Fig. 2

Elevated protein synthesis, p-Akt and p-ERK levels in FMRP-deficient neural progenitors and neurons. a Schematic overview of differentiation protocol to derive homogeneous population of NPCs using rapid neural induction protocol. b Control and FMR1KO hESC-derived NPCs showing comparable level of NPCs markers expression, Nestin and Pax6. The number of positive cells for PAX6 and NESTIN was quantified over the total number of cells (stained with DAPI). Scale bar = 25 μm. c Basal protein synthesis was determined in control and FMR1KO NPCs using the SUnSET assay, showing significant elevation of protein synthesis in FMR1KO NPCs compared to control (CON). Values shown as mean ± SEM based on n = 3 replicates per genotype; *p < 0.05 compared with control was determined by two-tailed unpaired Student’s t-test. Puromycin labelling was performed in NPCs for 30 min, after which equal amounts of protein were loaded on the gel for immunoblot analysis with anti-puromycin antibody. As a control, cycloheximide (CHX) was added to the cells and incubated for 15 min prior to Puro labelling. d Schematic overview of differentiation protocol to differentiate NPCs into neurons. e Control and FMR1KO neurons express post-mitotic neuronal markers, MAP2 and TUJ1. Scale bar = 25 μm. f Basal protein synthesis was assayed in neurons by SUnSET method, showing significantly elevated level of puromycin expression in FMR1KO compared to the control. Values shown as mean ± SEM based on n = 3 replicates per genotype; *p < 0.05 compared with control was determined by two-tailed unpaired Student’s t-test. g The levels of phosphorylated Akt, total Akt, phosphorylated ERK and total ERK in NPCs were determined by immunoblot. Protein loading was determined by the level of Calnexin. Representative immunoblot was shown for each sample. Quantification of protein level was performed by ImageJ. Values shown as mean ± SEM based on n = 3 replicates per genotype; *p < 0.05, **p < 0.01 compared with control was determined by unpaired Student’s t-test. h The levels of phosphorylated Akt, total Akt, phosphorylated ERK and total ERK in neurons were determined by immunoblot. Protein loading was determined by the level of Calnexin. Representative immunoblot was shown for each sample. Quantification of protein level was performed by ImageJ. Values shown as mean ± SEM based on n = 3 replicates per genotype; *p < 0.05, **p < 0.01 compared with control was determined by unpaired Student’s t-test