Fig. 4

AdipoR2 silencing causes increased dihydroceramide levels (data from Exp_190226). (A-C) and (D-F) shows the levels of SFA, MUFA and PUFA in phosphatidylcholines (PC) and phosphatidylethanolamines (PE) of HEK293 cells grown in basal media (containing BSA only) or in the presence of 200 μM palmitic acid (PA; conjugated to BSA) and treated with Non-target siRNA (NT) or AdipoR2 siRNA. The sum of five sphingolipid species are as box plots G-K) while their fatty acid composition is shown as a heat map (L). (M) and (N) show principal component analysis plots of the four treatments (vehicle-treated samples are clustered to the left in panel M) and of the lipid species that showed significant differences among treatments (panel N). Note that the strongest effect of AdipoR2 siRNA is the increase in dihydroceramides (see panels H, L and N). In the box plots, boxes indicate the 25th to 75th percentile while the whiskers indicate the data points still within 1.5 of the box range. For A-K, significant differences from the NT siRNA control were determined using Student’s t-tests with *P < 0.05, **P < 0.01 and ***P < 0.001. For the heat map, the amount of each lipid species was normalized to the average of the NT + 200 μM PA treatment and the heat maps show fold differences from the mean across all treatments (each column is a replicate); only lipid species with significantly different levels among siRNAs in a given culture condition are included (ANOVA, q < 0.05)