Chapter 494
Suplementary material
Figure S1
Figure S1. Analysis of correlations between cell viability and ThiolRedox Status (TRS) in HDFs treated with 0 mM, 2 mM or 4 mM H2O2 during one, one hour and a half, and two hours. Linear correlation between cell viability and TRS (R2 = 0.90). The data of VB-48 is expressed as Mean Fluorescence Intensity (MFI). Each plotted data point corresponds to the mean values of three and four independent experiments performed with two different control cell lines (NHDF-01, NHDF-02), respectively (total n = 7).
Figure S2
Figure S2.Analysis of mitochondrial membrane potential (MMP). Density plots show the red fluorescence (mitochondrial located) in the y-axis and the green fluorescence (cytoplasmic located) in the x-axis. MMP analysis has been plotted in logarithmic scale. CCCP co-incubation with JC-1 was used to establish the gate to differentiate polarized from depolarized MMP. Nonviable cells have been excluded based on DAPI staining.
Figure S3
Figure S3. MitoSOX staining detected by confocal laser scanning microscopy (CLSM). HDFs were incubated with 5 mol/L MitoSOX during 20 minutes at 37°C, to analyse the distribution pattern of MitoSOX in HDFs. Scale bar =20 m
Figure S4
Figure S4. Histograms of mitochondrial superoxide levels (MSL) by the probe MitoSOX.(A) HDFs from healthy individuals were incubated in the absence or presence of 2 mmol/L H2O2 or 4 mmol/L H2O2 during one, one hour and a half, and two hours. Solid line: 0 mmol/L H2O2; Dashed line: 2 mmol/L H2O2; Dotted line: 4 mmol/L H2O2. (B) Fluorescence intensities of HDF from VLCADD patients and healthy individuals in the absence or presence of 2 mmol/L H2O2 or 4 mmol/L H2O2 during one hour. Solid line: NHDF; Dashed line: P1; Dotted line: P2. Non-viable cells were gated out based on RedDot2 uptake. X-axis indicates MitoSOX fluorescence that correlates to the MSL. Fluorescence is quantified as the mean intensity per cell and it is plotted in logarithmic scale.