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Fig. 1. Quantitative analysis of the perinuclear clustering of endogenous Lamp1 from wild-type and BLOC-deficient fibroblasts. (A-C) Low-magnification fluorescence images of cells stained with anti-Lamp1 were acquired using a fluorescence microscope equipped with a digital camera. Images were processed to calculate areas (in pixels) and mean fluorescence intensities (in arbitrary units) of (A) `whole cell', (B) `nucleus' and (C) `nucleus + perinuclear region', which were drawn (yellow lines) by an operator who was unaware of the identity of the sample. Bar, 20 µm. (D) Plot of the ratio between background-corrected, mean fluorescence values of the regions defined as perinuclear (i.e. `nucleus + perinuclear region' minus `nucleus') and cytoplasmic (i.e. `whole cell' minus `nucleus') as a function of the ratio between the areas of `nucleus + perinuclear region' and `whole cell'. Filled circles correspond to values derived from the cell shown in A-C. Open circles correspond to the analysis of another cell in which Lamp1 staining was relatively less concentrated in the perinuclear area. For each cell, the perinuclear clustering index (PCI) was calculated as the slope obtained by linear regression of the data within the range of relative area sizes 0.15-0.25 (gray in D), multiplied by –1 to render positive values. (E) PCI of immortalized skin fibroblast lines derived from mutant murine strains deficient in BLOC-1 (pallid), BLOC-2 (cocoa) and BLOC-3 (pale ear), as well as from a wild-type control strain. For each cell line, the PCI of 20 randomly selected cells was averaged. Bars represent mean±standard error of three independent cell lines per strain. *P<0.05 (ANOVA followed by Tukey's test).
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