Fig. 4. Transient inhibition of epigenetic factors in NPCs drives faster maturation in neurons.

a, Temporal expression of epigenetic hits from gene knockout studies by RNA-seq (n = 3 independent experiments). b, Experimental paradigm. NPCs were treated transiently (d12 to d20) with epigenetic inhibitors. Neuronal maturation was assessed by western blot and Ca²⁺ imaging. c, Normalized expression of NEFH and STX1A by western blot. EZH2i, DOT1Li: n = 5; EHMT1/2i: n = 4; KMT5i: n = 2; KDM1Ai (STX1A), n = 3; KDM1Ai (NEFH) n = 4 (independent experiments). d, Amplitude and frequency of spontaneous Ca²⁺ spikes. DMSO: n = 292; EZH2i: n = 317; EHMT1/2i: n = 331; DOT1Li: n = 309; KDM1Ai: n = 282; KDM5i: n = 326; KMT5i: n = 326; EZH2i + DOT1Li: n = 221 (neurons from 2 independent experiment) e, Synchronous firing rate. DMSO, EZH2i, EHMT1/2, DOT1Li: n = 6; KDM1A: n = 2; KDM5i, KMT5i: n = 3 (FOV from 2 independent experiments). f, Representative traces of normalized GCaMP6m signal. g,h, Representative traces at d50 (g) and number of evoked action potentials (h) per injected current. DMSO: d30, n = 7; d50, n = 11. EZH2i: d30, n = 8; d50, n = 12 (neurons). Error bands indicate s.e.m. i,j, Representative traces (i) and amplitude and frequency (j) of mEPSCs (+40 mV, d60–d75). DMSO: n = 5; EZH2i: n = 8 (neurons). k, Representative images and quantification of juxtaposed SYN1–PSD95 puncta. n = 6 FOV from 2 independent experiments. Scale bar, 25 μm. l, Normalized expression (to d25 DMSO) of ‘relaxed’ monotonic upregulated transcripts by RNA-seq in neurons from EZH2i and DMSO-treated NPCs. n = 3 independent experiments. m, Experimental paradigm for transient EZH2i in forebrain organoids and representative image of Ca²⁺ imaging at d55. Scale bar, 50 μm. n, Amplitude and frequency of spontaneous Ca²⁺ spikes in organoids. DMSO: n = 222; EZH2i: n = 297 (4–8 organoids per condition from 2 batches). Data are mean ± s.e.m. Two-tailed ratio-paired t-test (c); Welch’s one-way ANOVA with Games–Howell’s correction (d); one-way ANOVA with Dunnett’s correction (e); mixed-effect model with Tukey’s correction (h); Kolmogorov–Smirnov test (j); two-tailed unpaired t-test (k); two-way ANOVA with Šidák correction (l); two-tailed Welch’s test (n).

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