Nt sirna

MRC-5 cells were seeded in triplicate in 12-well plates at 6 × 10⁴ cells per well. The following day, cells were treated with either 5 μl of PBS (mock), 5 μl of IFN-α2a (PBL Interferon), 5 μM human IFITM1 SMARTpool siRNA (catalog number L-019543-00; Dharmacon), or 5 μM nontargeting pool siRNA (NT siRNA) (catalog number D-001810-10; Dharmacon). Transfections were carried out using the Dharmafect reagent according to the manufacturer’s guidelines.

hUCB-MSCs were grown until 70% confluence. The cells were transfected with MT3-MMP (25 nM), GPR40 (100 nM), or non-targeting (Nt) siRNA (25 or 100 nM) (Dharmacon, Lafayette, CO, USA) using HiPerFect Transfection Reagent (Qiagen, Valencia, CA, USA) for 24 h according to the manufacturer's instructions. The sequences of GPR40 siRNA were 5′-CGCUCAACGUCCUGGCCAU-3′, 5′-GUGACCGGUUACUUGGGAA-3′, and their complement. The sequences of MT3-MMP siRNA were 5′- ACAGGGUGAUGGAUGGAUA-3′, 5′-CAAUGUGGAGGUUUGGUUA-3′, and their complement. The sequences of Nt siRNA were 5′- UAGCGACUAAACACAUCAA-3′ and its complement. The siRNA knockdown of MT3-MMP efficiently sustained for 5 days (Supplementary Figure S7a) and did not show any effect on the cell viability (Supplementary Figure S7b).

Cells were transfected with a validated nontargeting siRNA (NT-siRNA) or with ON-TARGETplus SMART pool against human eEF2K siRNA (L-004950–00–0005) obtained from Dharmacon, Inc. Transfections were performed with Lipofectamine 3000 reagent, according to the manufacturer’s instructions. Cell treatments were performed 72 h after transfections. After the siRNA transfection, the reduction in protein expression was assessed by Western blot analysis.

ON-TARGETplus SMART pool siRNAs for CRM1/XPO1 and a non-targeting control (NT siRNA) were purchased from Dharmacon (Horizon Discovery). In 24-well plate, cells were seeded with 40%–50% confluence, left overnight for adherence, and then transfected with siRNAs (50 nmol/L) using Lipofectamine RNAiMAX (Invitrogen) transfection reagent. After 48 hours of transfection, the cells were infected with different multiplicity of infection (MOI) of vMyx-GFP for 1 hour, washed to remove the unbound virus, and incubated with complete media. At the indicated timepoints, cells were either observed under a fluorescence microscope to monitor and record the expression of fluorescent proteins or harvested and processed for titration of progeny virions.

Cells were grown in 70% confluence and transfected for 24 hr with DRP1, Gα_q, Gα_i, Gα₁₂, RhoA, Rac1, Cdc42 and PKCζ siRNA (25 nM; Dharmacon, Lafayette, CO, USA), or NT siRNA (25 nM; Dharmacon, Lafayette, CO, USA) as a negative control using Turbofect Transfection reagents (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

HT-1080 human fibrosarcoma cells (ECACC, Salisbury, UK) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Lonza, Basel Switzerland), containing 10% FBS (Gibco, ThermoFisher, UK), penicillin/ streptomycin (P/S) (PAA). Rheumatoid arthritis synovial fibroblasts derived from three patients were cultured in DMEM supplemented with 20% FBS and P/S [54] (link). HT-1080 cells were transfected with plasmid constructs using Trans-IT2020 (Mirus Bio, Madison WI, USA) according to the manufacturer's instructions. Gene silencing was performed by transfection of SMARTpool ON-TARGETplus siRNA (Dharmacon, Thermo Fisher, Waltham, US) using INTERFERin (Polyplus-transfection, New York, NY, USA) according to the manufacturer's instructions. Non-targeting siRNA (NT-siRNA) was purchased from Dharmacon (ThermoFisher). Gene silencing effectiveness was tested by Western blotting (WB) or RT-PCR. Cells were subjected to the experiments after 72 h of transfection.

Logarithmically growing cells were seeded in 6‐well plates in complete growth medium and grown to 70% confluence. For transient transfection, commercially available ON‐TARGET‐plus siRNAs for human aurora kinase A (siRNA‐1, J‐003545‐26‐0002 and siRNA‐2, J‐003545‐27‐0002); aurora kinase B (siRNA‐1, J‐003326‐21‐0002 and siRNA‐2, J‐003326‐22‐0002); aurora kinase C (siRNA‐1, J‐019573‐11‐0002 and siRNA‐2, J‐019573‐13‐0002); and non‐target control siRNA (NT‐siRNA, D‐001830‐01‐05) sequences were used and transfected following the manufacturer's protocol (Dharmacon, Lafayette, CO, USA). pCMV‐6 vector (catalog number PS100001) and PDK4 (catalog number CW306843) expression plasmids obtained from Origene (Rockville, MD, USA) were used for overexpression analysis as reported previously [24 (link), 28 (link)].