Plasmid construction and lentiviral transduction

Complementary shRNA oligonucleotides directed against ITGAV or ITGA5 (Supplementary table 1) were designed with Dharmacon siDesign-CENTER software, annealed and cloned into the BglII-HinDIII sites of pENTR-THT²⁷. The sequence verified THT-shRNA cassette was recombined into the lentiviral RNAi destination vector pHR-Dest-SFFV-Puro as previously described²⁷. For constitutive ITGAV or ITGA5 overexpression, ITGAV-cDNA isoform 2 (pEF1-human ITGAV-V5-His6⁵³) was cloned into the BamHI/NotI site of pHR-SIN-CSGW-ΔNot⁵⁴ replacing the eGFP cDNA. KpnI-digested and blunt ended ITGA5-cDNA from pEGFP-N3-ITGA5⁵⁵ was cloned into the blunt ended BamHI/NotI site of pHR-SIN-CSGW-ΔNot. For survivin overexpression, survivin-cDNA (survivin variant 1 ({"type":"entrez-nucleotide","attrs":{"text":"NM_001168","term_id":"1519314839","term_text":"NM_001168"}}NM_001168)) was cut from pLIB-survivin-IRES-YFP⁵⁶ (provided by M. Ausserlechner) using EcoRI and NotI and cloned into the BamHI/NotI site of pHR-SIN-CSGW-ΔNot. EcoRI and BamHI sites were blunt ended using a DNA-polymerase-I Klenow-fragment. 1.5 μg of sequenced verified plasmids were then co-transfected with 0.9 μg pSPAX2 packaging and 0.9 μg pMD-G VSV-G pseudotyping plasmids using calcium phosphate transfection⁵⁷. Twenty-four and 48 hours post transfection, sterile filtered supernatants were diluted 1:2 with fresh PM4 medium and supplemented with 1 μg/ml polybrene for infection; 48 hours after infection ASCs were selected for puromycin resistance (1 μg/ml) and analyses were performed five days after transduction. All reagents were obtained from Sigma Aldrich, Germany.