Morpholino oligo

Plasmid transfections were performed on HEK293 cells using Lipofectamine 2000 as per the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). RIOK3 and GFP control FlexiTube siRNAs (Qiagen Germantown, Germantown, MD, USA) nos. Hs_RIOK3_4 and Hs_RIOK3_8) and HiPerFect Transfection Reagent were used for siRNA knockdown experiments in HEK293 cells according to the manufacturer’s instructions. Since HEK293 cells lack TLR3 expression for stimulation of innate immunity via poly (I:C), which stimulates both the MDA5 and the RIG-I pathways, and 3p-hpRNA, which stimulates only the RIG-I pathway, we transfected 1 μg/mL poly (I:C) (Tocris/BioTechne, Minneapolis, MN, USA) or 3p-hpRNA (Invivogen, San Diego, USA) into HEK293 cells using Lipofectamine 2000 according to the manufacturer’s instructions (Thermo Fisher Scientific). Morpholino oligos (Gene-Tools, Philomath, IN, USA) were transfected using Endo-Porter according to the manufacturer’s instructions (Gene-Tools).

The two siRNAs specifically targeting sVEGFR1-i13 were: sVEGFR1-i13(1) sense, 5′-UAACAGUUGUCUCAUAUC-3′; anti-sense, 5′-UGAUAUGAGACAACUGUUA-3′ and sVEGFR1-i13(2) sense, 5′-UCUCGGAUCUCCAAAUUU-3′; anti-sense, 5′-UAAAUUUGGAGAUCCGAGA-3′. Transfection of siRNA oligonucleotide duplexes was performed using JetPrime reagent (Ozyme). β1 integrin silencing was performed using SMARTpool siGENOME ITGB1 siRNA (M-004506-00-0020, Thermoscientific Dharmacon). Transfection of smartpools was performed using Oligofectamine RNAiMax (Invitrogen). The scrambled siRNA oligonucleotides used as a control for all RNA interference experiments were as follows: forward 5′-UCGGCUCUUACGCAUUCAATT-3′ and reverse 5′-CAAGAAAGGCCAGUCCAAGTT-3′. Cells were analysed 72 h post-transfection. Morpholino oligos were obtained from Gene Tools and resuspended in sterile water as a 200 µM stock solution. Sequences of control and sVEGFR1-i13 (MoFL2) Morpholino oligos were as follows: 5′- GATCCATCCCTCTGTTAAGACCTAG-3′ and 5′- TTTTTGTTGCAGTGCTCACCTCTGA-3′, respectively. For delivering, MGH7 or H2170, cells were seeded at 0.5 × 10⁶ cells/well in six-well plates for 24 h, treated with 10 µM of control or MoFL2 morpholino, then scraped using a rubber blade scraper and transfered to another culture plate. Cells were analysed at 24, 48 and 72 h after transfection.

Plasmid transfections were performed on HEK293 cells using Lipofectamine 2000 as per the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, USA). Morpholino oligos were synthesized by Gene Tools and transfected using Endo-Porter according to the manufacturer’s instructions (Gene Tools).

Morpholino oligos (Gene Tools, Philomath, OR, USA) as antisense oligodeoxynucleotides were used for loss-of-function studies. The base composition of the antisense oligodeoxynucleotide was the 25-mer morpholino, 5′CTG GCA GGT TCA CTG GTC ACA ATT A 3′ (Xarhgef3.2 MO), which targeted the 5′ UTR region of the Xarhgef3.2.L mRNA. The specificity of MO was examined by means of Western blot detection of the Flagged protein after injection of the 5′UTR-Xarhgef3.2-3Flag construct. The MO was against the 5′UTR region of 5′UTR-Xarhgef3.2 (including the endogenous target). The addback 5′-3Flag version was tagged in its 5′ region and its message would not bind the MO and thus would be expressed. The MO was confirmed to block target 5′UTR-Xarhgef3.2-3Flag without affecting amounts of endogenous Xarhgef3.2 RNA.