Figure 1: MiRNAs expression analysis in neuronally differentiated adipose stem cells (ASCs) using the DAVID database and effects of Wnt signaling pathway inhibition by ICG-001 assessed by immunofluorescence, Western blotting and qRT-PCR.

(a) Immunofluorescence photomicrograph image of βIII-TUBULIN (red) and HOECHST (HOE, blue) staining in ASCs of non-treated cells (-RA group) and RA-treated cells (+RA group) for 15 days (D 15). (b) Western blotting of OCT4, SOX2, βIII-TUBULIN and MAP2 in the –RA and +RA groups at different time points and GAPDH is used as control. (c) Proteins expression levels, quantified by determining the gray value. (d) The expression ratios and evaluation, based on the mathematical model (using Fold-change and Z-test method), for all-known miRNAs that are detected between -RA and +RA groups. (e) Top KEGG pathways are summed up from the DAVID database based on P-value. (f) Expressions of key gene (Wnt3a, Tcf4, Lef1, β-Catenin and Axin2) in Wnt signaling pathway are detected by qRT-PCR between -RA and +RA groups (** p < 0.01, n = 4). (g) Western blotting analysis of Wnt signaling pathway marker proteins (FZD and phosphorylated FZD in the cytoplasm and the β-CATENIN in the nuclear) in control group (non-treated), RA group (RA-treated for 15 days) and anti-Wnt/RA group (pre-treated by the inhibitor of Wnt signaling pathway, ICG-001 and the group is incubated with RA for 15 days), GAPDH is used as control. (h) The proteins expression levels, quantified by determining the gray values (n = 2). (i) Western blotting analysis of the stem cell stemness marker (OCT4 and SOX2) and neural cell marker (βIII-TUBULIN) expression in control group, RA group and anti-Wnt/RA group and GAPDH is used as control. (j) Proteins expression levels, quantified by determining the gray value (n = 2).