Plv ef1 mcs ires bsd vector

The human or mouse fused EBI3-P35 genes were amplified by PCR using commercial IL-35-overexpressing plasmids (InvivoGen) as the templates. Then, the fused IL-35 genes were cloned into pLV-EF1-MCS-IRES-Bsd vectors (Biosettia). Lentiviruses were produced in 293T cells for the stable transfection of the cell lines, per the manufacturer's instructions, and an empty vector was transfected into cells to be used as controls. A total of 1 × 10⁵ tumour cells in 2 ml medium with 8 μg ml⁻¹ polybrene were infected with 1 ml lentivirus supernatant. 48 h later, blasticidin (InvivoGen) was added for selection.
For the cell lines with stable knockdown, shRNA sequences were designed with Biosettia's shRNA designer (http://biosettia.com/support/shrna-designer). Three recommended sequences for each of the ICAM1, P35 and EBI3 genes were synthesized and cloned into the pLV-hU6-EF1α-puro or pLV-mU6-EF1α-puro vectors (Biosettia). Then, the lentiviruses were produced in 293T cells. Scrambled sequences were transfected into the cells to be used as controls. Tumour cells were simultaneously infected with sh-P35 and sh-EBI3 viruses to knock down the IL-35 expression. Of the three stable cell lines, the most efficient one was used for the relevant assays.

The complete coding sequence of the human DCBLD2 gene (NM_080927.4) was cloned into pLV-EF1-MCS-IRES-Bsd vectors (Biosettia, San Diego, CA, USA). The lentiviruses were produced in 293T cells according to the manufacturer’s instructions. Then, the cells were infected with lentivirus for 24 h and cultured for 48 h, followed by selection using 2 μg/mL blasticidin. For the cell lines with stable knockdown, shRNA sequences were designed with BLOCK-iT™ RNAi Designer (https://rnaidesigner.thermofisher.com/rnaiexpress/, accessed on 27 March 2018). Five recommended sequences for DCBLD2 were synthesized and cloned into pLV-H1-EF1α-puro vectors (Biosettia, San Diego, CA, USA). The cells infected with RNAi lentiviruses were collected for western blot analysis and RT-PCR. Of the five stable cell lines created, two cell strains with the highest RNAi efficacy were used for subsequent assays. shDCBLD2-1#, 5′-GCATCAAATTTGGTGACTTTG-3′; and shDCBLD2-2#, 5′-GCAAGAGAACAGTTGGAAACC-3′.