Journal: Oncotarget

Article Title: Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF

doi:

Figure Lengend Snippet: A. Human lung and colorectal cancer cells were plated into 96-well plates at a density of 3 × 10 3 cells/well on day 0. Under serum free culture media (black bar), cells were incubated with CCD-18co colonic fibroblast CM (white bar) and/or CPT-11 for 48 h. Inhibition of cell proliferation was determined by MTT assay. Significant differences were evaluated using an unpaired two-tailed Student's t -test. (Error bars denote the standard deviation [SD]) (* p < 0.05, ** p < 0.01 and *** p < 0.001). B. Induction of apoptosis in colorectal cancer cells. Cells were stained with PI and Annexin V-FITC, followed by flow cytometry analysis. Cells were treated with CPT-11 under serum free media or CCD-18co colonic fibroblast CM. The apoptotic cells were determined by Annexin V-FITC positive staining.

Article Snippet: The human colorectal cancer cell lines, HCT116, HT29 and DLD-1 and lung cancer cell line 226Br were cultured in RPMI1640 (Welgene, Seoul, Korea) containing 10% fetal bovine serum (FBS; Welgene) and antibiotics (100 mg/L penicillin and 100 mg/L streptomycin; GIBCO-BRL Life Technologies; Gaithersburg, MD, USA).

Techniques: Incubation, Inhibition, MTT Assay, Two Tailed Test, Standard Deviation, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Humanized anti-hepatocyte growth factor (HGF) antibody suppresses innate irinotecan (CPT-11) resistance induced by fibroblast-derived HGF

doi:

Figure Lengend Snippet: A. HGF secreted by cancer cells (HCT-116 and DLD-1) and colonic fibroblasts (CCD-18co) were measured. Cells were cultured with serum free medium for 24 h and HGF concentrations were determined by ELISA. B. CM from fibroblast activates c-MET receptors. HCT-116 and DLD-1 cells were cultured with serum free media or CCD-18co CM for 1 h. Cells were collected, and the indicated proteins were detected by western blotting. C. Colonic fibroblast cells promote CPT-11 resistance of colorectal cancer cells (HCT-116 and DLD-1). Cancer cells were cultured with (white bar) or without (black bar) CCD-18co cells, in the presence or absence of CPT-11 (1.25-20 μM) for 48 h, and inhibition of cell proliferation was determined by MTT assay. Significant differences were evaluated using an unpaired two-tailed Student's t -test. (Error bars denote the standard deviation [SD]) (* p < 0.05 and *** p < 0.001). D. Co-culture with colonic fibroblast CCD-18co cells increases c-MET receptor activation in colorectal cancer cells. HCT-116 and DLD-1 cells were co-cultured with CCD-18co cells for 24 h. Lysates were analyzed for c-MET activation by western blotting. E. Inhibition of HGF production from fibroblast by transfection with HGF siRNA. Colonic fibroblast cells were transfected with 10 nM HGF siRNA or scramble siRNA. After transfection, cells were collected and lysates were submitted to Western blotting to quantify HGF. F. HCT-116 and DLD-1 cells were cultured with CM from HGF siRNA transfected fibroblast for 48 h in the presence or absent of CPT-11. Cell viability was determined by MTT assay. Significant differences were evaluated using an unpaired two-tailed Student's t -test. (Error bars denote the standard deviation [SD]) (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Article Snippet: The human colorectal cancer cell lines, HCT116, HT29 and DLD-1 and lung cancer cell line 226Br were cultured in RPMI1640 (Welgene, Seoul, Korea) containing 10% fetal bovine serum (FBS; Welgene) and antibiotics (100 mg/L penicillin and 100 mg/L streptomycin; GIBCO-BRL Life Technologies; Gaithersburg, MD, USA).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Inhibition, MTT Assay, Two Tailed Test, Standard Deviation, Co-Culture Assay, Activation Assay, Transfection