Fig. 1.

Caco2 cells display a biphasic secretion of PCSK9 at the basolateral side during their differentiation into enterocytes. Caco2 cells were maintained on polycarbonate micropore membranes inserted into Transwells. The medium was changed every two days. A and B: Basolateral supernatants were collected every two days from day 4 to day 22 in two or more independent experiments done in triplicates. A: PCSK9 was measured in supernatants by ELISA. B: Proteins from supernatants (300 μl) were precipitated with acetone, and apoB and PCSK9 were analyzed by Western Blot (WB). C: Caco2 cells were recovered at day 8 (d8) and at day 16 (d16) after a 2-h secretion assay, and mRNAs were extracted. Sucrase isomaltase and ApoB expression relative to that of cyclophilin was analyzed by RT-qPCR in samples from nine independent experiments. Scatter dot plots + bars (median ± interquartile) are presented. ∗∗∗P< 0.001; ∗∗∗∗P< 0.0001. D and E: Day 8 (d8) and day 16 (d16) after Caco2 cells seeding, the PCSK9 content in supernatants from basolateral and apical sides after a secretion assay of 2 h was measured (C) by ELISA, scatter dot plots + histograms represent median ± interquartile. ∗∗P value < 0.01, or analyzed (D) by WB after acetone protein precipitation, the right blot showing apical PCSK9 secretion has been voluntarily overexposed to facilitate band visualization. Results are from six and three independent experiments, respectively. PCSK9, proprotein convertase subtilisin/kexin type 9.