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. 2016 Dec 22;8(1):1–10. doi: 10.1016/j.stemcr.2016.11.011

Figure 3.

Figure 3

l-Proline Mimics bFGF/Activin A Treatment in ESCs

(A and B) DRESCs were passaged three times in VitC (100 μM), plated at low density and treated with either l-Pro (1 mM) or F/A and analyzed at days 3 and 5. Representative FACS plots (A, left panels) and quantification (A, right panel) of mCherry±/eGFP± cell distribution. Quantification of eGFP intensity mean (B). Data are mean ± SEM; ∗p < 0.001 (n = 3 independent experiments).

(C) Schematic representation of eGFP/mCherry-positive cells' distribution in colonies from l-Pro- and F/A-treated DRESCs.

(D) Venn diagram showing the overlap between differentially expressed genes in mCherry+/eGFP+ (yellow) cells from l-Pro-treated (day 5) and F/A-treated (days 3 and 5) DRESCs. Relative expression of each gene was normalized to untreated cells.

(E) Heatmap representation of genes deregulated in l-Pro-treated (day 5) and F/A-treated (days 3 and 5) cells compared with control.

(F) Western blot analysis of phospho-Erk/Erk (upper panels) and phospho-Smad2/Smad2 (lower panels) in l-Pro-induced cells (PiCs) treated with ±bFGF (12 ng/mL) and ±Activin A (20 ng/mL) (15 min), respectively. Anti-Gapdh antibody was used as loading control. Densitometric analysis (arbitrary densitometry units [ADU]) is shown as pErk/Erk and pSmad2/Smad2 ratio. Data are mean ± SEM; ∗p < 0.01 (n = 3 independent experiments).

(G) Projection to latent structure-discriminant analysis (PLS-DA) score plot showing the projection of all the spectral classes analyzed (−l-Pro, green boxes; and +l-Pro, red boxes). The model quality is evaluated by the goodness of fit and the goodness of prediction, represented by parameters R2Y = 0.98 and Q2 = 0.96. Classes are separated along the first principal component t[1], which represents interclass variation. The second component t[2] accounts for intraclass variation.

(H) Schematic representation of the oxidative phosphorylation (blue) and glycolysis (red) metabolic pathways.

(I) Representative phase-contrast images of ESC and PiC colonies (day 5), and ±2-DG (50 mM; 16 hr), stained with crystal violet. Scale bars, 200 μm.

(J) Tetramethylrhodamine (TMRE) mean intensity in ESCs, EpiSCs, and PiCs at day 5. Data are mean ± SEM; ∗p < 0.05 (n = 3 independent experiments).

See also Figure S3.