Fig. 2: SCYL1 regulates Golgi structure and endolysosomal positioning.

a Generation of SCYL1 KO cells. Two SCYL1 CRISPR/cas9 KO clones targeting exon 3 (KO1) or exon 1 (KO2) of the SCYL1 gene ORF, were generated in MCF-7 cells. GAPDH was used as loading control. Equal protein amounts were loaded. One representative western blot out of n = 3 biological replicates is shown. *: nonspecific background signal. b SCYL1 KO cells display an enlarged Golgi. Representative fluorescence micrographs from WT and SCYL1 KO MCF-7 cells (clone KO1) are shown. Scale bar, 10 µm. c Quantification of b. The ratio between the Golgi apparatus volume, labeled with GOLGIN-97/GOLGA1 antibodies, and the total cellular volume, detected with the cell body detection tool (Imaris), was computed in individual cells. Violin plots were generated using InstantClue (white dots within violins denote medians; top and bottom of violins represent the 25th and 75th percentiles; thick vertical lines within violins show the interquartile range (IQR), whereas thin vertical lines represent the rest of the distribution extending on the 1.5xIQR rule)⁷⁵. Single-cell values are indicated with white small dots. An unpaired two-tailed Student’s t-test was used to compare WT and SCYL1 KO values: p = 8.05e-09 (****). n = 3 biological replicates with at least 36 individual cells measured per replicate. Error bar = SEM. d Expression proteomics comparing SCYL1 KO and WT MCF-7 cells. Scatter plot showing protein abundance ratios of SILAC-labeled SCYL1 KO and WT cells of two biological replicates. Normalized ratios were Log₂-transformed. Pearson’s correlation coefficient r = 0.74. Red dots: significantly upregulated proteins (Log₂ ≥ 0.5 in both replicates) in SCYL1 KO compared to WT cells; blue dots: significantly down-regulated proteins (Log₂ ≤ 0.5 in both replicates) in SCYL1 KO compared to WT cells. Upper left corner: Venn diagram quantifying common proteins significantly dysregulated in both SCYL1 KO replicates compared to WT (t-test two-sided, FDR = 0.05, BH-corrected). Raw data are provided in Supplementary Data. e Proteins involved in vesicular transport and secretion are increased in SCYL1 KO cells. GO term enrichment of upregulated (red) and down-regulated (blue) proteins in the SCYL1 KO proteome, as highlighted in d. A complete list of all significant GO terms is displayed in Supplementary Data 1. f, g Loss of SCYL1 leads to a perturbation of early and late endosome (f) and lysosome (g) distribution. Fluorescence micrograph from WT and SCYL1 KO MCF-7 cells in normal growth conditions (DMEM). Representative pictures of n = 3 replicates are shown. Scale bar = 10 µm (h). Quantification of f, g. The distance from nuclear envelope to RAB5, RAB7 and LAMP2 foci was computed and averaged per individual cell. All results for WT and SCYL1 KO cells were assembled in a violin plot (see panel c for details). Single-cell values are indicated with white small dots. An unpaired two-tailed Student’s t-test was used to compare WT and SCYL1 KO populations; RAB5: p = 0.38 (ns); RAB7: p = 0.00044 (***); LAMP2: p = 1.88E-05 (****). 10 individual cells were individually measured within three technical replicates per two biological replicates (n = 6). Error bar, SEM. All source data are provided as Source Data File.