Effects of p13 on ROS production in HeLa cells. HeLa cells were transfected with plasmids expressing p13-GFP or mt-GFP and cultured in standard (4.5 g/L) or low (1 g/L) glucose medium and loaded with MitoTracker Red H₂-MTR. MTR signals were analyzed by confocal microscopy and quantitated in regions of interest (ROI) using the Histogram software tool in a time-lapse experiment of 30 minutes. (A-B) Plots show the fractional rate of accumulation of MTR (ΔF/F₀, as described in “Analysis of ROS production in living cells”) over time. Data represent mean and SE of ΔF/F₀ recorded in at least 60 cells per time point per sample in 3 independent experiments. (C) Representative end-point fluorescence recordings and an example of the quantitation of the MTR signals over the indicated ROI. This analysis was carried out on at least 30 cells in randomly selected fields, with data collected after 30 minutes from a total of 3 experiments. (D) Presents the resulting mean MTR fluorescence values and SE bars. The effects of p13 on ROS were specifically triggered in response to glucose deprivation (P < .01 by Student t test).