, a2 OGD/R injury had no significant effect on cytoplasmic PKCe levels but significantly increased plasma membrane levels of PKCe and its Ser729-phosphorylated activated state. SalB and CBX reduced PKCe levels in the plasma membrane and increased them in the cytoplasm. Conversely, the OGD/R group’s Ser368-phosphorylated Cx43 levels were decreased in the plasma membrane and increased in the cytoplasm. SalB reversed these effects, but CBX reduced Ser368-phosphorylated Cx43 levels. b1, b2 The OGD/R group exhibited decreased cytoplasmic levels of Thr308-phosphorylated PKB, though SalB reversed this effect. There were no significant changes in the plasma membrane levels. Furthermore, the OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Ser373-phosphorylated Cx43. SalB reduced the plasma membrane levels but further increased the cytoplasmic levels. CBX had little effect on the levels of Ser373-phosphorylated Cx43 and Thr308-phosphorylated PKB. c1, c2 The OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. The OGD/R group also exhibited elevated plasma membrane levels of Src, which may be related to the elevated Tyr416-phosphorylated Src levels. SalB increased the plasma membrane levels of Src’s Tyr527-phosphorylated deactivated form but did not significantly affect plasma membrane levels of Tyr416-phosphorylated Src. CBX significantly reduced cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. Furthermore, the OGD/R group exhibited increased cytoplasmic and plasma membrane levels of Tyr265-phosphorylated Cx43. SalB reversed this effect, but CBX achieved only non-significant reduction of the plasma membrane levels. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. *p < 0.05, **p < 0.01, and ***p < 0.001