Fig 2: Heat-labile toxin alters the transcription of multiple brush border SLC genes.a Heatmap indicating key SLC genes modulated by heat-labile toxin (LT) compared to enzymatically inactive E112K LT mutant (mLT), or untreated (ø) Caco-2 cells (left) and human small intestinal (ileal) enteroids (Hu235D, right). Bars indicate absolute log2 fold change values + SE. Comparisons were made with DESeq293. *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 10-4, *****p = 10-5. Real-time qRT-PCR confirming LT-mediated modulation of genes in ileal (Hu235D) enteroids encoding b the major thiamine transporter SLC19A3 and c the SP1 cis-regulatory element. Data reflect two independent experiments with two replicates each. d Uptake of [3H]-thiamine by Hu235D cells is impaired following LT treatment. Data presented in b–d are from two independent experiments with n = 3 replicates each. (*<0.05, **<0.01 by Mann–Whitney two-tailed comparisons).

Fig 3: Effect of hypoxia on chamber-induced hypoxia on level of expression of hypoxia-inducible factors (HIFs) in human colonic epithelial NCM460 cells.A, schematic representation of the [i] SLC44A4, [ii] SLC19A2, and [iii] SLC19A3 minimal promoter regions depicting predicted binding sites for HREs. B, NCM460 cells were exposed to chamber-induced hypoxia for 16 h followed by examination of HIF expression. Level of expression of HIF-1α and HIF-2α protein (immunoblot) in hypoxia-exposed cells compared with those exposed to normoxia (control) condition. All protein results were normalized relative to GAPDH. Statistical significance of all proteins (n = 3; ∗p < 0.05) was evaluated using the Student's t test. HRE, hypoxia-responsive element.

Fig 4: Effect of chemically induced hypoxia on uptake of TPP and free thiamin by human colonic epithelial NCM460 cells. Cells were treated with the hypoxia-mimetic agent DFO (250 μM) for 48 h. A, effect of hypoxia on [i] colonic carrier–mediated [3H]-TPP uptake and [ii] level of expression of cTPPT mRNA. B, effect of hypoxia on [i] colonic carrier–mediated [3H]-thiamin uptake; [ii] and [iii] level of expression of THTR-1 and THTR-2 mRNA, respectively. All mRNA results were normalized relative to GAPDH, and comparison was made relative to simultaneously performed controls (normoxia). Statistical significance of uptake (n = 4; ∗p < 0.05) and mRNA (n = 4; ∗∗p < 0.01) was evaluated by the Student's t test. cTPPT, colonic TPP transporter; DFO, desferrioxamine; THTR-1, thiamin transporter-1; THTR-2, thiamin transporter-2; TPP, thiamin pyrophosphate.

Fig 5: Tissue distribution of SLC19A3 expression. The expression analysis was performed in a panel of human tissues from GTex (A) and in paired subcutaneous and omental adipose tissue biopsies (B, C). Expression analysis was performed by RNA sequencing (A), microarray (B) and real-time PCR (C). Data are presented as mean expression (with SD in B and C). Analysis was performed in 5 and 27 patients, respectively in B and C