Fig. 7. Target deconvolution using unbiased quantitative proteomics in AP4B1^KO SH-SY5Y cells and AP-4-HSP patient-derived hiPSC neurons treated with BCH-HSP-C01.

a–c Differential protein enrichment analysis. Statistical testing was done using protein-wise linear models and empirical Bayes statistics using the limma package in R (Ritchie et al.). Proteins were considered as differentially enriched with an FDR <0.05 and a log₂ fold change >0.3. PCA plots show the top 500 variable proteins. Differentially enriched proteins are shown in volcano plots colored in black. Proteins with the most consistent enrichment profiles across all experimental conditions (see Supplementary Fig. 8) are colored and labeled in red. a SH-SY5Y cells: 8141 unique proteins were analyzed. b hiPSC-derived neurons: 7386 unique proteins were analyzed. c Integrated analysis of SH-SY5Y cells and hiPSC-derived neurons: 5357 unique proteins were analyzed. The dot plot summarizes dysregulated Reactome pathways of the pooled analysis. Pathways were considered differentially expressed with an FDR <0.05. d The RAB protein family members RAB1B, RAB3C and RAB12 showed the most consistent profiles in response to BCH-HSP-C01 treatment and were selected for further analysis. LFQ intensities in SH-SY5Y cells (AP4B1^WT and AP4B1^KO pooled; 12 independent experiments per condition; exception: RAB12 in BCB-HSP-C01 treated SH-SY5Y cells was not detectable in 3 samples, which is why quantification is based on 9 independent experiments), and hiPSC-derived neurons (controls and patients pooled, 6 independent experiments per condition) are shown. Box plots show medians (center), upper and lower quartiles (hinges) and 1.5 x IQR (whiskers). Statistical testing was done using pairwise t-tests. P-values are two-sided and were adjusted for multiple testing using the Benjamini-Hochberg procedure. e Correlation of LFQ intensities of RAB3C and RAB12 in AP4B1^WT (n = 11 samples) and AP4B1^KO (n = 10 samples) SH-SY5Y cells, as well as control (n = 6 samples) and patient (n = 6 samples) hiPSC-derived neurons are measured by the Pearson correlation coefficient (r).