Fig. 4: Activation of TP53-mediated apoptotic pathway following Hnrnpu KO.
From: Heterogeneous nuclear ribonucleoprotein U (HNRNPU) safeguards the developing mouse cortex
A Time-dependent accumulation of TP53 (CM5 antibody) in neurospheres treated with control CRISPR/CAS9 plasmid (px330) or Hnrnpu sgRNA’s and plated at indicated time. Acquisition of the intensity of the CM5 signal was done in identical imaging parameters for all images. B Measurements of the dynamic accumulation of TP53. Cells were identified DAPI, and the average intensity in each spot was normalized to a non-specific background (upper panel). The accumulative number of cells in all fields of view is plotted against time (lower panel). C–F Low magnification images (Dapi, blue) of coronal sections (E18) of control (Hnrnpu +/+Emx1Cre/+) and mutant (Hnrnpufl/flEmx1Cre/+) brains. Inserts indicate the location of high magnification images (D, F). Anti-TP53 (CM5, Red) fails to react with sections from control cortices. D Reveals stabilization of TP53 in cells throughout mutant cortices (F). G Normalized expression levels (left panel) and alternative splicing (right panel) in the mutant (Hnrnpufl/flEmx1Cre) and control (Hnrnpu+/+Emx1Cre) E13 cortices, showing elevated expression of Tp53 targets and misrepresentation of alternative splice variants of genes in the TP53 pathway n = 3 biological repeats (each measured in triplicates). Error bars ±SEM. H Schematic representation of functional connections between differentially expressed (ellipsoid) and abnormal spliced (star) gene products presented in C. color code: Red-Strong activation, Pink- overexpression, Blue- downregulation, White- no change in expression levels. n = 4 biological repeats I-I’) Effective protection from apoptotic cell death of CRISPR/CAS9 sgRNA (Hnrnpu sgRNA, Red) Non-electroporated neurospheres (gray) and control (px330, blue) Electroporated neurospheres were treated with solvent only (DMSO), Q-VD-OPh (50 μM), Z-VAD-fmk (50 μM), Necrostatin-1 (Nec1, 1 μM) and Pifithrin-μ (pFT-mu, 5 μM). Bars indicate the number of EdU+ cells per 100 μM2 of the neurospheres surface area, following 30 min exposure to EdU. Ordinary one-way ANOVA with Tukey’s multiple comparison test was used for data analysis. P values: **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars (±SEM) are indicated F Images of neurospheres treated with either DMSO or indicated small molecules. EdU incorporation (Click chemistry, magenta) and Cleaved Caspase, Asp175 (CC3, Green), are presented. Size bars units are μM. Source data are provided as a Source Data file.
