Ppackh1 packaging plasmid mix

shRNA vectors were constructed using a pSIH-H1-copGFP shRNA vector (System Biosciences) by inserting synthetic oligonucleotides. The following four unique shRNA constructs were then prepared using synthetic oligonucleotides: (1) 5′-AGAGACAGAACCTCCTTCCAGCTACTCAA-3′; (2) 5′-AGGAGTGTCTGGTGACTGAAGTACAGGTC-3′; (3) 5′-AGGCGTCAACAGTACGAGTGTCTTCACTA-3′; and (4) 5′-GTGCGGAGAATCTGACCTGCTGGATTCAT-3′. Pseudoviral particles were generated by cotransfecting a pPACKH1 Packaging Plasmid Mix (System Biosciences) and a mixture of the four pSIH vectors into HEK293TN cells in a 6-well plate using Lipofectamine and Plus Reagent (Life Technologies, Carlsbad, CA, USA), as described previously [16 (link),17 (link)]. For controls, a pSIH-H1-copGFP vector inserted with a scrambled sequence (5′-TAGCGACTAAACACATCAA-3′) that had no homology with any human mRNA (shCont) and no vector [Lenti (−)] was used. Supernatants were collected after 72 h from the cultured medium and used for infection.

NKL reporter cells (NKL-κB-GFP) that express GFP under the control of NF-κB transcription response elements were generated by transducing NKL cells with lentiviral κB-GFP construct (System Biosciences). Lentiviral particles were produced by simultaneous transfection of 293TN packaging cell line (System Biosciences) with pGreenFire-NF-κB-EF1-Puro vector and pPACKH1 packaging plasmid mix (System Biosciences). NKL cells were transduced with lentivirus supernatant that encodes κB-GFP in the presence of 10 μg ml⁻¹ polybrene and 200 U ml⁻¹ rIL-2 and then selected with 1 μg ml⁻¹ puromycin (2 days after transduction). Thereafter, NKL transductants showing GFP upregulation upon TNF-α treatment were further selected by FACS-sorting and grown as pure cultures. To assess NF-κB activation, NKL-κB-GFP cells were stimulated with TNF-α or plate-immobilized mAbs specific for NK receptors, and GFP expression in the reporter NKL cells was analysed by flow cytometry.

For stable transduction of iBMSCs or ASCs with miR-181a overexpression, miR-181a antagomiR, PER3 shRNA and PPARG shRNA lentiviral vectors HEK 293 T cells were cotransfected with the lentiviral expression vector along with pPACKH1 Packaging Plasmid Mix (System Biosciences) according to the manufacturer provided protocol. Both Scp-1 and PASC-1 cells were then transduced with the lentivirus according to the manufacturer provided protocol. For selection of the iBMSCs or ASCs transduced with miR-181a overexpression (both p000 and p181a vectors) and miR-181a antagomiR (both cmiR and antimiR vectors) RFP FACS was used. For selection of the iBMSCs transduced with PER3 shRNA (both shscram and shPER3 #1 & #2 vectors) and PPARG shRNA (both shscram and shPPARG vectors) GFP FACS was used. For the experiments involving PPARG knockdown the Scp-1 p000 cells were transduced with shscram vector (Scp-1 p000-shscram) and the Scp-1 p181a cells were either transduced with either shscram (Scp-1 p181a-shscram) or shPPARG vectors (Scp-1 p181a-shPPARG). These cells were sorted for the dual RFP/GFP+ cells. All cell lines selected with FACS were analyzed every 3–5 passages to make sure that the RFP and/or GFP+ population was >95% with re-sorting done as needed.

For stable transduction of FTSECs with miR-181a overexpression, miR-181a antagomiR, and RB1 shRNA lentiviral vectors HEK 293T cells were cotransfected with the lentiviral expression vector along with pPACKH1 Packaging Plasmid Mix (System Biosciences) according to the manufacturer provided protocol. FTSECs (passage 5 or earlier) were then transduced with the lentivirus according to the manufacturer provided protocol. For selection of the FTSECs transduced with miR-181a overexpression (both p000 and p181a vectors) RFP flow cytometry was used. For cells transduced with miR-181a antagomiR (both cmiR and antimiR vectors) hygromycin selection was used. Post transduction, cells were selected with 500 μg/mL hygromycin in DMEMF12 for 48 h. After recovery, selection was maintained by treatment with 500 μg/mL hygromycin in DMEMF12 for 48 h every three passages. miR-181a antagomiR transduction efficiency was assessed by qRT-PCR. For selection of the FTSECs transduced with RB1 shRNA GFP flow cytometry was used. All cell lines selected with flow cytometry were analyzed every 3–5 passages to make sure that the RFP or GFP + population was >95% with resorting done as needed.