Quantification of pAkt/Akt ratios of immunofluorescence images

Quantification of pAkt/Akt ratio images was carried out as described previously (Pacitto et al., 2017). pAkt and Akt images were corrected for shade, bias and background (Pacitto et al., 2017; Hoppe, 2012). A binary image map was created from the corrected Akt (denominator) image. The corrected Akt images and binary images were combined using the ‘Logical AND’ command of MetaMorph and the resulting image saved as ‘AND image.’ The corrected pAkt image was divided by the AND image, multiplied by 100 and saved as ‘Ratio image’. These ratio images were used to quantify the average pAkt/Akt ratio value in single cells. Ratio images were thresholded to exclude background areas, and cellular subregions were selected manually. Using the ‘region measurements’ command of MetaMorph, the integrated intensity of the pAkt/Akt ratio and threshold area for a target cell were logged in Excel. The integrated intensities were divided by the threshold areas to yield relative intensities as the average pAkt/Akt ratio. More than 10 cells (Fig. 4 and Figs S2 and 3) or 20 cells (Fig. 5) were observed for each condition. One-way ANOVA was applied for the statistics.