Figure 4. CSE cause cell death and intracellular oxidative stress.

(A). Live/dead cell assay to estimate hemichannel mediated cell death under oxidative stress. Early cell death was observed in CSE treated cells (red fluorescent cells; EtBr homodimer dye) at the end of 10 hrs (F) compared to control (E), βGA pretreated cells (G) and MFA pretreated cells (H), where more live cells (green fluorescent cells; Calcein AM dye) are seen. Cell death in F was predominantly apoptotic as evident from the morphology of the dying cells viz., cell shrinkage, fragmentation into membrane bound apoptotic bodies etc., (inset in panel F). The histogram shows the % of live cells at the end of 10 hrs in all four categories. Error bar indicates SE of the mean (*p<0.012). Scale bar: 20 µm. (B). Oxidative stress molecules enter into the cells through open hemichannels as detected by ROS sensitive dye (Carboxy-H₂DCFDA). Cx-expressing cells (MC and L2), showed increase in fluorescence after 10 min treatment with CSE (B,G) and H₂O₂ (C, H) compared against their corresponding controls (A,F). Similarly, βGA pretreatment decreased such change with CSE (D,I) as well as with H₂O₂ (E,J). Cx-deficient N2A cells showed no significant change from their control (K) under same treatment conditions (L,M). Scale bar: 20 µm. Histogram summarizes the mean fluorescence intensities of different treatment conditions of all three cell lines subjected to oxidative stress. Error bar indicates SE of the mean (*p<0.0007, **p<0.0015).