Mission packing mix

The following double strand oligos (only sense strands indicated) were cloned into the pLKO.1 plasmid at AgeI and EcoRI sites and sequence verified before use. shPRKCA: 5′-CCGGCGAGGTGAAGGACCACAAATTCTCGAGAATTTGTGGTCCTTCACCTCGTTTTTTG-3′, shPKN2: 5′-CCGGGTCCACGTCAAAGTATGATATCTCGAGATATCATACTTTGACGTGGACTTTTTG-3′. A MISSION non-target shRNA control vector served as the scrambled control (Sigma-Aldrich, SH002). Lentivirus were produced by cotransfection of 293T cells with above constructs and the MISSION packing mix (Sigma-Aldrich) using FuGENE HD transfection reagent (Promega). Cells were incubated with lentivirus for 24 hours before selection with puromycin (Gibco). Gene knockdown was confirmed by western blotting analysis.

The shRNA sequences that specifically target HSP90, PKM2, and HMGCR were cloned into the lentiviral vector pLKO.1 plasmid (Addgene, 10,878) and verified by sequencing.
shHSP90-1:5′-CCGGGTTATCCTACACCTGAAAGAACTCGAGTTCTTTCAGGTGTAGGATAACTTTTTG-3′,
shHSP90–2:5′-CCGGTACTTGGAGGAACGAAGAATACTCGAGTATTCTTCGTTCCTCCAAGTATTTTTG-3′,
shPKM2–1:5′-CCGGGTTCGGAGGTTTGATGAAATCCTCGAGGATTTCATCAAACCTCCGAACTTTTTTG-3′,
shPKM2-2:5′-CCGGGCCCGAGGCTTCTTCAAGAAGCTCGAGCTTCTTGAAGAAGCCTCGGGCTTTTTTG-3′,
shHMGCR-1:5′-CCGGCTATGATTGAGGTCAACATTACTCGAGTAATGTTGACCTCAATCATAGTTTTTG-3′,
shHMGCR-2:5′-CCGGGGTTCTAAAGGACTAACATAACTCGAGTTATGTTAGTCCTTTAGAACCTTTTTG-3′.
The empty plasmid pLenti-CMV and the plasmid containing PKM2 (PKM) cDNA (accession number NM_182470), HSP90 cDNA (accession number NM_007355) were obtained from Public Protein/Plasmid Library. Lentiviruses were established by transfecting the lentiviral vectors and packaging with the MISSION packing mix (Sigma) into human embryonic kidney cell HEK293T cells following the manufacturer’s protocol. ACHN and 786-O cells were stably transduced with lentivirus for cDNA expression or mRNA knockdown and selected with puromycin for 7 days before further analysis.