Fig. 7: Ire1α-mediated regulation of protein translation in the developing cortex.
From: Protein translation rate determines neocortical neuron fate
a Images of representative primary cortical neurons prepared from Ire1αf/f (CTR) or Ire1αf/f; Emx1Cre/+ (cKO) cortices after ribopuromycilation assay to label stalled ribosomes. b Quantification of ribosome stalling in (a). c Representative Western blotting results using the control and Ire1α cKO E18.5 cortical lysates. d Quantifications of the protein level from c. Representative images of immunostaining against EGFP and CTIP2 in E18.5 coronal cortical sections of wild-type embryos after IUE at E12.5 with gRNAs and Cas9 nickase to achieve indicated genotypes. f Quantification of neuronal cell identity in the experiment in (e). g–j Representative Western blotting results using the control and Ire1α cKO cortical lysates at indicated developmental stages and quantification of protein levels. Note that the quantification for Ire1α levels in (h) is identical with Fig. 5l. Coomassie brilliant blue, CBB. k Representative Western blotting results of HEK293T cells after 4 h pulse with indicated compound. l-m Representative Western blotting of CHX pulse experiment in HEK293T treated with indicated compounds and quantification. n Representative Western blotting results of endogenous Ire1α co-immunoprecipitation (IP) from E12.5 and E14.5 cortical homogenates (H). j Interaction between Ire1α and indicated proteins was quantified relative to the amount of immunoprecipitated Ire1α. Graphs represent data points and averages ± S.D. Thick lines on violin plots represent median, thin lines represent quartiles. CBB, Coomassie Brilliant Blue stain to visualize proteins in SDS-PAGE gels. Statistics for b, d, f, h, j and o D’Agostino-Pearson normality test; for b unpaired t-test, ncells for CTR = 22 and for cKO=32 from three independent cultures, p < 0.0001; d Mann–Whitney test, ncortices for CTR = 4 and for cKO=4, for eEF-2, p = 0.0286; for eEF-2-P, p = 0.0286; for eIF4A1, p = 0.0286; f unpaired t-test, for Satb2 counts, nbrains for CTR = 4 and for KO = 4, p = 0.0140, and for CTIP2 counts, nbrains for CTR = 4 and for KO = 4, p = 0.0691; h Mann–Whitney test, for Ire1α, ncortices for CTR = 12 and for cKO=10, p = 0.0169; for eIF4A1, ncortices for CTR = 12 and for cKO=10, p = 0.3463; for eEF-2, ncortices for CTR = 12 and for cKO = 10, p = 0.2276; for eEF-2-P, ncortices for CTR = 7 and for cKO = 7, p > 0.9999; j unpaired t-test, for Ire1α, ncortices for CTR = 10 and for cKO = 11, p = 0.0284; for eIF4A1, ncortices for CTR = 10 and for cKO = 11, p = 0.0494; for eEF-2, ncortices for CTR = 10 and for cKO = 11, p = 0.5561; for eEF-2-P, ncortices for CTR = 7 and for cKO=7, p = 0.8440; m two-way ANOVA with Šidák multiple comparisons test, n = 4 biological replicates, p < 0.0001; o unpaired t-test, ncortices for E12.5 = 4 and for cKO=4; for RPS6, p = 0.0.0007, for RPL7, p = 0.3751, for PABPC4, p = 0.7826. Statistical tests were two-sided. 0.01 <* p < 0.05; 0.001 <** p < 0.01; *** p < 0.001.
