Fig. 2.

CRISPR/Cas9 deletion of all three ORMDL isoforms increases SPT activity. A: ORMDL protein levels in A549 cells with CRISPR/Cas9-mediated KO of ORMDL3 alone in two clones (ORMDL3-KO1 and ORMDL3-KO2) or in KO of all three ORMDL3 isoforms (ORMDL3-TKO) as determined by Western blotting compared with control (CTL1 and 2). (n= 4). B: SPT activity was determined by incorporation of [3H]serine into de novo–synthesized sphingolipids in the indicated clones. The SPT inhibitor myriocin was used as a negative control. Data are the means ± SEM (n = 6). Each data point represents two independent experiments each with three separate biological replicates. ∗P < 0.01 compared to CTL. Statistical analysis by one-way ANOVA with Tukey’s post hoc test. C: Duplicate samples were analyzed by Western blotting with the indicated antibodies to demonstrate equal levels of the SPTLC1 subunit of the SPT complex. ND, not detected; SPT, serine palmitoyltransferase.