Inhibition of SPT disrupts nuclear morphology in human cells. (a) De novo synthesis pathway of sphingolipids in humans. See Fig. S1 b for a more detailed description of the pathway. Chemicals used in our experiments are highlighted in red: Myr, myriocin; Fum B1, fumonisin B1. SPTC1 and SPTLC2 are the main subunits of SPT; LCBs, long-chain bases; CerS, ceramide synthase. (b) Representative immunofluorescence images of RPE-1 cells upon knockdown of scramble sequence (Control), SPTLC1, or SPTLC2. Green, anti-alpha-tubulin; purple, Hoechst 33342 (DNA); and red, anti-lamin B1. Scale bar, 5 µm. Some images are also shown in Fig. S4 c. (c) Representative images of cells treated with 5 µM myriocin or 10 µM fumonisin B1. Immunofluorescence markers as in b. Scale bar, 5 µm. Some images are also shown in Fig. S4 c. (d) Percentage of the nuclear phenotype of RPE-1 cells. Cells were treated with 5 µM myriocin or 10 µM fumonisin B1 (n = 200 cells). Abnormal refers to irregular nuclear shape relative to normal round nuclei. (e) Circularity (ImageJ) of 200 nuclei for each condition. *P < 1e-4, one-way ANOVA. (f) Representative images of RPE-1 cells in lipid-depleted media treated with 250 nM Torin, 10 µM TOFA, 10 µM cerulenin, 40 µM C75, or 1 µM lovastatin cells for 24 h. Blue, Hoechst 33342 (DNA); green, anti-lamin B1 (NE). Scale bar, 5 µm. (g) Circularity (ImageJ) of 200 nuclei for each condition in Fig. 5 f. *P < 1e-4, one-way ANOVA. (h) FACS profiles of cells in each condition in Fig. 5 f.
