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. 2013 Oct 14;8(10):e77098. doi: 10.1371/journal.pone.0077098

Figure 4. Inhibition of miR-221/222 in differentiating GICs increases Nestin expression and decreases GFAP and TUJ1 levels.

Figure 4

miR-221 and miR-222 inhibition in GN1C cells growing in differentiation medium was carried out with specific anti-miRs and was confirmed by q-RT-PCR 14 days after transfection, compared to cells transfected with anti-miR negative control (anti-C-) (A, B). The expression of miR-221 and miR-222 was normalized with respect to RNU6B as 2-ΔCt. The expression of the progenitor marker Nestin (NES) as well as of astrocytic (GFAP) and neuronal (TUJ1) differentiation markers was also measured by q-RT-PCR and normalized with respect to GAPDH and to the cells transfected in parallel with the anti-miR negative control as 2-ΔΔCt (C, D). Transfections were carried out in triplicate. Dotted lines indicate the expression level of GN1C cells transfected with anti-miR negative control. The corresponding protein expression levels of Nestin, GFAP and TUJ1 were visualized by immunofluorescence (E) using antibodies conjugated to FITC (green) or Texas Red (red) counterstained with DAPI (blue). Images were acquired at 20X magnification with a 739CCD camera coupled to an Axio Imager Z1 microscope (Carl Zeiss Inc.) using the Isis Imaging System software. Quantification of mean intensity for Nestin, GFAP and TUJ1 fluorescence is displayed (F). * indicates p value <0.05 using unpaired t-test or Mann-Whitney U test for statistical analysis and the Holm-Bonferroni correction for multiple comparisons.