Fig. 5: Loss of DDHD2 increases ROS-induced apoptosis sensitivity and delays ROS clearance in MEFs. | Cell Death & Disease

Fig. 5: Loss of DDHD2 increases ROS-induced apoptosis sensitivity and delays ROS clearance in MEFs.

From: Loss of DDHD2, whose mutation causes spastic paraplegia, promotes reactive oxygen species generation and apoptosis

Fig. 5

a WT and DDHD2 KO MEFs were treated with 2 µM antimycin A and 1 µM rotenone (AR) or 0.1 mM PQ for 16 h, followed by staining with TUNEL. The number of TUNEL-positive cells was determined. b DDHD2 KO MEFs and ones with stable expression of DDHD2-WT-mCherry or DDHD2-S351A-mCherry were treated with AR for 16 h, followed by staining with TUNEL. The number of TUNEL-positive cells was determined. c WT MEFs were transfected with a plasmid encoding FLAG (Empty), FLAG-DDHD2 WT or one of the indicated constructs. At 24 h after transfection, the cells were treated with 0.1 mM PQ for 1 h, followed by incubation with 2.5 µM CellROX Green for 30 min and analysis. The fluorescence intensity of CellROX Green was quantified and is expressed as the ratio relative to that in MEFs with FLAG vector transfection. No significant difference in the intensity of CellROX staining was observed between cells transfected with FLAG and SPG-associated mutant constructs. d WT and DDHD2 KO MEFs were incubated with 2 μM antimycin A and 1 μM rotenone (AR) for 3 h, washed, and then incubated without AR for 1 h. To detect ROS, cells were incubated with 2.5 µM CellROX Green for 30 min. The fluorescence intensity of CellROX Green relative to that observed just after 3-h AR treatment was determined. The scale bars for whole cell panels are 10 μm. Data in this figure represent means ± SEM (N = 3). *P < 0.05, **P < 0.01, Student’s t-test

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