Fig 2: UTX Is Upregulated in Earlier Stages of Neural Differentiation of hESCs(A) Schematic illustration of neural differentiation of hESCs. hESCs, human embryonic stem cells; EB, embryoid bodies; hNSCs, human neural stem cells; hNPCs, human neural progenitor cells. Scale bar, 50 µm.(B) Real-time PCR analysis showing higher mRNA levels of UTX in earlier stages of neural differentiation of hESCs. GAPDH is used as an internal control for real-time PCR.(C) Western blotting indicating upregulated protein levels of UTX at earlier stages during neural differentiation of hESCs. ß-Tubulin is used as a loading control.(D) Representative images of H3K27me3 immunostaining at different stages of neural differentiation of hESCs. DAPI, a nuclei marker; Nestin, an NSC marker; DCX, an NPCs marker; MAP2, a mature neuron marker. Scale bar, 50 µm.(E) Western blotting showing protein levels of H3K27me3 and H3K27ac upon neural differentiation of hESCs. Histone 3 was used as a loading control.Results are presented as mean ± SEM; n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: eCB enhancement reduces SM levels in cultured ASM-KO neurons AMean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle or with the indicated concentrations of AEA (***P < 0.0001, n = 3 independent cultures, one-way ANOVA, Bonferroni post hoc).BMean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, the inhibitor of NSM GW, AEA, or the combination of GW and AEA (***P < 0.0001, n = 3 independent cultures, one-way ANOVA, Bonferroni post hoc).CWestern blot against NSM and GAPDH (used as loading control) and graph showing mean ± SEM NSM protein levels in extracts from cultured neurons treated with vehicle or with 50 µM AEA (*P = 0.0230, n = 6 independent cultures, Student's t-test).DMean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, or with JNJ, PF, or URB in the presence or absence of AEA (**P JNJ = 0.0013, *P PF = 0.0143, ***P URB = 0.0002, **P JNJ + AEA = 0.0069, **P PF + AEA = 0.0077, ***P URB + AEA < 0.0001, n = 3 independent cultures, one-way ANOVA, Bonferroni post hoc).EMean ± SEM SM levels, expressed as percentage of vehicle values, in cultured neurons from ASM-KO mice treated with vehicle, with PF, or with SR141716 + PF (*P PF = 0.0109, *P SR + PF = 0.0343, n = 3 independent cultures, one-way ANOVA, Bonferroni post hoc).FImmunofluorescences against the dendritic marker MAP2 of cultured neurons from ASM-KO mice treated with vehicle, AEA, JNJ, PF, or URB. Scale bar, 50 µm.GMean ± SEM number of apoptotic cells measured by TUNEL assays in cultured neurons from ASM-KO mice treated with H2O2 (as positive control), vehicle, AEA, JNJ, PF, or URB (n = 3 independent cultures). Source data are available online for this figure.

Fig 4: The aiFACS technique. (A) Schema of the instrumental apparatus: BD FACSAria III implemented with the aiFACS device. The sorter fluidics is modified to allow the injection of a pharmacological agonist. Two syringes, one containing D-PBS (in gray) and the other one containing the agonist (in green), are connected to their respective tubing: the D-PBS tubing (in gray) and the agonist tubing (in green). These are further connected to a downstream Y-shaped connector that enters the flow cell (the chamber in which the cells are aligned to pass one-by-one through the light beam for sensing). The sample is connected to the sorter through a tubing (in blue) having the same diameter as the agonist tubing (in green). A peristaltic micropump (P) allows control of the speed of solution injection and synchronization to the speed of sample flow in the sorter. The incubation time between each cell of the sample and the agonist is also controlled (red tubing). (B) Time versus Fluo-4 AM biparametric graph showing the response of the cells to different stimuli in real time. At time t0, the opening of the D-PBS valve starts the perfusion, and the baseline levels of fluorescence (in the red rectangle) are obtained with continuous perfusion. At time t1, the D-PBS valve is closed, and the one of the agonist is opened. The magnitude of the cellular calcium response to the KCl agonist (65 mM final) is shown in the light-blue rectangle. At time t2, ionomycin is added (6.5 µm final) to KCl as a positive control of stimulation. The maximal response of the cells is displayed in the green rectangle. (C) Addition of beads to the agonist solution allows real-time detection of the agonist presence. Bead fluorescence is shown in purple. (D) aiFACS allows viable recovery of stimulated cells. (Upper panels) Discrimination of cells based on scatter parameters ([FSC] forward scatter; [SSC] side scatter) before sorting (55.3% of the total population; left panel). The presort viability is determined by labeling the cells with DAPI (95.5% of the cells in the red region; right panel). (Lower panels) Discrimination of cells based on scatter parameters (FSC and SSC) after sorting (90.9% of the total population; left panel). The viability of the cells after KCl stimulation and aiFACS sorting is determined by reanalyzing the DAPI staining (99.7% of the cells in the red region; right panel). (E) Sorted neurons are viable and can grow neurites when plated on L-ornithine–coated glass coverslips, cultivated for up to 6 d in vitro (DIV 6) in complete neurobasal medium, and analyzed by immunocytochemistry (MAP2 staining). 63× magnification; scale bars, 10 µm.

Fig 5: UTX Loss-of-function Inhibits Neural Differentiation of hESCs(A) Immunostaining of neuron markers MAP2 and Tuj1 at day 40 of neural differentiation. Scale bar, 20 µm.(B) Quantitative analysis showing reduced percentages of MAP2+ and Tuj1+ neurons derived from UTX KO hESCs.(C) qRT-PCR analysis showing that the expression levels MAP2 and Tuj1 were significantly lower in UTX KO cells at day 40 of neural differentiation. Results are presented as mean ± SEM; n = 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.