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1.
Figure 5

Figure 5. From: TGF-?3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells.

Graph demonstrating the fibril densities obtained from the TEM images for HCK and HCF constructs' ±T3. HCKs showed higher density of fibrils per unit area when compared with HCFs, both ±T3, with T3 stimulation of the HCKs causing the density to become significantly greater than with the HCFs (**P < 0.01).

Dimitrios Karamichos, et al. Invest Ophthalmol Vis Sci. 2013 October;54(10):6612-6619.
2.
Figure 1

Figure 1. From: TGF-?3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells.

Schematic representation of the method used to quantify the fibril density from TEM images with ImageJ software. (A) The original TEM image with random 0.5 μm × 0.5 μm area selected (white box); (B) selected area magnified; (C) magnified image converted to a binary black/white image; (D) collagen fibrils identified by edge detection method (blue spots); (E) outlines of each detected area; (F) fitted ellipses to detected area; and (G) final highlighted ellipses showing the collagen fibrils. The fibril density was then calculated by counting the fibrils within the selected area.

Dimitrios Karamichos, et al. Invest Ophthalmol Vis Sci. 2013 October;54(10):6612-6619.
3.
Figure 6

Figure 6. From: TGF-?3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells.

Confocal images of the immunolocalization of Col I (AD), Col V (EH), Col III (IL), and TSP-1 (MP) in HCK and HCF constructs ±T3 stimulation. Both Col I (AD) and V (EH) were present throughout the constructs under all conditions. Upon T3 stimulation, the amounts of Col I (C, D) and V (G, H) remained unchanged; however, the collagen seemed to be more aligned when compared with No T3 [A, B] and [E, F]. Little, if any, Col III expression was apparent in, both the HCKs and HCFs ± T3 stimulation (IL). Also, little, if any, TSP-1 expression was present in HCKs ± T3 stimulation (M, O); however, high levels were apparent with HCFs without T3 stimulation (N). Upon T3 stimulation, the expression of TSP-1 decreased (P).

Dimitrios Karamichos, et al. Invest Ophthalmol Vis Sci. 2013 October;54(10):6612-6619.
4.
Figure 2

Figure 2. From: TGF-?3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells.

(A) Quantitative reverse transcriptase PCR analysis of both HCKs and HCFs for keratocan expression. Four conditions were tested: No VitC, VitC, T3, and VitC+T3. Under all conditions HCKs expressed significantly higher levels of Keratocan when compared with HCFs (P < 0.01). (B) Representative images showing the morphology for both HCKs (a, b) and HCFs (c, d) in two-dimensional (2D) (a, c) and 3D (b, d) cultures. HCKs maintained their dendritic morphology (a, b), whereas HCFs showed a more fibroblastic, elongated morphology (c, d). Red = Phalloidin, Blue = TOPRO-3. Scale bars: 50 μm.

Dimitrios Karamichos, et al. Invest Ophthalmol Vis Sci. 2013 October;54(10):6612-6619.
5.
Figure 3

Figure 3. From: TGF-?3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells.

Quantitative reverse transcriptase PCR analysis of both HCKs and HCFs for (A) Col I, (B) Col V, and (C) Col III expression. Four conditions were tested: No VitC, VitC, T3, and VitC+T3. HCKs showed significant upregulation of Col I and V (∼4–6-fold, P < 0.01) when T3 was present independent of VitC stimulation [A] and [B], respectively). On a smaller scale HCFs also showed upregulation of both Col I and V (∼2-fold, P < 0.05). HCKs expressed Col I and V at higher levels compared with HCFs under all conditions. Col III (C), on the other hand, was significantly upregulated in HCFs under all conditions when compared with HCKs (∼6–53-fold, P < 0.01).

Dimitrios Karamichos, et al. Invest Ophthalmol Vis Sci. 2013 October;54(10):6612-6619.
6.
Figure 4

Figure 4. From: TGF-?3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells.

Graph of (A) the average construct thickness, (B) total number of cells per unit area (millimeter squared), and (C) the ratio of constructs' thickness over cell per unit area for both HCKs and HCFs stimulated with VitC ± T3. (A) Both HCK and HCF constructs' thicknesses were significantly upregulated (3- and 1.6-fold, respectively; P < 0.001) when stimulated with T3. HCFs total thickness was higher (2–4-fold) under all conditions when compared with HCKs. (B) HCFs had a higher number of cells per unit area ± T3 stimulation when compared with HCKs. A significant decrease in number of cells per unit area following T3 stimulation (P < 0.001) is shown for HCKs compared with VitC only. (C) The ratio of construct thickness/cells per unit area shows the difference in ECM secretion between HCKs and HCFs ± T3. The amount of ECM per cell was dramatically increased in HCKs upon T3 stimulation (∼11-fold) compared with that of the HCFs (∼3-fold), suggesting a greater ability of the HCKs to increase ECM production than HCFs.

Dimitrios Karamichos, et al. Invest Ophthalmol Vis Sci. 2013 October;54(10):6612-6619.

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