Journal:

Article Title: p21 WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

doi:

Figure Lengend Snippet: NaBu and TSA induce p21 mRNA expression in HT-29 cells in an immediate-early fashion. (A) Dose response for p21 induction by NaBu. HT-29 cells were treated with various concentration of NaBu, 0–20 mM for 24 hr, and p21 mRNA expression was examined by Northern blot analyses. (B) Time course for induction of p21 by NaBu. Cells were treated with 5 mM NaBu for varying lengths of time, from 0 to 24 hr, and p21 induction was examined by Northern analysis. (C) p21 induction by NaBu is not blocked by protein synthesis inhibition. HT-29 cells were treated concomitantly with 5 mM NaBu and the protein synthesis inhibitor, cycloheximide (CHX, 10 μg/ml), for 48 hr. (D) Time course for p21 induction by TSA. Cells were treated with 0.3 μM TSA for varying lengths of time. (E) p21 induction by TSA is not blocked by protein synthesis inhibition. Cells were treated concomitantly with 0.3 μM TSA and 10 μg/ml CHX for 6 hr. Representative Northern blots (n = 4) are depicted in each figure. The actin control is shown in the lower panel. Twenty micrograms of total RNA was loaded in each lane, with equal loading verified by ethidium bromide staining.

Article Snippet: For stable transfections, HT-29 cells were transfected with an expression plasmid encoding the neomycin resistance gene (pSV2-neo, ATCC) and/or the human p21 gene (pCMV-Cip1, ATCC).

Techniques: Expressing, Concentration Assay, Northern Blot, Inhibition, Control, Staining

Journal:

Article Title: p21 WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

doi:

Figure Lengend Snippet: Overexpression of histone deacetylase (HDAC1) blocks induction of the p21 promoter by NaBu and TSA. (A) NaBu and TSA induce p21 promoter activity. HT-29 cells were transiently transfected with CAT reporter plasmids under the control of various amounts of the mouse p21 promoter (0–4.7 kb upstream from the transcriptional start site). Transfectants were treated without or with 5 mM NaBu or 0.3 μM TSA, and total cellular protein was examined for CAT expression. Results are shown for the 0-kb (pCAT), 1.4-kb (p211.4CAT), and 4.7-kb (p214.7CAT) p21 promoter constructs and are expressed as fold CAT induction compared with the untreated negative control, arbitrarily taken as 1. (B) Cotransfection of HDAC1 blocks p21 promoter induction by NaBu and TSA. Transfections were performed with the p214.7 CAT −/+ HDAC1, and cells were treated without or with 5 mM NaBu or 0.3 μM TSA. The results are expressed as fold CAT induction compared with the untreated negative control (arbitrarily taken as 1). All results are shown as mean ± SEM (n ≥ 4, in all cases).

Article Snippet: For stable transfections, HT-29 cells were transfected with an expression plasmid encoding the neomycin resistance gene (pSV2-neo, ATCC) and/or the human p21 gene (pCMV-Cip1, ATCC).

Techniques: Over Expression, Histone Deacetylase Assay, Activity Assay, Transfection, Control, Expressing, Construct, Negative Control, Cotransfection

Journal:

Article Title: p21 WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

doi:

Figure Lengend Snippet: NaBu, TSA, and p21 overexpression inhibit growth of HT-29 cells. (A) NaBu and TSA inhibit growth of HT-29 cells. Cells were plated at equal density in six-well plates. At 80% confluence, cells were treated without or with 5 mM NaBu or 0.3 μM TSA for 24 hr. 3H-thymidine (1 μCi/ml) was added for the last 6 hr of treatment, and incorporation was measured by scintillation counting. Treatment results are expressed as percent change compared with untreated negative control, arbitrarily taken as 1. (B) p21 overexpression inhibits growth of HT-29 cells. Cells were stably transfected with an expression plasmid containing the human p21 gene, and overexpression of the gene in pooled stable transfectants compared with the parental and NEO controls (n = 5 each) was determined by Northern analyses (see Inset). 3H-thymidine incorporation was measured in exponentially growing cells under basal conditions, and results are expressed as percent change compared with parental control, arbitrarily taken as 100%, and are shown as mean ± SEM (n ≥ 4, in all cases).

Article Snippet: For stable transfections, HT-29 cells were transfected with an expression plasmid encoding the neomycin resistance gene (pSV2-neo, ATCC) and/or the human p21 gene (pCMV-Cip1, ATCC).

Techniques: Over Expression, Negative Control, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Northern Blot, Control

Journal:

Article Title: p21 WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

doi:

Figure Lengend Snippet: p21 is required for NaBu-induced growth inhibition of colon cancer cells. (A) p21 mRNA is induced early in HCT116 +/+ cells. Cells were treated at 80% confluence with 1 mM NaBu or 0.15 μM TSA (higher concentrations of either agent led to cell death), and p21 mRNA expression was examined by Northern analysis. (B) p21 deletion prevents growth inhibition by butyrate and TSA. HCT116 +/+ and −/− cells were treated with 1 mM NaBu or 0.15 μM TSA for 24 hr, and 1 μCi/ml 3H-thymidine was added for the last 6 hr of treatment. Incorporation was measured by scintillation counting. (C) HCT116 −/− cells are growth-inhibited by serum starvation and postconfluent growth. HCT116 +/+ and −/− cells were serum-starved for 24 hr or grown to 2 weeks postconfluence, and 3H-thymidine incorporation was measured. Cells grown in standard McCoy’s 5A medium with 10% fetal bovine serum and that were preconfluent were used as controls. Results are expressed as percent change compared with control groups (arbitrarily taken as 1) and are shown as mean ± SEM (n ≥ 4, in all cases).

Article Snippet: For stable transfections, HT-29 cells were transfected with an expression plasmid encoding the neomycin resistance gene (pSV2-neo, ATCC) and/or the human p21 gene (pCMV-Cip1, ATCC).

Techniques: Inhibition, Expressing, Northern Blot, Control