NUDT6 is suppressed in the presence of catechins. (A, left panel) HCT-116 cells were treated with 50 µM of epicatechin gallate (ECG) for 24 h, and RT-PCR was performed for NUDT6; ATF3, activating transcription factor 3; NAG-1, NSAID-activated gene-1; NR4A1, nuclear receptor subfamily 4, group A, member 1; ID2, inhibitor of DNA binding 2; ID1, inhibitor of DNA binding 1. (A, right panel) Gel densitometry results from three independent experiments for the respective genes from left panel. Significance was set at p=*0.05, **0.01, and ***0.001. (B) Different human colorectal cancer cells were treated with ECG (50 µM) for 24 h, and NUDT6 RNA transcripts were measured by RT-PCR. (C) HCT-116 cells were treated with ECG at different time points and at different doses. (D) HCT-116 cells were treated with different catechins (50 µM each), and NUDT6 gene expression was determined by RT-PCR.

Involvement of 3’UTR sequences in EGCG-induced NUDT6 mRNA suppression. (A) Schematic diagram of the pSV40-LUC-NUDT6 3’UTR construct. (B) The construct was transfected (0.5 µg) along with pRL-null vector (0.05 µg) into HCT-116 cells, and the cells were treated with different doses of EGCG (10, 50 and 100 µM). After 24 h treatment, luciferase activity was measured. All other values were normalized to the control (Veh) value set as 1.0. Tukey’s multiple comparison test was used to express results as mean ± SD of three independent transfections, and different letters indicate significant difference (p< 0.05). (C) After transfection, HCT-116 cells were treated with 4 different catechins (EGCG, ECG, EC and EGC at a dose of 50 µM) and luciferase activity was measured. The values obtained from vehicle-treated samples (Veh) were defined as 1.0, and data was analysed using Tukey’s multiple comparison test. (D) HCT-116 cells were plated in a 12-well plate, transfected with the construct mentioned in Fig. 3A, pretreated with inhibitors for the ERK (U0126, 5 µM), p38MAPK (SB203580, 15 µM) and JNK pathways (SP600125, 30 µM) for 30 min and then treated with EGCG (50 µM) for 24 h. The y-axis represents luciferase activity measured by relative luciferase unit (RLU). **p<0.01 and ***p<0.001, based on Student t tests. All experiments were performed in triplicate with columns representing means and bars representing SD.

Post-transcriptional regulation of NUDT6. (A) EGCG facilitates NUDT6 mRNA degradation. HCT-116 cells were incubated for 24 h with vehicle or EGCG at a concentration of 50 µM and subsequently treated with actinomycin D (5 µM). At the indicated times, total RNA was isolated, and NUDT6 transcripts were measured by RT-PCR. The relative level of NUDT6 mRNA was calculated, and the results were plotted as the percentage of the mRNA level present at time 0 of actinomycin D treatment (bottom panel). (B) HCT-116 cells were pretreated with 10 µg/ml cycloheximide and DMSO for 1 h in serum-free media followed by treatment with EGCG (50 µM) for 24 h. The data are representative of three independent experiments. ***p< 0.001.

Characterization of Biological activity of NUDT6 in human colorectal cancer cells. (A) The stable cell lines pcDNA3.1/NUDT6 and pcDNA3.1/CONTROL were made as described in materials and methods section. These clones contained tag proteins, V5 and histidine. Western blot was carried out using anti V5 antibody to confirm the integration of plasmid. Actin served as a loading control. (B) Cell growth curve for CONTROL and NUDT6 cells. CONTROL and NUDT6 cells were plated into a 96-plate (1 day before treatment) and either vehicle or 5 µM staurosporine added at 0 day. Cell growth was measured using the CellTiter96 Aqueous One Solution Cell Proliferation Assay. Values are expressed as mean±SD of six replicates. *p<0.05, **p<0.01, ***p<0.001 versus control cells. (C) Soft agar assay. The stable cell lines were grown for 2–3 weeks in 0.4% soft agar and stained with p-iodonitrotetrazolium violet solution. The results are representative of three different experiments. The data represent mean ± SD. (D) Micro-impedance measurement of HCT-116 cells with stable cell lines. The corresponding normalized resistance (R) and reactance (X) were measured in HCT-116 cells over a 3.5-day period. The subscripts c and n indicate cell-covered and naked scans, respectively. For the sake of clarity, symbols represent 20 data intervals. The starting cell concentration used here was 2.4 × 10⁴ cells/ml.