Journal: International Journal of Cancer. Journal International du Cancer

Article Title: Triptolide reverses hypoxia-induced epithelial–mesenchymal transition and stem-like features in pancreatic cancer by NF-κB downregulation

doi: 10.1002/ijc.28583

Figure Lengend Snippet: NF-κB is involved in hypoxia-induced progression. ( a ) Cells were incubated under normoxic (N) or hypoxic (H) conditions, followed by isolation of nuclear protein extracts 24 hr later. DNA binding was analyzed by EMSA using a biotin-labeled oligonucleotide probe for the NF-κB promoter consensus sequence. Specific (NF-κB) and unspecific shifts (UNSP) are marked. Competition with a tenfold excess of unlabeled oligonucleotide (10×) served as a control for specificity of binding. ( b ) Cells were transfected with siRNA oligonucleotides encoding a nonsense (NS) sequence or a specific sequence for inhibition of c-Rel (sicRel). Three days later, proteins were prepared, and the expression of c-Rel was evaluated by Western blot analysis. β-Actin served as a control for equal loading. The bar indicates 50 µm. ( c ) Three days after transfection with nonsense or c-Rel siRNA, the confluent cell layers were scratched, and then, they were cultured under hypoxic conditions. Closure of the wounded region was monitored 6 and 24 hr after scratching by microscopy and documented by photographs. The bar indicates 100 µm. ( d ) Likewise, the expression of EMT-related proteins Slug and Vimentin was detected by double immunofluorescence staining of cells exposed to hypoxia for 48 hr. DAPI counterstaining was used for detection of nuclei. Mouse anti-CD44 mAb/Alexa Fluor 594 IgG (red) and rabbit polyclonal anti-CA IX/Alexa Fluor 488 IgG (green) were used as secondary antibodies. Tissue sections were analyzed under ×400 magnification using a Leica DMRB fluorescence microscope. The bar indicates 25 µm. ( e ) Western blot analysis of Twist2 expression was performed using protein extracts derived from siRNA-transfected cells cultured under normoxia or hypoxia for 3 days.

Article Snippet: Nonsense or c-Rel siRNA oligonucleotides were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Incubation, Isolation, Binding Assay, Labeling, Sequencing, Control, Transfection, Inhibition, Expressing, Western Blot, Cell Culture, Microscopy, Double Immunofluorescence Staining, Fluorescence, Derivative Assay