Fig. 5: BAF A12T is associated with enhanced protein synthesis and translation errors.

A–B Differential gene expression analysis in wild-type compared to NGPS1 and NGPS2 cell lines reveals enrichment for biological processes associated with RNA processing and translation. P-values were adjusted for multiple testing via the False Discovery Rate (FDR; Benjamini-Hochberg) approach. Gene Ontology Analysis was performed on genes with a False Discovery Rate value less than 0.01. C Nascent protein synthesis assay using HPG incorporation followed by labeling using a “click” reaction with Alexa fluor 488 (AF 488) in WT, NGPS2 or NGPS2 corrected (C) or in WT fibroblasts expressing a BAF WT or BAF A12T construct (D). AF 488-HPG intensity was quantified using the high-content microscope in 4 (C) or 3 (D) independent experiments, each measuring 1000 cells in 3 wells per cell line, graphed as a Superplot with data from different experiments indicated in different colors and larger symbols indicating each experiment’s average. AF 488-HPG intensity was normalized to the WT cell line. Mean values were analyzed using nested one-way ANOVA (C) or paired two-tailed t-test (D), with Tukey’s method used for multiple comparisons in (C). E Immunofluorescence images of nucleolin used to identify and outline the nucleoli (yellow) in the indicated cell lines using the HCS Studio software. F–G Average nucleolar area quantified in NGPS2 and NGPS2 corrected cells (F) or in WT fibroblasts expressing BAF WT or BAF A12T (G). F and (G) are superplots as in (C–D), and statistical comparisons were made using paired two-tailed t-tests. H Translation error rate measured as an increased read-through using a dual luciferase assay in 3 independent repeats (Superplot of the data). Mean values were analyzed using one-way ANOVA with Dunnett’s multiple comparison testing. Results are derived from the ratio hFluc/hRluc, given in fold induction.