Shrna plasmid a

To overexpress Prx1, SCC15 cells were transfected with pEZ-M02-PRX1 (GeneCopoeia, USA) or control plasmid (GeneCopoeia, USA) with Lipofectamine™2000 (Invitrogen, USA) to 50% confluence on a 6-well plate. After transfection for 48 h, stably transfected cells were selected using G418 (200 μg/ml) for 10 days. To knock down Prx1, SCC15 cells were transfected with Prx1 shRNA Plasmid (Santa Cruz Biotechnology) using Lipofectamine™2000 (Invitrogen, USA). The shRNA Plasmid-A (Santa Cruz Biotechnology) was used as a control. The efficiency of Prx1 knockdown was determined by RT-PCR and western blot analysis.

SCC15 cells were transfected with shRNA Prx1 plasmid (Santa Cruz Biotechnology, Santa Cruz, CA, United States) using Lipofectamine 2000 (Invitrogen, Life Technologies Corp., Carlsbad, CA, United States). The plasmid was constructed according to standard techniques, and the target sequence for the Prx1 shRNA was: 5′-CGAAGCGCACCAATTGCTCA-3′. The shRNA Plasmid-A (Santa Cruz Biotechnology) was used as a vector control. The efficiency of Prx1 knockdown was determined by reverse transcription polymerase chain reaction (RT-PCR) and western blotting after selection with puromycin.

Recently, our laboratory established and published stable lentiviral mediated DSPP, MMP20, combined DSPP–MMP20 silenced OSC2 cell lines with scrambled (ShC) counterparts as controls [33 (link)]. ShRNA Plasmid A also used as negative control was obtained from Santa Cruz Biotechnology (cat. #sc-108060; Santa Cruz, CA, USA). The stably silenced phenotype of these cell lines was validated (75% silencing) [33 (link)], cultured as a monolayer in DMEM/F12 medium containing 10% FBS (Invitrogen, Carlsbad, CA, USA), supplemented with 1% Penicillin/Streptomycin and 500 ng/mL Hydrocortisone (Sigma Aldrich, St. Louis, MO, USA), and maintained in the presence of 5% CO² humidified air at 37 °C.