Figure 3.

Effects of TGF β 1, 2, 3 and neutralizing antibody anti-TGF-βs on hCECs cultivated in presence of FBS. After cell seeding in 384-well plates, the hCECs were cultured in culture medium based on BGM that contained 8% FBS for 4 weeks. BGM only (control) or BGM containing 10 ng/mL of TGF-β1, 2, or 3 or 10 µg/mL of neutralizing antibody was applied. (A) Illustrative images. Cell lateral membranes were stained in green by NCAM and the nuclei were counter-stained in blue using DAPI. A higher magnification (X40) was added in order to show the cell morphology. The images were taken in the center of each well. Images of the same magnification were acquired with the same parameters. The scale bar was 200 µm for X10 images and 50 µm for X40 images. (B) Assessment of standardized ECD. ECD was assessed by nuclei counting using DAPI staining. The ECD data were standardized by calculating the ratio with controls. (C) Assessment of EndMT by normalized fluorescence intensity of NAM. The IF images of NCAM were acquired with an FITC filter using the same parameters. Normalized fluorescence intensity represents the fluorescence intensity per cell/cell perimeter as described in the Materials and Methods. The data were also standardized by calculating the ratio with the control. For B and C, at least two cultures from different donors were used (n ≥ 16 wells). Significant differences by comparison to controls were denoted by red stars on the corresponding bars: *** p < 0.0001, ** p < 0.001, * p < 0.005.