Figure 3: Oxygen–glucose deprivation in vitro induces rapid and robust formation of actin stress fibres in endothelial cells. | Nature Communications

Figure 3: Oxygen–glucose deprivation in vitro induces rapid and robust formation of actin stress fibres in endothelial cells.

From: Rapid endothelial cytoskeletal reorganization enables early blood–brain barrier disruption and long-term ischaemic reperfusion brain injury

Figure 3

(a) HBMECs were exposed to 1-h OGD and the expression of total β-actin, polymerized F-actin and soluble G-actin was examined by western blotting at 1–6 h after OGD. The ratio of F-actin to G-actin in HBMECs was quantified as a measure of stress fibre formation. Data represent four independent experiments. F-actin expression was elevated after OGD, whereas G-actin was downregulated, resulting in an increased F/G-actin ratio. (b) Phosphorylation of ADF/cofilin (pADF/cofilin) and expression of total ADF/cofilin (tADF/cofilin) in HBMECs were assessed at 1–6 h after OGD. β-Actin was used as an internal loading control. pADF/cofilin was quantified and expressed relative to non-OGD controls (Con). Data represent four independent experiments. (c) Phosphorylation of MLC (pMLC) was evaluated at 0–3 h after OGD by western blotting and expressed relative to non-OGD controls. Data represent four independent experiments. *P≤0.05, **P≤0.01 versus non-OGD. (d) HBMECs were treated with vehicle or the ROCK inhibitor Y27632, or infected with lentiviral vectors carrying the scrambled short hairpin RNA (shRNA) sequence (ROCKsc) or ROCK-targeting sequence (ROCKt). Cells were then subjected to 1 h of OGD and pMLC was examined by western blotting at 1 h after OGD. Data represent four independent experiments. *P≤0.05, **P≤0.01. (e–i) HBMECs were infected with control empty lentivirus (Lenti), or lentiviral vectors carrying HA-tagged WT ADF (ADF), HA-tagged constitutively active mutant ADF (ADFm), MLC-targeting shRNA (MLCt), ROCK-targeting shRNA (ROCKt) or non-targeting scrambled sequences (MLCsc or ROCKsc). In separate cultures, HBMECs were treated with vehicle or Y27632. Cells were subjected to 1-h OGD. (e) HBMECs were stained at 1 or 3 h after OGD for F-actin+ stress fibres (green) and the AJ protein VE-cadherin (red), and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclear labelling. Scale bar, 30 μm. OGD-induced stress fibre formation was significantly attenuated by ADFm overexpression, MLC knockdown, ROCK knockdown or ROCK inhibition. (f–i) The ratio of F-actin to G-actin was quantified at 3 h after OGD. Data represent four independent experiments. (f) *P≤0.05, **P≤0.01 versus Lenti. #P≤0.05 versus ADF. (g) **P≤0.01 versus MLCsc. (h) *P≤0.05 versus ROCKsc. (i) **P≤0.01 versus vehicle.

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