Plv hu6 ef1α puro

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.