Journal: PLOS ONE
Article Title: Quantitative imaging and semiotic phenotyping of mitochondrial network morphology in live human cells
doi: 10.1371/journal.pone.0301372
Figure Lengend Snippet: (A) Fluorescent representative imaging of nuclei using Hoechst staining (blue; HOE), membranes with CellMask dye (green; CMG) and cellular distribution of the mitochondria-specific dye MitoTracker Red FM (red; MTR) after irradiation with increasing doses of UVB (50 to 400 mJ/cm 2 ). Pictures were taken using live-cell confocal microscopy 6h after irradiation. Single, overlapping images and zoom inserts are presented. Scale bar = 10μm. (B) After analysis of a minimum of 300 cells with MitoTouch software, the distribution of normalized morphological features (Vn) is represented by a MitoSpider plot. Significant differences with respect to the control condition, n = 3 independent experiments, are notified by small (*: p<0.05), medium (**: p<0.01) or large (***: p<0.001) circles (t-test). Fibroblasts are sensitive to increasing UVB irradiation as shown by the purple broken lines in relation to the reference red circle (no UVB irradiation). Profiles are similar until 200 mJ/cm 2 , and slightly modified with 300 and 400 mJ/cm 2 as reported by the MitoSpider plots and the associated MitoSigils. (C) Projection of the samples onto the 2D space generated by the first two Principal Components (a). PCA2 segregates irradiated samples from untreated ones, independently of the irradiation dose. Correlation of each feature to the first two Principal Components (b). Projection of the samples onto the 3D space generated by the first three Principal Components (c). Projection of the samples onto the 2D space generated by the first two LDA discriminant axis (d). LDA1 splits irradiated samples from untreated ones, while LDA2 highlights the irradiation dose gradient. Correlation of each feature to the first two LDA discriminant axis (e). Note that features parallel to LDA1 have an explanatory role in the observed segregation between treated and untreated samples, whereas those parallel to LDA2 in the categorization of irradiated samples depending on their UVB dose. Projection of the samples onto the 3D space generated by the first three LDA discriminant axis (f). UVB doses are shown in the inset.
Article Snippet: During UVB irradiation and microscopy, NHDF were cultured in phenol-free Fibroblast Basal Medium-2 (C-23225) with supplementation of Growth Medium 2 SupplementMix (C-39325) and NHEK in phenol-free Keratinocyte Basal Medium (C-20216) and Growth Medium 2 SupplementMix (C-39016) (PromoCell, Germany).
Techniques: Imaging, Staining, Irradiation, Confocal Microscopy, Software, Control, Modification, Generated
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