Figure 2. Knockdown of PNPLA1 causes impairment of acylceramide production.

Keratinocytes were infected with lentivirus harbouring control shRNA or shPNPLA1 and were differentiated for 7 days. (a) Total RNAs prepared from differentiated keratinocytes were subjected to RT-PCR using primers specific for PNPLA1 and GAPDH. Uncropped scanned photographs of the electrocataphoresis gels are provided as Supplementary Fig. 2. (b) Cells were labelled with [³H]sphingosine for 6 h at 37 °C in the presence of 10 μM linoleic acid. Lipids were extracted, separated by normal-phase TLC and detected by autoradiography. AcylCer, acylceramide; Cer, ceramide; Acyl-GlcCer, acyl-glucosylceramide; GlcCer, glucosylceramide; SPH, sphingosine; SM, sphingomyelin. Two independent experiments gave similar results, and a representative result is shown. (c) Lipids were extracted and subjected to LC-MS/MS analysis. Acylceramides were detected in the MRM mode and quantified using MassLynx software. The graph depicts the amount of each acylceramide species with the indicated FA chain-length. The inset represents the total acylceramide levels. Values represent the means±s.d.s of three independent experiments. Statistically significant differences compared to control cells are indicated (two-tailed Student's t-test; *P<0.05; **P<0.01).