Fig. 6: Overexpression of CERS2 transcript (AS1) decreases expression of very-long-chain ceramides in BT-474 cells. | Cell Death & Disease

Fig. 6: Overexpression of CERS2 transcript (AS1) decreases expression of very-long-chain ceramides in BT-474 cells.

From: Alternative splicing of ceramide synthase 2 alters levels of specific ceramides and modulates cancer cell proliferation and migration in Luminal B breast cancer subtype

Fig. 6

A Change in expression of all ceramide synthases (CERS1-6) (mean ± SD, n = 4) in BT-474 cells transfected with empty vector, BT-474 (+Vector), full-length protein-coding CERS2 OE (PC), and alternatively spliced transcript coding CERS2 OE (AS1) constructs. Expression was normalized to β-actin. B Immunoblot showing the expression of CERS2 in BT-474, BT-474 (+Vector), CERS2 OE (PC), and CERS2 OE (AS1) cells by western blot analysis. Protein corresponding to PC transcript is marked by blue asterisk, and to AS1 transcript by red asterisk. C Heat map showing the average fold change in levels of ceramides (C16:0, C18:0, C20:0, C22:0, C24:0, and C24:1) in BT-474 cells transfected with CERS2 OE (PC) and CERS2 OE (AS1) with respect to BT-474 cells. D–F Absolute quantification of ceramides (mean ± SD, n = 4) show significant decrease in C22:0 (D), C24:0 (E), and C24:1 (F) ceramides in CERS2 OE (AS1) cells as compared to CERS2 OE (PC) cells. G–I Fold change (mean ± SD, n = 4) in deuterium-labeled ceramides (pmol/mg protein) in HEK 293T cells transfected with CERS2 OE (PC) and CERS2 OE (AS1) constructs using HEK 293T cells as a control shows significant decrease in C24:0 Ceramide (Cer)-d7 (G) and C24:1 Cer-d7 (H) ceramides with no significant change in C16:0 Cer-d7 (I) ceramides in CERS2 OE (AS1) cells as compared to CERS2 OE (PC) cells. Data in A, D–I were analyzed using ANOVA test.

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