Short-chain fatty acids induce cell cycle inhibitors in colonocytes Jingping Wang, Eileen A. Friedman  Gastroenterology  Volume 114, Issue 5, Pages 940-946 (May 1998) DOI: 10.1016/S0016-5085(98)70313-0 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Cell cycle analysis of (A) asynchronous U4 cells cultured in parallel but left untreated or (B) U4 cells by cytofluorography after 24 hours of treatment with a mixture of SCFAs mimicking wheat bran fiber (WBF) digestion products found in the colon. S-phase cells are indicated in brackets. Thirty-five percent of untreated cells were in S phase, whereas the percentage of S-phase cells was only 14% in the wheat bran fiber–treated culture. A mean of 7634 ± 739 (SE) cells was analyzed for each figure. Gastroenterology 1998 114, 940-946DOI: (10.1016/S0016-5085(98)70313-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Western blots showing abundance of TGF-β1 and the cdk inhibitor p21cip1 after treatment of U4 cells for 24 and 72 hours with the following optimal concentrations of SCFAs for inducing growth arrest: 40 mmol/L ammonium acetate (A), 5 mmol/L sodium butyrate (B), 15 mmol/L sodium propionate (P), a mixture of these three SCFAs (OP); and in addition, treatment with mixtures of SCFAs mimicking digestion products of pectin (PC), wheat bran (W), oat bran (O), and cellulose (C) as described in Table 1. CT, control untreated cells. Gastroenterology 1998 114, 940-946DOI: (10.1016/S0016-5085(98)70313-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Analysis of cdk-2/cyclin complexes immunoprecipitated from U4 cells treated for 24 and 48 hours with either a mixture of SCFAs mimicking digestion products of wheat bran (WB) or with 5 mmol/L butyrate (B); decreased kinase activity shown was caused by an increased abundance of cdk inhibitors in the cdk-2/cyclin E complex. (A) Immune complex kinase reaction using histone H1 as substrate; and (B–E) Western blots of immunoprecipitated cdk-2/cyclin complexes for the (B) cdk inhibitor p21cip1, (C) cdk inhibitor p27kip1, (D) cyclin E, and (E) cdk-2, the control for equal immunoprecipitations. Gastroenterology 1998 114, 940-946DOI: (10.1016/S0016-5085(98)70313-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Analysis of cdk-4/cyclin complexes immunoprecipitated from U4 cells treated for 24 and 48 hours with a mixture of SCFAs mimicking digestion products of wheat bran (WB); decreased kinase activity shown was caused by an increased abundance of cdk inhibitors in the cdk-4/cyclin D1 complex. (A) Immune complex kinase reaction using histone H1 as substrate; (B–E) Western blots of cdk-4/cyclin complexes for the (B) cdk inhibitor p21cip1, (C) cdk inhibitor p27kip1, (D) cyclin D1, and (E) cdk-4, the control for equal immunoprecipitations. Gastroenterology 1998 114, 940-946DOI: (10.1016/S0016-5085(98)70313-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Western blot showing abundance of the cdk inhibitor p21cip1 after treatment of U4 cells for 0, 6, 12, 24, and 48 hours with 5 ng/mL TGF-β1 and 6, 24, 48, and 72 hours with either 5 mmol/L sodium butyrate or a mixture of SCFAs mimicking digestion products of wheat bran; the latter induced a 30-fold increase in p21 levels. (B) Western blots showing abundance of the cdk inhibitors p21cip1 and p27kip1 after treatment of U4 cells for 72 hours with 0–20 ng/mL TGF-β1, with TGF-β1 inducing only a modest 2–3-fold increase in p21 and p27 levels. Gastroenterology 1998 114, 940-946DOI: (10.1016/S0016-5085(98)70313-0) Copyright © 1998 American Gastroenterological Association Terms and Conditions