Extended Data Fig. 6: PLA2G15-deficient cells and lysosomes accumulate BMP.
From: PLA2G15 is a BMP hydrolase and its targeting ameliorates lysosomal disease
a,b, Targeted analyses of BMP lipids in PLA2G15-deficient cells reveal their accumulation in lysosomes and whole cells. a, Heatmap representation of log2-transformed changes in BMP abundances in lysosomes (IP) and whole cells (WC) of PLA2G15-deficient HEK293T clone1 compared to wildtype control (n = 3 WT and n = 3 PLA2G15 KO). Data are the ratio of the mean of each BMP. Significant changes from the graph are represented by *p < 0.05. P values were calculated using two-tailed unpaired t-tests and are presented in Supplementary Table 1. b, Fold changes in total BMP abundance. The sum of all abundances of measured BMPs was used to generate these values. Data are mean ± s.d. Statistical analysis was performed using two-tailed unpaired t-tests. **p = 0.0013, ****p = 0.000051. c, Generation of PLA2G15-deficient clones 2 and 3. d, Schematic for fluorescently labeled PLA2G15 uptake experiments. Labeled recombinant PLA2G15 proteins were supplemented to PLA2G15 KO HEK293T cells for 48 h and imaged to validate delivery. e, Representative images of labeled PLA2G15 wildtype and mutant proteins (Alexa488) and lysosomes (Lysotracker) show successful uptake and trafficking in PLA2G15 KO HEK293T cells (left). In the merged images, green and magenta represents the protein and Lysotracker channel respectively and white represents the colocalized spots. Scale bar = 5 µm. Intensity showing that labeled proteins colocalize with Lysotracker (right). Each experiment was repeated three times. f, Recombinant PLA2G15 does not rescue elevated levels of around a fifth of BMPs resulting from PLA2G15 loss. Fold changes in the levels of BMPs in PLA2G15 KO HEK293T cells after supplementation with wildtype or mutant PLA2G15 (n = 3 WT, n = 3 PLA2G15 KO, n = 3 PLA2G15 KO + PLA2G15 and n = 3 PLA2G15 KO + PLA2G15 S198A). Data are mean ± s.d. Statistical analysis was performed using two-tailed unpaired t-tests. **p = 0.0012, ***p < 0.001 and ****p < 0.0001. g, Targeted analyses of BMP lipids reveal that a deficiency in PLA2G15 increases levels of most BMPs in HeLa cells. Data are mean ± s.d. (right) (n = 3 WT and n = 3 PLA2G15 KO). Statistical analysis was performed using two-tailed unpaired t-tests. *p < 0.05, **p = 0.0017, ***p < 0.001 and ****p < 0.0001. Individual BMP species and their statistics are presented in Supplementary Table 3. h, Targeted analyses show minimal phospholipid alterations in PLA2G15-deficient mouse tissues (top), HEK293T cells and lysosomes (bottom). Heatmap representation of log2-transformed changes in total lipid class in PLA2G15-deficient mouse tissues (mouse brain, kidney and liver) compared to their control counterparts (n = 6 WT and n = 6 Pla2g15-/-) and PLA2G15-deficient HEK293T cells compared to wildtype cells (n = 3 PLA2G15 KO and n = 3 WT). Data are the ratio of the mean of each lipid class. Significant changes from the graph are represented by *p < 0.05. P values were calculated using two-tailed unpaired t-tests. All measured individual lipid species, total lipid classes and their statistics are presented in Supplementary Table 5. i,j, Loss of PLA2G15 has no effect on BMP synthesis and lysosomal biogenesis. i, Immunoblot analysis of lysosomal markers (LAMP2 and Cathepsin B), the BMP synthase CLN5 and a loading control (Vinculin). j, BMP synthesis in HEK293T cells using labeled phosphatidylglycerol (D5-PG) at the indicated time points. n = 3 WT and n = 3 PLA2G15 KO. Data are mean ± s.d. Gel source data are provided in Supplementary Fig. 1. Panel d was created using BioRender (https://biorender.com).
