Retinoic acid treatment induces differential translation in SH-SY5Y human neuroblastoma cells. a Schematic of experimental design and data acquisition work-flow. b Immunocytochemistry performed on Non-Diff and RA-Diff SH-SY5Y cells with antibodies against neurofilament (red) and β-actin (green). Nuclei were DAPI stained (blue). c β-actin expression was decreased in RA-Diff cells. Individual cell fluorescence was quantified and represented as a corrected total cell fluorescence (CTCF) for β-actin; n = 119 for Non-Diff and n = 118 for RA-Diff. d Primary neurite length measured by neurofilament staining; n = 109 for Non-Diff and n = 100 for RA-Diff. e FMRP expression by immunoblot before and after RA treatment, quantified in F); n = 4 for both conditions. For panels C), D), and f Student’s t test, ****p ≤ 0.0001. Graphs represent mean ± S.E.M. g Differential mRNA abundance based on Non-Diff versus RA-Diff TPM. Transcripts were defined as significantly up-regulated (cyan) or down-regulated (gold) in the RA-Diff condition based on rank-change in abundance compared to the Non-Diff condition. h Volcano plot of transcripts with differential translation by translational efficiency (TE) by Riborex analysis. Significantly up-regulated genes (cyan) and down-regulated genes (gold) in RA-Diff cells are defined by an absolute log2-normalized fold-change cutoff of ±1 (vertical lines), and a multiple testing corrected p-value cutoff of 0.1 (horizontal line). i Gene sets (biological process) with significantly downregulated TE in RA-Diff cells. Genes upregulated in RA-Diff cells is shown in (j). The top five biological processes with significant change using a multiple testing corrected p-value cutoff of 0.05 (vertical line) are shown on the graph. k Plot shows normalized mRNA reads (grey) and RPF (cyan/gold) over the 5’leader (thin line, left), and CDS (thick line, middle). The axon guidance gene, PLXNA2, is representative of a transcript with higher translational efficiency and RPF in the RA-Diff condition.