Lipofectamine

The shRNA against OPN was conducted by delivery of shRNA plasmid DNA into cells using the lipofectamine method with a ratio of 5 μg DNA per 30 μL lipofectamine (Qiagen, Hilden, Germany). Cells transfected with plasmid of luciferase gene serve as the vector control. After DNA transfection, cells are grown in regular medium for 48 hours and transfection efficacy is evaluated by luciferase bioactivity.

Approximately 2 × 10⁶ cells/well were seeded in 6-well plates. After 24 h of culturing, cells were transfected with plasmids such as 809 pcDNA3-GFP-PTEN (Plasmid #10759) [24 (link)] or empty vector pCMV-PTEN (Plasmid #28298) by using 8 µL of the transfection reagent, Lipofectamine (Qiagen). After 24 h of incubation at 37°C in 5% CO₂, cells were subsequently subjected to in vitro assays.

A synthetic scrambled miR mimic and a miR‐34a mimic (catalog numbers SI03650318 and MSY0000542, respectively; Qiagen) were transfected into primary mouse lung fibroblasts with HiPerFect (Qiagen) or MLg cells with Lipofectamine^® 2000 or 3000, as per manufacturer's instructions. Locked nucleic acid (LNA) oligonucleotides (purchased from Exiqon) included a scrambled (inert) sequence (5′‐ACGTCTATACGCCCA‐3′); an antimiR directed against miR‐34a (5′‐AGCTAAGACACTGCC‐3′) and miRCURY LNA™ microRNA Target Site Blockers (herein referred to as target site blockers) directed to target the interaction between the two miR‐34a binding sites in the mouse Pdgfra 3′‐UTR and miR‐34a: 5′‐TTGGCAGTATTCTCCA‐3′ (TSB1) and 5′‐AGGCAGTGATACAGCT‐3′ (TSB2) (see Fig 3A). In vitro, synthetic oligonucleotides were transfected into MLg cells with Lipofectamine^® 2000. When combined, synthetic microRNA mimics and LNA target site blockers were applied together as a cocktail, at a final cumulative concentration of 160 nM (Fig 4B). In vivo, both target site blockers (applied as a cocktail of a 1:1 mixture of TSB1 and TSB2) and a scrambled or miR‐34a‐specific antimiR were all applied by intraperitoneal injection at a dose of 10 mg/kg at P1 and P3, in ddH₂O.