Fig. 3: NCT and NFT induce fat clearance and act through SPNS2.

For A–C T6PNE cells were treated with 0.25 mM palmitate and 10μM NCT or NFT for 2 days. A Photomicrographs of representative wells stained for fat with Oil Red O (upper panels) or Nile Red (lower panels). B Quantification of Nile Red staining was done on a per cell basis using a Celigo imaging cytometer (N = 14). C Triglyceride (TG) level was normalized to cellular protein measured by BCA and fold change was calculated relative to DMSO control (N = 6). D Candidate genes induced by NCT that have a role in fat metabolism were screened for a role in NCT-induced fat clearance using siRNAs. T6PNE cells were transfected with scrambled or target siRNAs. Two days later, DMS O or NCT plus palmitate (0.25 mM) was added for 2 days, followed by harvesting or analysis of the level of fat by Nile Red. Shown are representative photomicrographs. N = 4 biological replicates. E Quantification of Nile Red-positive cells processed as in D. The percent of total cells with Nile Red staining greater than a predetermined threshold was determined for each siRNA and normalized to the scrambled siRNA for that gene to calculate the fold change using a Celigo imaging cytometer. N = 4 biological replicates. F SPNS2 and S1PR3, but not SPHK2, are required for NCT-induced fat clearance. Cells were treated with siRNAs to the indicated genes plus NCT and palmitate as in panel A, followed by staining with Oil Red O. Shown are representative photomicrographs. G Quantification of cellular fat detected by Nile Red staining was done as in panel E. N = 4 biological replicates. Values represent the mean ± SE. *p < 0.05, **p < 0.01 (vs DMSO for A–C or scrambled siRNA for D–G). Scale bar = 100 μm.