Fig. 2

Inhibition of Huwe1 increased the expression of Gadd45b at 24 h after reperfusion. Neuron cells were treated with lentivirus shRNA-Huwe1 or shRNA-Gadd45b on DIV 3, and then exposed to OGD 3 h and reperfusion for 24 h on DIV 7. Cells were prepared for western blot and qPCR. a The GFP-expressing lentiviral vectors cells were observed under an invert fluorescent microscope. It showed GFP (green) expressed in neurons. DAPI (blue) was used for cell nucleus staining. b ShRNA-Huwe1 (LV-shHuwe1) obviously decreased the protein expression of Huwe1 at 24 h, compared to the lentivirus-GFP-scramble control group (LV-GFP-ctrl), as seen by western blotting. c ShRNA-Huwe1 also decreased the mRNA level of Huwe1 at 24 h, as seen by qPCR. d ShRNA-Huwe1 increased the protein expression of Gadd45b at 24 h. e ShRNA-Huwe1 had no effect on the mRNA level of Gadd45b at 24 h. f ShRNA-Gadd45b (LV-shGadd45b) decreased the protein expression of Gadd45b at 24 h compared to the lentivirus-scramble control group (LV-ctrl). g ShRNA-Gadd45b decreased the mRNA level of Gadd45b at 24 h. h-i ShRNA-Gadd45b did not affect the protein and mRNA levels of Huwe1 at 24 h, as seen by western blotting (h) and qPCR (i). Tubulin was used as an internal control for western blot; GADPH was used as an internal control for qPCR. *p < 0.05 versus control, ^# p < 0.05 versus LV-GFP-ctrl or LV-ctrl. Scale bar = 10 μm