Fig 1.
RA induces hESC differentiation and concomitant upregulation of HAND1 and GATA-4 expression.
(A) Immunofluorescence analysis of OCT4 (green) in RA-treated hESC. Cell nuclei were labeled with DAPI (blue). Scale bar: 100 μm. (B) Flow cytometric analysis of OCT4, SSEA-3, NANOG, SOX-2, GATA-4 and HAND1 expression in RA-treated hESC on day 3 and 5. Untreated hESC (grown in mTeSR1) harvested at the identical time-points were used as controls. Average Fold Change based on Median Fluorescence Intensity (MFI) values was calculated in relation to corresponding control (mTeSR1) samples. Statistical significance with P-values less than 0.05 are labeled with “*” (C) Flow cytometric analysis of RA-treated and control (mTeSR1) hESC cells co-stained with antibodies recognizing OCT4 and GATA-4 or HAND1. Overlays of RA-treated (red) and control (mTeSR1, blue) hESC populations are presented. (D) Flow cytometric analysis of RA-treated and control (mTeSR1) hESC co-stained with GATA-4 and HAND1-specific antibodies. The percentages of cell populations in each quadrant are indicated on the density plots.
Fig 2.
The expression of laminin α5, β1, β2 and γ1 chains in differentiating RA-treated hESC.
(A) Immunofluorescence analysis of laminin (LM) chains α5, β1, β2 and γ1 and OCT4 in RA-treated hESC. Laminin chains (red) and OCT4 (green) were detected with appropriate antibodies. Cell nuclei were labeled with DAPI (blue). Scale bar: 100 μm. (B) Multilayer confocal microscopy was used to visualize the LM β1 chain (red) localization and OCT4 (green) expression in RA-treated hESC. Cell nuclei were labeled with DAPI (blue). Scale bar: 20 μm. (C) Immunoprecipitation of laminin-511 and -521 from RA-treated hESC. The protein complexes were immunoprecipitated using laminin α5 chain-specific antibody. The laminin α5, β1, β2 and γ1 chains were detected by Western blot analysis using corresponding antibodies.
Fig 3.
Laminin α5 chain expression increases in differentiating hESC.
Flow cytometric analysis of RA-treated (red population) and control (mTeSR1, blue population) hESC stained with antibodies recognizing laminin (LM) α5 chain and SSEA-3 (A) or HAND1 (B).
Fig 4.
hESC express a diverse range of laminin chains.
(A) Western blot analysis of indicated laminin (LM) chains in hESC. The lysates of JEG-3 (α1 chain), A431 (α2, α3, β1-β3, γ1- γ3), A549 and JAR (α5) cells and human platelets (α4) were used as controls. See the text for a detailed explanation. (B) Flow cytometric analysis of laminin (LM) chains α1, α2, α3 and α5 in hESC on day 3 and 5 of RA treatment. Untreated hESC (mTeSR1) were used as controls. Average Fold Change based on Median Fluorescence Intensity (MFI) values was calculated in relation to corresponding control (mTeSR1) samples. Statistical significance with P-values less than 0.05 are labeled with “*”.