Fig. 3

Gsr expression increases in ESCs transfected with a shRNA vector targeting Nanog. a R1 ESCs were transfected with pLKO.1-puro derived vectors targeting the transcription factor Nanog (shNanog) or eGFP (shGFP), as indicated under each bar. Then, transfected cells were selected with puromycin for 48 h and RNA was extracted. Nanog and Gsr mRNA levels were analyzed by RT-qPCR. Gene expression was normalized to the geometrical mean of Gapdh and Pgk1 expression and referred to the control condition (shGFP). Results are shown as mean ± SEM of four independent experiments. Different letters indicate statistically significant differences between treatments (p < 0.05). b R1 ESCs were co-transfected with shNanog and a vector encoding the fusion protein H2B-mCherry. 48 h after transfection, Nanog was visualized by immunofluorescence and nuclei were counterstained with DAPI. Transfected cells were identified by mCherry fluorescence detection. The figure corresponds to a representative image that shows that Nanog intensity signal is lower in transfected cells respect to non-transfected cells, evidencing Nanog downregulation by shNanog. Scale bars: 10 μm