Fig 2: Olig1 and Olig2 are critical to miR-219-mediated neural differentiation. (a, b) Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (a) and western blot (b) in ESCs transfected with Olig1/Olig2-encoding plasmids for 48 h. (c, d) ESCs were transfected with siRNAs that were targeted to Olig1/Olig2 for 12 h and treated with RA or miR-219 mimics for another 36 h. Relative levels of Nestin, Map2, and Tuj1 were detected through qRT-PCR (c) and western blot. (e) Immunofluorescence shows the abundance of Oct4, Nestin, and Map2, as well as the morphologies of ESCs transfected with Olig1/Olig2 or miR-291 mimics. **P<0.01; NS, no significance

Fig 3: Identification of ß-catenin downstream genes that contribute to the negative regulation of cAMP-induced differentiation in GSCs.A Volcano plot and heat map show the distribution of DEGs in dbcAMP-treated GSCs before and after the knock-down of ß-catenin. B Function and pathway enrichment of DEGs in dbcAMP-treated GSCs before and after the knock-down of ß-catenin. C Heat map and Venn diagram demonstrating the overlap of top downregulated DEGs in the two gene sets. D Validation of the expression of four DEGs by using qRT-PCR. E Limiting dilution assays of GSC1 and GSC11 cells after treatment with specific siRNA targeting APELA, CARD16 and MSLN. F Quantification of MAP2 and TUJ1 in GSCs treated with the combination of dbcAMP and knock-down of CTNNB1, APELA, CARD16 or MSLN, by using flow cytometry. (db, dbcAMP; Data are shown as the mean ± SD. N.S., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Fig 4: Direct reprogramming of DM1 patients’ skin fibroblasts into neuronal like cells.(a) Differentiation process of DM1 skin fibroblasts into neuronal like cells. The top left image shows DM1 skin fibroblasts before adding lentivirus co-expressing shRNA against PTBP and puromycin resistant gene. The top middle image shows lentivirus transduced cells successfully passing the selection process by adding 5 µg/mL puromycin to the culture medium for 2 days. The top right image shows expansion of cell bodies at 6 DPI. The bottom left image shows cells with neuronal like morphology of expanded cell bodies and distinct neurites at 8 DPI. The bottom right image shows bipolar and multipolar neurons with extended neurites at 10 DPI. Scale bar, 50 µm. (b) Co-immunostaining of control (top panel) and DM1 (bottom panel) hiNeurons with a neuron specific ß-III tubulin antibody (TUJ1) (green) and MAP2 (red) at 10 DPI. DAPI was used to stain nuclei (blue). On the right: merged images of immunostaining with TUJ1, MAP2 and DAPI. Scale bar, 50 µm. (c) Graph shows the percentage of positive TUJ1-stained cells (TUJ1+) at 10 DPI, quantified by dividing the number of TUJ1+ cells by the total number of nuclei stained by DAPI. Data are shown as mean ± SEM; n = 3 for each group, a total of 90 images were analyzed per group (11,510 nuclei were analyzed per ctrl group and 14,668 nuclei per DM1 group). ns, not significant compared to control group by unpaired t-test. (d) Graph displays the percentage of MAP2 positive cells at 10 DPI, quantified by dividing the number of MAP2+ cells by the total number of TUJ1+ cells. Data are shown as mean ± SEM; n = 3 for each group, a total of 90 images were analyzed per group (9,941 TUJ1+ cells were analyzed per ctrl group and 16,286 TUJ1+ cells per DM1 group). ns, not significant compared to control group by unpaired t-test. (e) Additional staining for neural markers. Fluorescent images display positive co-staining of DM1 hiNeurons with TUJ1 (green) and anti GABA or glutamate or NeuN (red) antibodies at 15 DPI. The bottom panel shows lack of synapse formation at 10 DPI in DM1 hiNeurons co-stained with TUJ1 (green) and SYN1 (red) antibodies. DAPI was used to stain nuclei (blue). On the right merged images. Scale bar, 50 µm. Ctrl, control; DM1, myotonic dystrophy type 1; hiN, human induced neurons; DPI, days post viral-infection; TUJ1, ß-III tubulin antibody; MAP2, microtubule-associated protein 2; SEM, standard error of the mean; GABA, gamma-aminobutyric acid; NeuN, neuronal nuclear antibody; SYN1, synapsin 1 antibody.

Fig 5: Morphine-induced neuronal differentiation is mediated by MOR. (a) Cultured rat NSCs express both NSC marker nestin and MOR but express negligible levels of glial marker GFAP and neuronal marker DCX/NeuN. Scale bar represents 20 µm. (b) FCM analysis of NSCs reveals the effect of morphine treatment on neuronal differentiation. (i) NSCs are detected by CD24. Representative FCM histograms are shown. (ii) Quantification of differentiation using fluorescence intensity. ***p < 0.001, ****p < 0.0001; one-way ANOVA. (c) Morphological analysis of NSCs showing effect of morphine treatment on dendritogenesis. (i) Representative images of cell morphology of NSCs after morphine treatment. Cells are labeled by neuronal differentiation marker MAP2 (red) and Hoechst (blue). Scale bar represents 20 µm. (ii) Chronic morphine treatment increases the number of cells with complex dendrites. *p < 0.05, **p < 0.01; one-way ANOVA with Tukey’s multiple comparisons test. (iii) Chronic and higher doses of morphine treatment increases total neurite length of NSCs. **p < 0.01; one-way ANOVA with Tukey’s multiple comparisons test. All data are shown as mean ± s.e.m.