Fig. 3: Orthogonal assays in AP4B1^KO SH-SY5Y cells confirm 5 active compounds.

a Overview of the orthogonal screen of 16 active compounds in differentiated AP4B1^KO SH-SY5Y cells. b Baseline differences in ATG9A ratios of AP4B1^WT vs. AP4B1^KO SH-SY5Y cells were quantified from 160 AB4B1^WT and 158 AB4B1^KO wells from 5 assay plates. Means are shown as black dots; whiskers represent ±1.5 x IQR. Statistical testing was performed using the Mann-Whitney U test. P-values are two-sided. c–g Dose-response curves for ATG9A ratios in AB4B1^KO cells treated with different compounds. Data points represent per well means from 3 different assay plates. Black dots and error bars represent mean ± 1 SD. Dashed lines show mean Z-scores for positive (green) and negative (salmon) controls. Shaded areas represent ± 1 SD. h Representative images of the intracellular ATG9A distribution for individual compounds. The merged image shows beta-3 tubulin (gray), DAPI (blue), the TGN46 (red) and ATG9A (green). The TGN46 and ATG9A channels are further separately depicted in greyscale. Scale bar: 10 µm. i Baseline differences of DAGLB ratios in AP4B1^WT vs. AP4B1^KO cells were quantified from 192 AB4B1^WT and 192 AB4B1^KO wells from 4 assay plates. Means are shown as black dots; whiskers represent ±1.5 x IQR. Statistical testing was done using the Mann-Whitney U test. P-values are two-sided. j–n Dose-response curves for DAGLB ratios in AB4B1^KO cells treated with different compounds. All data points represent per well means from 4 different assay plates. Black dots and error bars represent mean ± 1 SD. Dashed lines show mean Z-scores for positive (green) and negative (salmon) controls. Shaded areas represent ± 1 SD. o Representative images of the intracellular DAGLB distribution for individual compounds. The merge shows beta-3 tubulin (gray), DAPI (blue), the TGN46 (red) and DAGLB (green). The TGN46 and DAGLB channels are further separately depicted in greyscale. Scale bar: 10 µm.