Figure 6

CHIP functions as an anti-necroptotic factor in OGD-challenged cells (A) 24 h after transfection of N2a cells with PGPU6-GFP-shNC or PGPU6-GFP-si-CHIP, cells were challenged with OGD for 4 h. After 12 h of recovery, the population of PI positive cells and apoptosis cells were determined using flow cytometry using the AnnexinV-FITC Apoptosis Detection Kit. (B) The population of PI positive cells. Bars represent the mean ± SEM of nine independent experiments. Significant differences ^bp < 0.05 vs. Control, ^ap < 0.05 vs. shNC. (C) The population of apoptosis cells. Bars represent the mean ± SEM of nine independent experiments. Significant differences ^ap < 0.05 vs. ShNC, ^bp < 0.05 vs. OGD (D,E) Representative western blot assessing of CHIP, RIPK3, p-RIPK3, p-MLKL in transfected cells were tested after transfection and OGD challenging. Bars represent the mean ± SEM of nine independent experiments. Significant differences *p < 0.05 vs. ShNC, ^#p < 0.05 vs. Control. (F) q-RT-PCR analysis of CHIP mRNA levels in transfected and OGD insulted cells. *p < 0.05 vs. ShNC. (G) q-RT-PCR analysis of RIPK3 mRNA levels in transfected and OGD insulted cells. *p < 0.05 vs. Control. Related blots are shown in Supplementary Fig. S5.