Dharmafect 4 reagent

Knockdown of target genes was established by a reverse transfection using smartpool siRNAs according to the manufacture’s protocol (Dharmacon, Pittsburgh, PA, USA) using Dharmafect 4 reagent and with final siRNA concentration of 50 nM.

IKE was purchased from Med Chem Express (HY-114481). Ferrostatin-1 was purchased from Sigma (SML0583). TRIzol reagent (15596026) was purchased from Thermo fisher scientific. Silencer Select siRNA to Zeb1 (assay id s229971) was purchased from Ambion. Dharmafect4 reagent was purchased from Dharmacon. The siRNA targeting TAZ and YAP was synthesized by Dharmacon and sequence was described previously (20 (link)). LM332 was purchased from Biolamina. We purchased mirVana miRNA inhibitor and hsa-miR-200 (a, b, and c) from Invitrogen.

For MDA-MB-231 and BT549 cells, miRIDIAN miRNA mimics (Dharmacon, Lafayette, CO, USA) were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol for transfecting siRNA. Mimics (final concentration of 50 nM) were mixed with 1 % Lipofectamine 2000 (v/v) diluted in Opti-MEM transfection medium (Invitrogen) and incubated for 20 minutes. The mimics were added dropwise onto cells in growth medium at a final concentration of 50 nM. Fresh medium was replaced 24 h post transfection. A non-radioactive cell proliferation assay (Promega, Madison, WI, USA) was used to assess the number of viable cells. Three biological replicates were conducted.
MDA-MB-436 and HS578T breast cancer cells were reverse transfected with 40 nM of either miR-184 mimic or scrambled using Dharmafect 4 reagent (Dharmacon) following manufacturer’s instructions; SiTox (Dharmacon) was used as a positive control. The following day, medium was changed and cells were left in culture for an additional 72 h. To assess viability, CellTiter-Glo (Promega) was added directly into the cell medium (1:2 ratio) and left to incubate for 10 minutes; luminescence was read using FLUOstar Omega plate reader (BMG Labtech, Ortenburg, Germany). The experiment was performed in three biological replicates.

All the cell lines were transfected with a negative control siRNA or a specific siRNA using DharmaFECT 4 reagent (GE Dharmacon, Lafayette, CO). A mixture of siRNA (15 nM final concentration) and DharmaFECT 4 reagent (12.5 nM final concentration) was incubated for 20 minat room temperate before being applied to the cells.

HeLa cells were plated in a six-well plate and grown to confluency. Cells were transfected with 20 pmol of siRNA (ON-TARGETplus siRNA SMARTPool, Dharmacon) and DharmaFECT 4 reagent (Dharmacon) according to manufacturer’s instructions. Cells were incubated for 48–72 h at 37 °C and 5% CO₂, and siRNA knockdown was validated by western blotting.

The full-length coding sequence of psoriasin [GenBank: AJ012825.1] was cloned into pcDNA3.1 [37 (link)] and transfected into TU167 cells by Lipofectamine (Invitrogen, Burlington, ON, Canada). Stable clones were selected by zeocin, as a selection marker. Several clones were developed, and immunoblot determined protein expression of psoriasin. TU167-mock was generated by transfection with the empty vector. JMAR cells were also transfected with protein-specific siRNA (Qiagen, Mississauga, ON, Canada) of psoriasin. siRNA was conducted using Dharmafect-4 reagent (Dharmacon).