FIGURE 5.

S1P lyase (SPL) modulates autophagy independently from GPR55 and maintains the mitochondrial membrane potential. (A) Western blotting (WB) analysis of the proteins involved in autophagy in cell lines with different expression levels of SPL or GPR55 as compared to control cell lines. (B) WB quantification by densitometry using the NIH ImageJ software. The bands from a total of three replicates of the same set of samples were used, and the protein levels or the phosphorylation rates were quantified. (C) Mitochondrial membrane potential was measured in SPL‐inhibited cell lines with or without addition of PE (5 μM) in the presence or absence of glucose. (D, E and F) The metabolites (taurine, glucose 6‐phosphate, mannose 6‐phosphate) were measured using GC‐MS in SPL‐overexpressing cell lines, in which levels were increased compared to control cell lines. (*) indicates the difference between the control and SPL‐overexpressing cell lines, (#) indicates the difference between the EV‐transfected control and SPL‐overexpressing cell lines. (G) Mitochondrial membrane potential was measured in GPR55‐inhibited cell lines with or without SPL overexpression by adding lysophosphatidylinositol (LPI) 18:1 (1 μM), lysophosphatidylglycerol (LPG) 18:1 (1 μM), PE (5 μM) and Hexa (10 μM). There were no changes with the addition of any of the compounds in glucose‐present condition. (*) indicates differences with HR control cell line, (#) indicates differences with SPL overexpressing HR control cell line. Differences between the two groups were statistically analysed by the unpaired Student's t‐test, while differences among more than two groups were statistically analysed by one‐way ANOVA. EV, empty vector transfected control cell line; HR, homologous recombination vector only transfected control cell line; SPL, SPL overexpressing cell line; SPL HR, homologous recombination vector transfected SPL overexpressing cell line; Cr/Cas SPL, SPL knock out cell lines by using CRIPR/Cas9 system; Cr/GPR55, GPR55 knock out cell lines by using CRIPR/Cas9 system in SPL overexpressing cell line; Cr/GPR55 LPI, or LPG, or PE, or He, GPR55 knock out cell lines by using CRIPR/Cas9 system with addition of the LPI, or LPG, or PE, or He. SPL Cr/Cas GPR55: GPR55 knock out cell lines by using CRIPR/Cas9 system in SPL overexpressing cell line; SPL Cr/Cas GPR55 + LPI, or LPG, or PE, or He, GPR55 knock out cell lines by using CRIPR/Cas9 system in SPL overexpressing cell line with addition of the LPI, or LPG, or PE, or He