Sirna buffer

siRNAs [SMARTpool ON-TARGETplus Non-targeting Control Pool, SMARTpool ON-TARGETplus Human SLC27A1, SMARTpool ON-TARGETplus Human SLC27A2, SMARTpool ON-TARGETplus Human SLC27A4)] were purchased from Dharmacon and resuspended in siRNA Buffer (5X siRNA Buffer, Dharmacon) to generate 20 μM stocks. For transfection, 5 μL of Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific) was combined with 250 μL Opti-MEM and incubated for 5 minutes. The RNAiMAX complex was then combined with siRNAs diluted in Opti-MEM (2 μL of siRNA with 250 μL of Opti-MEM) in a gentle dropwise fashion. The siRNA-RNAiMAX complex was then incubated for 20 minutes at room temperature and added to wells with 5E5 cells suspended in 1.5 mL RMPI-1640 (containing 10% FBS). 18-24 hours post-transfection, designated wells were infected with VSVΔ51-GFP for 40 minutes. The virus inoculum-containing medium was removed and replaced with a CTL medium or ACM (50% v/v).

C2C12 cells were seeded at a density of 7900 cells/cm² in a 12-well plate (Greiner Bio-One, 665180, Alphen aan den Rijn, The Netherlands) in antibiotic-free growth medium (DMEM, 1% glucose (Gibco, 31885, Waltham, MA, USA), 10% fetal bovine serum (Biowest, S181B, Nuaillé, France)) at 37 °C, 5% CO₂ and allowed to adhere overnight. SiRNA with a final concentration of 25 nM was prepared according to manufacturer’s protocol. Then, 50 nM siControl, 25 nM siAcvr1b + 25 nM siControl, 25 nM siTgfbr1 + 25 nM or 25 nM siAcvr1b + 25 nM siTgfbr1 was added to the medium of the cells. We used 2 µL DharmaFECT1 per well. The cells were treated with siRNA for 24 h in antibiotic-free growth medium. Subsequently, cells were treated with siRNA for 48 h in antibiotic-free differentiation medium (DMEM, 1% glucose (Gibco, 31885, Waltham, MA, USA), 2% horse serum (HyClone, 10407223, Marlborough, MA, USA)). The following reagents for transfection were obtained from Dharmacon (Lafayette, Colorado): ON-TARGET plus Non-targeting Pool (D-001810-10), DharmaFECT1 (T-2001), 5X siRNA Buffer (B-002000-UB-100), mouse ON-TARGET plus Tgfbr1 siRNA (J-040617-05), and mouse ON-TARGET plus Acvr1b siRNA(J-043507-08)

ON-TARGETplus Human NEK2 SMARTpool siRNA (Dharmacon, 4751) was used to deplete NEK2 protein from PEL cells (J-004090–18: GGAUCUGGCUAGUGUAAUU, J-004090–19: GCAGACAGAUCCUGGGCAU, J-004090–20: GGCAAUACUUAGAUGAAGA, and J-004090–21: GCUAGAAUAUUAAACCAUG). The ON-TARGETplus control pool siRNA (Dharmacon, D-001810–10–20) was used as the nontargeting control (NTC). The siRNAs were reconstituted in 5x siRNA buffer (Dharmacon, B-002000-UB-100) in water for a final concentration of 1x. The Amaxa Cell Line Nucleofector Kit (Lonza, VCA-1003) was used for nucleofection of PEL cells following the manufacturer's instructions. Two micrograms of siRNA was used per sample, each containing 1 × 10⁶ PEL cells in log phase. Transfected cells were incubated at 37°C for 48 hours before being used for protein or RNA extraction.

Huh7.5 (10⁶) cells were seeded and reverse transfected in 6 well plates, in DMEM-E2 minus for 24 hr, with 20 nM final concentration of small interfering RNA (siRNA) following the manufacturer protocols (Dharmacon, Inc., Lafayette, CO). siRNA, Dharmafect 4 Transfection reagent, 5X siRNA buffer and siGLO Transfection Indicator were obtained from Dharmacon. siRNA specific to MMP-9 (ON-TARGET plus Human MMP-9 siRNA “SMART pool”, Target sequences: GCAUAAGGACGACGUGAAU, GGACCAAGGAUACAGUUUG, GCGCUCAUGUACCCUAUGU, and GAACCAAUCUCACCGACAG). MMP-9 sequence-Scrambled siRNA was used as a negative control (ON-TARGET plus Non-Targeting siRNA, Target sequence: UGGUUUACAUGUUUUCCUA).

MISSION siRNA was purchased from Millipore Sigma. Duplexes targeting Mcl1 (NM_008562: SASI_Mm01_00048593, SASI_Mm02_00314161, SASI_Mm01_00048594) as well as Bcl2l1 (NM_009743: SASI_Mm02_00316924, SASI_Mm02_00316925, SASI_Mm02_00316926) were ordered and resuspended at a concentration of 25 μM in 5X siRNA buffer (Dharmacon) diluted to 1X RNase-free water (Thermo Scientific). siRNA was reverse transfected into ESCs (6 × 10⁵ cells/mL) at a final concentration of 75 nM using INTERFERin according to the manufacturer’s protocol (Polyplus Transfection). MISSION siRNA Fluorescent Universal Negative Control #1 conjugated to 6-FAM was used as both a transfection control and as a non-targeting siRNA control. After verifying KD at the protein level by Western blot, the best two siRNAs were chosen for further experiments. All shRNA and siRNA TRC/ID numbers, and shRNA sequences (or the target position where siRNA is predicted to bind) are listed in supplemental table S3.