The human colonic monocarboxylate transporter Isoform 1: Its potential importance to colonic tissue homeostasis  Mark Cuff, Jane Dyer, Mark Jones, Soraya Shirazi-Beechey  Gastroenterology  Volume 128, Issue 3, Pages 676-686 (March 2005) DOI: 10.1053/j.gastro.2004.12.003 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Inhibition of MCT1 abundance by RNAi using 21-nt siRNAs targeted to the MCT1 transcript. HT-29 cells were grown to 30% confluence, transfected with siRNA specific for human MCT1, and maintained under standard culture conditions until harvest. Mock transfections and transfections using siRNA unrelated to MCT1 each served as parallel experimental controls. MCT1 abundance was monitored by both immunofluorescence and Western analysis. (A) Representative immunofluorescence images showing MCT1 abundance within the plasma membrane 72 hours posttransfection; (i) mock-transfection, (ii) MCT1-specific siRNA, and (iii) unrelated siRNA control. (B) Western analysis at increasing times (36–96 hours) posttransfection. Con, unrelated siRNA control. 0, mock-transfected control. Gastroenterology 2005 128, 676-686DOI: (10.1053/j.gastro.2004.12.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Inhibition of MCT1 expression is reflected functionally as a decrease in butyrate transport. HT-29 cells were grown to 30% confluence, transfected with siRNA specific for human MCT1, and maintained under standard culture conditions until harvest at 72 hours. Mock transfections served as parallel experimental controls. Plasma membrane vesicles were prepared and [U-14C] butyrate uptake was determined as described in the Materials and Methods section. Values are means ± SEM of 3 experiments. Gastroenterology 2005 128, 676-686DOI: (10.1053/j.gastro.2004.12.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Butyrate modulation of target gene expression in HT-29 cells. Cells either were treated with NaBut (.2–10 mmol/L) for 36 hours or left untreated for the same time period. Total RNA was isolated and abundance of IAP, p21, CD1, bcl-xL, and bak mRNA was determined by Northern blotting. Data shown are representative of 3 independent experiments. β-actin served as an internal loading control. Gastroenterology 2005 128, 676-686DOI: (10.1053/j.gastro.2004.12.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Effect of inhibition of MCT1 expression on butyrate-induced modulation of target gene expression. HT-29 cells were mock-transfected or transfected with siRNA targeted to the MCT1 transcript. At 36 hours posttransfection, the cells either were maintained in standard culture conditions or treated with NaBut for the same time period (36 h). Total RNA was isolated and relative abundance of IAP, p21, CD1, bcl-xL, and bak mRNA was determined by Northern blotting. Left: Representative autoradiograms. Right: Combined densitometric analyses, each from 3 independent experiments. β-actin served as a loading control. Results are expressed relative to those obtained from mock-transfected controls, assigned a value of 1. (Error bars represent SEM.) Gastroenterology 2005 128, 676-686DOI: (10.1053/j.gastro.2004.12.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Cellular and functional consequences of inhibition of MCT1 expression. HT-29 cells either were mock-transfected or transfected with siRNA targeted to the MCT1 transcript. At 36 hours posttransfection, the cells either were treated with NaBut or maintained in standard culture conditions for the same time period (36 hours). Cells were harvested and cell-cycle distribution, IAP activity, and apoptosis were determined as described in the Materials and Methods section. (A) Cell-cycle distribution. Representative histograms showing DNA content. Axes represent cell number (ordinate) and fluorescence intensity (abscissa). (B) Relative proportion of cells in G1 phase of the cell cycle. Results are expressed relative to the mock-transfected control cell population, which is assigned a value of 1. (C) Relative IAP activity. Results are expressed relative to the mock-transfected control cell population, which is assigned a value of 1. (D) Proportion of apoptotic cells expressed as percentage of total cell population. Error bars represent SEM determined from combined analyses of 3 independent experiments. Gastroenterology 2005 128, 676-686DOI: (10.1053/j.gastro.2004.12.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions