Journal: Cancer Science

Article Title: TNF‐α enhances TGF‐β‐induced endothelial‐to‐mesenchymal transition via TGF‐β signal augmentation

doi: 10.1111/cas.14455

Figure Lengend Snippet: Quantification of Smad2/3‐activating factors in conditioned medium (CM) of HUAECs using HEK‐Blue TGF‐β reporter cells. (A) HUAECs were cultured in the absence (−) or presence (+) of 1 ng/mL TGF‐β2 in combination with 10 ng/mL TNF‐α for 60 h, followed by DMEM with 1% FBS, replacement and further culture for 16 h. (B) HEK‐Blue TGF‐β cells were cultured in the CM derived from HUAECs cultured for 16 h, followed by the measurement of absorbance at 640 nm, which represented colorimetric change of HEK‐Blue substrate by secreted alkaline phosphatase induced by Smad2/3 signal. (C, D) HEK‐Blue TGF‐β cells were cultured in the CM derived from HUAECs, in the absence (−) or presence (+) of 50 ng/mL of follistatin (C), control IgG (50 μg/mL) and anti‐TGF‐β (1D11: 50 μg/mL) (D), followed by the measurement of the absorbance at 640 nm representing colorimetric change of HEK‐Blue substrate by SEAP alkaline phosphatase induced by Smad2/3 signals. Values were normalized to the number of HUAECs responsible for secretion of Smad2/3‐activating humoral factors. Error bars represent standard deviation. * P <0.05; n.s., not significant

Article Snippet: HUAECs were treated with TGF‐β2 and TNF‐α for 60 h. The culture medium was then replaced with DMEM with 1% FBS, followed by incubation for additional 16 h. The CM was then collected and added to the pre‐seeded HEK‐Blue TM TGF‐β reporter cells, followed by incubation for 24 h. Fraction of the medium obtained from incubation with HEK‐Blue cells was then mixed with QUANTI‐Blue substrate (InvivoGen) and incubated for 30 min.

Techniques: Cell Culture, Derivative Assay, Standard Deviation