Psih1 puro stat3 shrna

Luciferase assays were performed with the dual luciferase assay kits (Promega, Madison, WI, USA) according to the manufacturer's instructions. In brief, p4xM67-TK-luc plasmid (Addgene plasmid 8688, Addgene, Cambridge, MA, USA) [32 (link)] containing four copies of the STAT-binding site (TTCCCGTAA) was transfected in 293T or MDA-MB-231 cells and then extracts were treated for 24 hours. EF.STAT3C.UBC.GFP and EF.STAT3DN.UBC.GFP (Addgene plasmids 24983 and 24984, Addgene, Cambridge, MA, USA) [33 (link)] were transfected into 293T or MDA-MB-231 cells, which were subjected to the luciferase assays. Luciferase assays were conducted in quadruplicate and independently repeated at least three times. Representative data were described as means ± standard deviations. For knockdown strategies, pSIH1-puro-STAT3 shRNA (Addgene plasmid 26596, Addgene, Cambridge, MA, USA) [34 (link)] was used.

The short hairpin RNA (shRNA)-expressing lentiviral vectors (pSIH1-puro-STAT3 shRNA: #26596, pSIH1-puro-control shRNA: #26597) (Addgene, Watertown, MA, USA) and cDNA-expressing lentiviral vectors (pCDH-puro-BCL-X_L: #46972 [Addgene], pCDH-CMV-MCS-EF1-Puro: #CD510B-1 [System Biosciences, Palo Alto, CA, USA]) were used to stably express shRNA or cDNA, respectively. To generate lentiviruses, HEK 293T/17 cells were transfected with lentiviral vectors and packaging plasmids (Lentiviral High Titer Packaging Mix: Takara Bio, Shiga, Japan) using TransIT®-293 transfection reagent (Mirus Bio, Madison, WI, USA). On the following day, the medium was replaced with fresh growth medium, and lentivirus-containing supernatants were harvested and concentrated by centrifugation using a Lenti-X™ Concentrator (Takara Bio). To establish shRNA- and cDNA-expressing stable cell lines, the cells were transduced with lentiviral particles using 1 μg/mL Polybrene® (Santa Cruz Biotechnology, Dallas, TX, USA) and then selected with 2 μg/mL puromycin (Sigma-Aldrich) for 10–14 days. Stable gene knockdown was confirmed by western blotting.