Fig. 2

a Cartoon showing the two-Stage ESC neural differentiation protocol. b Representative image showing morphology of day 6 and day 20 control and Adnp−/− ESC-derived neurospheres and neuronal cultures from three independent experiments with similar results. The white arrows pointing to the fiber-like neuronal structures. c The dynamic expression profile of representative neuroectodermal genes during control and Adnp−/− ESC neural induction. d Flow cytometry analysis for quantification of PAX6⁺ cells. Blue: isotype control; purple: experimental group using PAX6 antibody. e IF staining of neural progenitor marker NESTIN and ADNP for control, Adnp−/− ESC and FLAG-ADNP restoring Adnp−/− ESC-derived day 6 NPCs. Representative image from 3 to 5 random microscopic fields of three independent experiments with similar results. f Quantification of mean fluorescence intensity of NESTIN staining using ImageJ software. FLAG-ADNP rescued the fluorescence intensity of NESTIN staining in Adnp−/− ESC-derived neurospheres. g qRT-PCR analysis for Tubb3 (encoding TuJ1) and Gfap for day 19 control and Adnp−/− ESC-derived neuronal cell cultures (n = 3 per group). h IF staining of neuronal marker TuJ1 and glial marker GFAP for day 19 control and Adnp−/− ESC-derived neuronal cell cultures. The white arrows showing the neuronal fiber structures. i Quantification of mean fluorescence intensity of TuJ1 and GFAP staining using ImageJ for panel (h). j WB analysis of TuJ1 and GFAP levels in day 19 control and Adnp−/− ESC-derived neuronal cell types. WB were repeated at least two times, and shown were the representative data. qRT-PCR was based on three biologically independent experiments in (c, g). Mean fluorescence intensity was calculated based on three biologically independent experiments (n = 3–5 different regions of interest per group) in (f, i). Data are presented as mean values ± SEM and p values by two-tailed unpaired t-test are shown in (c, f, g, i).