Loss of VPS13C results in altered lysosomal lipid composition. (A) IB showing abundance of organelle markers in postnuclear supernatant (PNS) and lysosomal fractions (Lys) from WT and VPS13C^KO HeLa cells. Equal amounts of total protein were loaded in each lane. Note the striking enrichment of LAMP1 in lysosomal fractions. (B) Quantification of band intensities from A normalized to GAPDH to show relative enrichment. (C) Percentages of phospho- and sphingolipid classes in WT and VPS13C^KO lysosomal fractions normalized to total measured lysosomal lipid content. n = 4 biological replicates. (D) Concentrations of di-22:6- and di-18:1-BMP normalized to total protein in WT and VPS13C^KO HeLa total cell lysate. n = 3 biological replicates. (E) IB showing loss of VPS13C protein expression in two clonal VPS13C^KO i³Neuron lines after 14-d differentiation. GAPDH was used as a loading control. (F) Concentrations of di-22:6- and di-18:1-BMP normalized to total protein in WT and VPS13C^KO i³Neuron (day 14) total cell lysate. n = 3 biological replicates. For lipidomic data, *, q < 0.05; **, q < 0.01; ***, q < 0.001. For all other data, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 compared with WT control. Lipidomic data in C were compared using FDR. All other data were compared using two-sided t tests. Error bars represent ±SD. Source data associated with this figure can be found at https://doi.org/10.5281/zenodo.6416363.