Mir 34a mimic

The miR-34a mimic, inhibitor and a negative control mimic were purchased from Qiagen. Transfections of cells with mimics (50 nM) were performed using HiPerFect Transfection Reagent (Qiagen, Valencia, CA). Total RNA from samples were isolated after 48 h of transfection for quantification of miRNA and target genes' expression.

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.

The oligonucleotides including miRNA mimics, inhibitors, and their negative control oligos were purchased from GenePharma (Shanghai, China). Cells were transiently transfected with 50 nM miR-497 mimic, miR-34a mimic, miR-497 inhibitor, miR-34a inhibitor, CCNE1 siRNA (Qiagen, Germany), or siRNA negative control (NC siRNA; Qiagen) using Lipofectamine 2000 (Invitrogen).