Dual luciferase assay protocol

HeLa cells were co-transfected with pSTAT3-TA-luc (Beyotime) and pRL-TK (Promega) as a transfection efficiency control in a 6-well plate. After 12 h, cells were transferred to a 48-well plate with complete medium and cultured for 24 h. Cells were pre-treated with different concentration of compounds (in 0.05% DMSO) for 6 h in low FBS medium, and EGF (100 ng/ml) was subsequently added into the wells for 30 min to stimulate STAT3 expression and the cells were incubated for 30 min. In the IFN-α-induced comparative assay, HeLa cells were co-transfected with p-GAS-TA-luc (Biovector, Beijing, China) and pRL-TK in a 24-well plate for 6 h. Cell were cultured with complete medium for another 18 h. The indicated concentrations of compound 1 and S3I-201 were added to each well for 5 h, followed by the addition of 1000 U/ml IFN-α for 1 h to stimulate STAT3/STAT1 expression. Cell lysates were collected according to the dual luciferase assay protocol (Promega). Sample light output was analyzed using a luminometer and the resulting data were normalized relative to pRL-TK values.

The RNAPII inhibitor, α-amanitin (A2263 Sigma, St Louis, MO, USA) was dissolved in bi-distilled sterile water and filtered by 0.2 μm filter. The experiments were performed by using growing doses of a-amanitin, that is, 1, 2, 3, 5, 10 μg/ml on each point of 2000.000 cells/ml or 2 and 10 μg/ml on each point of 500 000–1000 000 cells/ml. After 16 h, the cells were lysed in 700 μl of Qiazol (Qiagen, Venlo, Netherlands) for the RNA extraction.
Transfection of DNA vectors (200–500 ng/50 000 cells) in HEK293T was performed with Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's procedures, whereas transfection of MEC-1, MEC-2, WaC3CD5 and K562 was performed using the Amaxa nucleofector (Lonza, Basel, Switzerland) according to the manufacturer's protocols (plasmids:3000 ng/1 000 000 cells; small interfering RNA 40 pmol/1 000 000 cells). siPOLR3A (sc-90684), siPOLR3G (sc-43507) and small interfering RNA -A as control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The efficiency of transfection was determined by cytofluorometry, messenger RNA expression and\or fluorescence microscopy. After transfection of luciferase reporter vectors (pGL3Ren-187_A and pGL3Ren-187_G), the luciferase assay was conducted according to manufacturer's dual luciferase assay protocol (Promega).