RT-PCR probes and their dilutions

<p>RT-PCR probes and their dilutions</p

TEM image showing mitochondrial structure in HCFs, T1DMs, and T2DMs, when cultured in our 3D model.

<p>(A) HCFs: Mitochondria appeared normal, with a double membrane, discrete cristae and little internal space. (B) T1DMs: Mitochondria appeared condensed and showed expanded internal spaces. (C) T2DMs: Smaller mitochondrial structure, dilated vacuoles and presence of endoplasmic reticulum were witnessed. (D) Quantification analysis of abnormal mitochondria cells percentage in HCF, T1DM, and T2DM constructs. The data is representative of 4 independent experiments, n = 4 (* = p≤0.05; ** = p≤0.01; *** = p≤0.001; **** = p≤0.0001). Scale bars = 500nm.</p

Western Blot antibodies and their dilutions

<p>Western Blot antibodies and their dilutions</p

Scratch wound healing and MTT assay.

<p>(A-C) HCF, T1DM and T2DM cells were scratched and the relative cell migration distance was quantified at 0hr, 4hr, 24hr and 48hr time points. (D) Scratch assay quantification showing significant increase in cell migration distance in both T1DM and T2DMs when compared to HCFs. (E) MTT assay quantification for HCF, T1DM, and T2DMs. Data was normalized to HCFs and fold regulation is plotted. One way ANOVA for total n≥3 data sets. (* = p≤0.05; ** = p≤0.01; *** = p≤0.001; **** = p≤0.0001).</p

Schematic representation and quantification of the metabolic flux modulation in glycolysis cycle for HCFs, T1DMs, and T2DMs, when cultured in our 3D model.

<p>Quantification of metabolites activity expressed along the glycolysis cycle. One way ANOVA was performed for total n≥4 data sets (* = p≤0.05; ** = p≤0.01; *** = p≤0.001; **** = p≤0.0001).</p

Western blot analysis and quantification of RALDH1 and RALDH3 in postnatal human ocular tissues.

<p><b>(A)</b> Cytosol fractions from ocular tissues of donors aged 14, 49, and 50 years were immunblotted with anti-RALDH1 or anti-RALDH3. <i>Top panel</i>: RALDH1 (∼ 55–57 kDa) was abundantly expressed in the lens and choroid, moderately expressed in the sclera, and faintly detected in the retina and RPE. <i>Bottom Panel</i>: RALDH3 was not detected in postnatal ocular tissues at any of the ages examined. 3 μg total protein/lane was loaded on the blot. 1.25 μg recombinant human RALDH3 (R3) was loaded as a positive control. <b>(B)</b> Abundance of RALDH1 was measured as the integrated optical density (IOD) per 3 μg total protein of RALDH1-immunopositive bands. <b>(C)</b> Average RALDH1 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 3). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).</p

Establishment of a 3D <i>In Vitro</i> Model to Accelerate the Development of Human Therapies against Corneal Diabetes - Fig 4

<p><b>Protein expression for (A) Col I, B) Col III, C) Col V, D) Col I/Col III ratio, and E) Col/Col V ratio, in HCFs, T1DMs, T2DMs</b>.(A-E) Quantification of protein bands and their ratios that are normalized to the loading control. And, n≥7 for HCFs, T1DMs and T2DMs. Error bars represent standard error of the mean. One-way ANOVA was performed (* = p≤0.05; ** = p≤0.01; *** = p≤0.001; **** = p≤0.0001).</p

Confocal images of RALDH1 expressing cells in postnatal human ocular tissue after immunolabeling with an anti-RALDH1 antibody.

<p><b>(A)</b> Low power magnification of ocular tissues demonstrating RALDH1 labeling <i>(red)</i> in the retina and choroid. <b>(B)</b> Negative control slide (non-immune IgG used in place of primary antibody) demonstrating no labeling in the retina and choroid and auto-fluorescence in the RPE. <b>(C, D)</b> Choroid sections demonstrating RALDH1 labeling in extravascular cells throughout the choroidal stroma (arrowhead). Upward arrow in (C-D) indicates orientation for the scleral side of the choroid. Nuclei were counterstained with DAPI (<i>blue)</i>. BV, blood vessel; C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; ILM, inner limiting membrane. Scale bars = 100 μm in A, B; 20 μm in C, D.</p

Western blot analysis and quantification of RALDH2 in postnatal human ocular tissues.

<p><b>(A)</b> Cytosol fractions from ocular tissues of donors aged 16–46 years were immunblotted with anti-RALDH2 or anti-RPE65. <i>Top Panel</i>: RALDH2 immunopositive bands (∼ 55–59 kDa) were present in the choroid (C), sclera (S), retina (R), and RPE (E) of postnatal human eyes. <i>Bottom Panel</i>: anti-RPE65 was used to identify the presence of RPE contamination in the retina, choroid, and sclera samples. 3 μg total protein/lane was loaded for each blot. <b>(B)</b> Relative abundance of RALDH2 was measured as the integrated optical density (IOD) per 3 μg total protein of RALDH2-immunopositive bands. <b>(C)</b> Average RALDH2 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 5). *<i>p</i> < 0.05, **<i>p</i> < 0.01 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).</p