Figure S1 - Reactive Oxygen Species Facilitate Translocation of Hormone Sensitive Lipase to the Lipid Droplet During Lipolysis in Human Differentiated Adipocytes

Macro to analyze HSL translocation with Image J software. For the pixel intensity around the lipid droplet, the confocal picture of the lipid droplets, detected using Lipotox red stain, was converted into a mask. This mask was then duplicated and one copy was dilated twice. The unaltered lipid mask was then subtracted from the twice dilated lipid mask and the resulting mask outlined the perimeters of the lipid droplets. This ‘peri-lipid’ mask was then applied to the HSL confocal picture and the number of pixels and intensity at each pixel could be extracted from the histogram for this picture. For the cytosolic pixel intensity, a mask was generated from the HSL confocal picture. This mask was then subtracted from the twice dilated lipid mask generated for the ‘peri-lipid’ analysis. The resulting mask, termed ‘HSL mask’, included all of the cytosol except that which was directly around each lipid droplet. This HSL mask was then applied to the HSL confocal picture and the number of pixels and intensity at each pixel could be extracted from the histogram for this picture. The index ‘translocation to the lipid droplet’ was then calculated using the average pixel intensities from the peri-lipid and cytosol analyses. (TIF)