Fig. 3: ROS accumulation in DDHD2 KO MEFs. | Cell Death & Disease

Fig. 3: ROS accumulation in DDHD2 KO MEFs.

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

Fig. 3

a Non-immortalized WT and DDHD2 KO MEFs were incubated with 2.5 µM CellROX Green for 30 min, and then analyzed by IF microscopy. The graph shows the ratio of the CellROX Green intensity of DDHD2 KO MEFs to that of WT MEFs. b WT MEFs, DDHD2 KO MEFs, and DDHD2 KO MEFs with stable expression of DDHD2-WT-mCherry or DDHD2-S351A-mCherry were incubated with 2.5 µM CellROX Green for 30 min, and then analyzed. The graph shows the ratio of the CellROX Green intensity of DDHD2 KO MEFs or those with stable expression of DDHD2 constructs to that of WT cells. c Lysates (20 µg) of WT and DDHD2 KO MEFs were analyzed by WB with antibodies against SOD1, catalase, and GAPDH. Three different preparations were analyzed. d DDHD2 KO MEFs were transfected with a plasmid encoding FLAG (Empty), FLAG-DDHD2 WT, FLAG-DDHD2-S351A, FLAG-DDHD2-W103R, FLAG-DDHD2-V220F, or FLAG-DDHD2-D660H. At 24 h after transfection, the cells were incubated with 2.5 µM CellROX Green for 30 min and analyzed. The fluorescence intensity of CellROX was quantified and is expressed as the ratio relative to that in WT MEFs. No significant difference in the intensity of CellROX staining was observed between cells transfected with FLAG and SPG-associated mutant constructs. The scale bars for whole cell panels are 10 μm. Data represent means ± SEM for four (a) and three (b and d) independent experiments, respectively. *P < 0.05, Student’s t-test

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