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. 2022 Jun 7;25(7):104542. doi: 10.1016/j.isci.2022.104542

Figure 4.

Figure 4

Determination of expression and activity of SGLT1 and MTP in monolayer IECs

(A and D) Monolayer IECs from hiPSOs and undifferentiated and differentiated Caco-2 cells were cultured in either collagen I-coated six-well plates or Transwells, as in Figure 1. mRNA levels of each gene were determined by qRT-PCR and normalized to 18S rRNA levels. Assays were performed in n = 3 independent biological replicates (mean ± S.D.). Statistical significance was determined by one-way analysis of variance with the Bonferroni test. ∗p < 0.05 (versus each group of IECs).

(B) Immunohistochemistry analysis of frozen sections of hiPSO-derived IECs stained with DAPI (blue) and an anti-SGLT1 (red) antibody. Scale bar, 20 μm.

(C) Monolayer IECs from hiPSOs and undifferentiated and differentiated Caco-2 cells were treated with 10 μM phlorizin or 1 μM sotagliflozin for 2 h before treatment with 100 μM AMG and 1 μM [14C]-AMG for 1 h. A scintillation counter determined radioactivity. Assays were performed in n = 3 independent biological replicates (mean ± S.D.). Statistical significance was determined by one-way analysis of variance with the Bonferroni test. ∗p < 0.05 (versus no inhibitor treatment group). No asterisk indicates no significance (p > 0.05).

(E) Monolayer IECs from hiPSOs were treated with or without 500 μM OA for 3, 8, 24, and 48 h in a basal medium with 20% FBS. Supernatants were collected from the apical or basolateral side. The same volume of the supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis was performed using an anti-ApoB antibody. Right, an illustration of Transwell insert.

(F and G) Monolayer IECs (F) or differentiated Caco-2 cells (G) were treated with or without 500 μM OA for 24 h in a basal medium with 20% FBS. Cells were after that treated with 0, 1, 10, 100, and 1000 nM CP346086 in the presence of 500 μM OA After 24 or 48 h, supernatants from the basolateral side were collected. The same volume of the supernatant was subjected to SDS-PAGE, and Western blot analysis was performed using an anti-ApoB antibody.

See also Figures S5 and S6.