(A) HCT116, SW480, WiDr and HT29 cells were maintained in DMEM with 1 mg/ml glucose and treated with 0.1, 1, 10 µg/ml cetuximab or 1, 3, 5 µM SAHA the cell survival was measured by MTT assay after 48 hours treatment (B) HCT116 and SW480 cells were serum-starved for 24 hours and then stimulated with 1 µM EGF for, 1, 5, 15, 30 and 60 minutes. (C) HCT116 and SW480 cells were pre-incubated with 0.5, 1 µM TSA or 5 µM SAHA for 24 hours and then stimulated with EGF for 5 minutes. (D) HCT116, SW480, A431 and MDA-MB468 cells were treated with 1 µM TSA for 24 or 36 hours Whole cell lysate was prepared and subjected to western blot analysis with antibodies specific for phospho-Akt, phosphor-ERK, Akt and Erk.

(A) HCT116 cells were transfected with Myc-EGFR as well as its vector control and transfected cells were treated with 5 µM SAHA for 24 hours. Cells were fixed by 70% ethanol and stained with propidium iodide, and fraction of cell cycle was analyzed by flow cytometer. (B) HCT116 cells were transfected with Myc-EGFR as well as its vector control and the transfected cells were treated with 5 µM SAHA for 24 hours. Whole cell lysate were prepared and subjected to western blot using Ab specific for EGFR, Myc, p21 and tubulin.

(A) Cells were treated with 1 µM Mithramycin A (MTM) for 18 or 36 hours, then the total cell lysate were prepared. Cells were transfected with 0, 400 or 800 pmole SP1 siRNA, then the total cell lysates were prepared and subjected to western blot analysis with antibodies specific for EGFR and SP1. (B) Cells were transfected with EGFR-Luc and then treated with 0.1, 1 or 5 µM Mithramycin A (MTM) for 16 hours. Luciferase activities were measured as described under “Material and Method”. The results were normalized to the Renilla luciferase activity and expressed as the mean ± SE. For three independent experiments performed in triplicate.

In the basal state, HDAC3, CBP and SP1 were both recruited to the promoter region and responsible for the transcription of EGFR (A). While treated with HDACi, the complex of HDAC3, CBP and SP1 were disrupted and dispersed from EGFR promoter, leading to the inactivation of EGFR transcription (B).

(A) HCT116 cells were treated with 1 µM TSA for 24, 36 or 48 hours. Whole cell lysate were prepared and subjected to western blot analysis with antibodies specific for SGLT1, EGFR and Actin. (B) Cells were cultured in DMEM with 1 mg/ml and treated with 1 µM TSA for 24 hours. The glucose content was measured as described in material and method (Triplicate samples were used in each group. The asterisk indicates a significant difference with p<0.05. Error bars indicate mean ± SE.) (C) Cells were cultured in DMEM with 1 mg/ml or 4.5 mg/ml glucose and treated with 1, 3 or 5 µM TSA for 24 hours. The glucose content was measured. (D) Cells were cultured in DMEM with 1 mg/ml glucose and treated with 1 or 3 µM TSA. After 24 or 48 hours of treatment, the glucose was adjusted to 4.5 mg/ml. The cell survival was measured by MTT assay after 72 hours treatment with TSA. Results were expressed as mean ± SE of three independent experiments performed in triplicate.

(A) HCT116 cells were treated with 1 µM TSA or 5 µM SAHA for 1 and 8 hours. Total RNA (2 µg) was used for RT-PCR and Real-time PCR as described. (B, upper panel) Cells were treated with 1 µM TSA or 5 µM SAHA for 6 hour, then transfected with EGFR-Luc. Luciferase activities were measured as described under “Material and Method”. (B, lower panel) Cells were treated with 1 µM TSA for 4 h before RNA synthesis was stopped by actinomycin D (5 µg/ml) for 0, 2, 4 or 6 h. RNA was prepared at indicated time points following the addition of actinomycin D and levels of EGFR mRNA were measured by real-time PCR. (C, upper panel) Cells were transiently transfected with Myc-tagged HDAC1, 2 or 3, respectively and total RNA (2 µg) was used for RT-PCR to detect the EGFR mRNA level. (C, lower panel) Cells were transfected with shLuc, shHDAC1, shHDAC2 or shHDAC3 by lentivirus as described under “Material and Method”. Cell lysates were harvested on day 5 after transfection and then subjected to western blot analysis with antibodies specific for EGFR, HDAC1, HDAC2 and HDAC3. (D) Lysates of paired human normal and malignant colon tissues were subjected to western blotting using anti-EGFR, anti-HDAC3 and anti-Actin antibodies. The correlation between EGFR and HDAC3 expression levels was evaluated by correlation coefficients.

(A) Illustration of the EGFR promoter and the ChIP primer. (B) Cells were treated with SAHA for 8 hours, then fixed, sonicated and subjected to Chromatin Immunoprecipitation using Ab specific against SP1 and acetylated histone H3. Histone H4 acetylation H3K4 dimethylation, CBP and HDAC3 on EGFR promoter was measured by ChIP assays as described under “Material and Method”.