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Suppression of ganglioside synthesis caused inhibition of the neuronal differentiation of hDPSCs
To investigate the role of gangliosides in neuronal differentiation of hDPSCs, we used lentivirus-mediated shRNA expression to knockdown Ugcg gene expression. hDPSCs were infected with four different Ugcg gene-targeting shRNAs. 72 h after infection, the efficient knockdown of Ugcg mRNA expression was observed in shRNA-3 infected beta interleukin 1 (163-171) ( Fig. 4A). FACS analysis for the GM3 expression of hDPSCs was conducted to determine if Ugcg knockdown reduced ganglioside synthesis. The GM3 expression was reduced by approximately 18% in response to the shRNA ( Fig. 4B).
Fig. 4.
Knockdown of UDP-glucose ceramide glucosyltransferase (Ugcg) affects neuronal differentiation of hDPSCs. (A) RT-PCR analysis of mRNA expression in Ugcg knockdown hDPSCs. Lane 1; control hDPSCs, and lanes 2–5; different Ugcg shRNA clones. GAPDH was used as a loading control. (B) FACS analysis of the expression of ganglioside GM3. (C) Immunostaining analysis of gangliosides, GM3 (FITC; green) and GD3 (FITC; green), and neuronal markers, Nestin (TRITC; red) and MAP-2 (TRITC; red). Expression of gangliosides and the neuronal markers (Nestin and MAP-2) was determined in the knockdown cells cultured in SFM + 2 mM BME at 5 h and cultured in 10% FBS + 2% DMSO + 200 μM BHA at 1 week. (For interpretation of color mentioned in this figure legend the reader is referred to the web version of the article.)