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. 2022 Jul 27;119(31):e2201096119. doi: 10.1073/pnas.2201096119

Fig. 7.

Fig. 7.

Roles of ceramide in mouse and zebrafish cilia. (A) Colocalization of ceramide and acetylated tubulin immunofluorescence in cilia of mouse IMCD3 epithelial cells and zebrafish lateral line neuromasts (rosette-like sensory organs that detect directional water flow) and olfactory placode (nose, the lateral edge of the anterior neural plate) (magenta, acetylated tubulin; green, ceramide; blue, DAPI). (Scale bar, 5 μm.) (B) Representative immunocytochemistry images in IMCD3 cells treated with ceramide biosynthesis inhibitors (5 μM myriocin, 10 μM FB1) with or without exogenous 10 μM sphingolipid mixture [sphingolipid mix contains sphingosine (C17 base), sphinganine (C17 base), sphingosine-1-P (C17 base), sphinganine-1-P (C17 base), lactosyl(β) C12 ceramide, 12:0 sphingomyelin, glucosyl(β) C12 ceramide, 12:0 ceramide, and 12:0 ceramide-1-P] or transferred with sptlc1 or sptlc2 siRNA (green, acetylated tubulin; blue, DAPI) for 48 h. (Scale bar, 5 μm.) (C) Quantifications in IMCD3 cells treated with ceramide biosynthesis inhibitors (5 μM myriocin, 10 μM FB1) with or without exogenous 10 μM sphingolipid mixture. n = 161 to 179 cells from three independent experiments. Values are the mean ± SD combined cell populations. Statistical significance was calculated by the Student’s t test using the means of biological replicates: *P < 0.05; **P < 0.01; ***P < 0.001; and N.S., not significant. (D) Quantifications in IMCD3 cells transferred with sptlc1 or sptlc2 siRNA. n = 115 to 200 cells from three independent experiments. Values are the mean ± SD combined cell populations. Statistical significance was calculated by the Student’s t test using the means of biological replicates: **P < 0.01 and ***P < 0.001. (E) Alignment of sequences flanking the dominant human diseases-associated residue C133 and V144 in SPTLC1. (F) Representative immunocytochemistry images in IMCD3 cells transfected with SPTLC1 related mutants (green, acetylated tubulin; magenta, SPTLC1; blue, DAPI). (Scale bar, 5 μm.) (G) Quantifications in IMCD3 cells transfected with SPTLC1-related mutants. n = 90 to 120 cells from three independent experiments. Values are the mean ± SD combined cell populations. Statistical significance was calculated by the Student’s t test using the means of biological replicates: *P < 0.05; **P < 0.01; ***P < 0.001; and N.S., not significant.