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. 2011 Dec 16;6(12):e28981. doi: 10.1371/journal.pone.0028981

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Aguer et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A. Representative light microscopy of myotubes derived from control subjects (Control) or obese type 2 diabetic patients (OBT2D), after 8 days of differentiation, stained by oil red O after palmitate treatment (0.6 mM for 16 h). The 4 Control and the 5 OBT2D cells showed a staining similar to the representative pictures. Arrows show reserve cells. Scale bar represents 30 µm. B. Merged picture of FAT/CD36 (H300), troponin T (TT) and dapi staining in OBT2D differentiated cells (for 8 days). Living cells were incubated for 1 h with an antibody against FAT/CD36 (H300, left panel) and for 1 h with a polyclonal secondary antibody conjugated to alexa 488 (green). After fixation and permeabilization, cells were incubated with an antibody against troponin T (TT) visualized using a secondary monoclonal antibody conjugated to alexa 546 (red). The 5 OBT2D cells showed a staining similar to the representative pictures. Arrows show reserve cells. Scale bar represents 30 µm. C. Left panel: Western blot analysis of the expression of total FAT/CD36 in proliferative (0), and after 2, 4, 6 and 8 days of differentiation of cells established from two control subjects (Control 1 and 2) and two obese type 2 diabetic patients (OBT2D 1 and 2). Troponin T (TT) and caveolin 3 were used as markers of myotube differentiation and α-tubulin as a loading control. Right panel: quantification by density analysis of the 2 controls and the 2 OBT2D. Data are presented normalized to α-tubulin protein expression where the value of Control cells after 8 days of differentiation has been arbitrary chosen as the reference value equal to 1. D. Representative immunofluorescence microscopy of cells established from obese type 2 diabetic patients (OBT2D) in proliferative (0), and after 2, 4 and 8 days of differentiation, treated by palmitate (0.6 mM for 16 h), incubated for the last hour with CD36-alexa488 antibody (green) and stained by oil red O (red) after fixation. The 5 OBT2D cells showed a staining similar to the representative pictures. Scale bar represents 30 µm. E. Percentage of inhibition of lipid content in Control (n = 4) and in OBT2D differentiated satellite cells (n = 5) after phloretin stimulation (400 µM for 30 min) followed by palmitate treatment (0.6 mM for 16 h). Data are means ±SEM. Each point was assayed in triplicate for each of the 9 independent cell cultures. *, p<0.05, OBT2D versus Control cells. F. Percentage of inhibition of lipid content in Control (n = 4) and OBT2D differentiated satellite cells (n = 4) after SSO stimulation (250 µg/ml for 30 min) followed by three PBS washes and by palmitate treatment (0.6 mM for 16 h). Data are means ±SEM. Each point was assayed in triplicate for each of the 8 independent cell cultures. *, p<0.05, OBT2D versus Control cells.