Fig. 5.

Effect of ETEC infection on expression of human thiamin transporter-1 and -2 (hTHTR-1 and -2) at the protein (A and B), mRNA (C and D), and promoter (E and F) levels. Confluent monolayers of Caco-2 cells were infected with ETEC (200 MOI for 3 h along with 6 h postincubation) and total protein was isolated by treating with RIPA buffer. Western blot analysis was performed using specific anti-hTHTR-1 (A) and anti-hTHTR-2 (B) polyclonal antibodies. Representative gel image is shown. *P < 0.05, compared with control. Quantitative real-time PCR analysis was performed using total RNA from Caco-2 cells treated with ETEC or nonpathogenic E. coli and hTHTR-1 (C)- and hTHTR-2 (D)-specific primers as described in Methods. *P < 0.05, compared with control. Effect of ETEC infection on activity of the SLC19A2 (E) and SLC19A3 (F) promoters. Caco-2 cells were transfected with SLC19A2 and SLC19A3-luciferase reporter plasmid constructs or a control pGL3-basic vector by use of Lipofectamine 2000. Cells were then infected (3 h) with ETEC or nonpathogenic E. coli (200 MOI) followed 6 h postinfection incubation. Luciferase assay was performed as described in Methods. Firefly luciferase activity was normalized relative to the activity of simultaneously expressed Renilla luciferase. All experiments were run on at least 3 separate occasions. Results of a representative experiment are shown. Values are means ± SE. *P < 0.05, compared with control.