Pspax2 12260

Envelop plasmid and packaging plasmid (pMD2.G, 12259; psPAX2, 12260) were provided from Addgene (MA, USA). Multiple shRNA sequences targeting VHL, FAM163A and negative control in the PLKO.1 lentiviral vector was designed and constructed by IGEbio (Guangzhou, China). The sequences of shRNA targeting VHL and FAM163A are listed. Overexpression sequence targeting VHL in PLVX-Flag lentiviral vector and negative control (PLVX-NC) were purchased from IGEbio. The transfections were performed according to the protocol provided by the manufacturer (PEI, Polysciences, PA, USA). The indicated cells infected with the lentiviral vectors were cultured with 10 μg/ml puromycin (Invitrogen, Thermo Fisher, Waltham, MA, US) for a week to establish the stably transfected NB cells. The targeting sequence for VHL was 5′-CCGGGCTCAACTTCGACGGCGAGCCCTCGAGGGCTCGCCGTCGAAGTTGAGCTTTTTGAATT-3′; the targeting sequence for FAM163A #A was 5′-CCGGGCACGACCTTCCCACGCATCCCTCGAGGGATGCGTGGGAAGGTCGTGCTTTTTGAATT-3′; the targeting sequencing for FAM163A #B was 5′-CCGGGAGGCCTTCACCAATCCAAGGCTCGAGCCTTGGATTGGTGAAGGCCTCTTTTTGAATT-3′.

70% confluent HEK293T cells were transfected with plasmids expressing either a control shRNA or a shRNA targeting Pla2g7 (Sigma‐Aldrich, Mission shRNA DNA clone pLKO.1 #SHCLND‐NM_013737) together with lentivirus packaging plasmids (Addgene psPax2 #12260, pMD2.G #12259 provided by Didier Trono) previously combined to an OptiMEM (Life Technologies #31985062)/lipofectamine (Thermo Scientific #11668019) mixture. The day after, cells were switched to normal media supplemented with 2% fatty acid free bovine serum albumin (Sigma‐Aldrich #A7030). Media containing lentivirus were collected 24 h later, filtered with 0.45 μm filters, and stored at −80°C until use.
50% confluent C26 cells were incubated for 24 h with lentivirus carrying either the control shRNA or the shRNA targeting Pla2g7. Transduced cells were selected using 3.5 μM puromycin (Thermo Fisher #A1113803) for 3 days. Once all non‐transduced cells had died, puromycin‐resistant cells were trypsinized and expanded in normal media. Growth rate of control C26 cells (C26‐shCTR) and C26 cells lacking PLA2G7 (C26‐shPla2g7) was estimated by comparing the number of cells during trypsination with the number of cells initially seeded.

Lentiviral ShIft88 #1 (TRCN0000178064), ShIft88 #2 (TRCN0000182620), ShKif3a #1 (TRCN0000339512), ShKif3a #2 (TRCN0000339514), and control shRNA plasmids were purchased from Sigma. psPAX2 (12260) and pMD2.G (12259) developed by Dr. Didier Trono were obtained from Addgene. Luciferase reporter plasmids were kindly provided by Dr. Takashi Ito, Osaka University (wild type and TonE-mutant TauT-Luc)^{15 (link)}, Dr. H Moo Kwon, University of Maryland (wild type and TonE-mutant HSP70-Luc)^{20 (link)}, and Dr. Joan D. Ferraris, NIH (GAL4dbd-548-1531, GAL4dbd, and AR-Luc)^{47 (link),48 (link)}. Backbone GAL4dbd contains no TAD but only expresses the GAL4dbd. pFR-Luc reporter (Stratagene) contains the yeast GAL4-binding site, upstream of a minimal promoter driving the firefly luciferase gene.

LKB1 knockdown in the KIF7-CC over-expression clones from PC3, C4-2B and 22Rv1 cells was performed by infection with lentivirus that expressed human LKB1-specific short hairpin RNAs (shH and shM) (61231 & 61242) or non-target control shRNA (shNTC) (1864), which were packaged with psPAX2 (12260) and pMD2.G (12259) from Addgene (Cambrige, MA; http://www.addgene.org) using lipofectamine 2000. Infected cells were selected by puromycin (2 μg/ml, Sigma).

shRNAs targeting Eftud2 (shRNA1 – Sigma Aldrich, #TRCN0000294567, shRNA2 – Sigma Aldrich, #TRCN0000306704), Sf3b4 (Sigma Aldrich, #TRCN0000379192), Txnl4a (Sigma Aldrich, #TRCN0000123687), Prpf8 (Sigma Aldrich, #TRCN0000109106) or non-targeting (nt-) shRNA (Addgene, 30323) were transfected with lentiviral envelope and packaging plasmids (Addgene, psPAX2 12260, pMD2.G 12259) into HEK293FT cells using Lipofectamine 2000 (Thermo Fisher Scientific, #11668027). Virus-containing medium from HEK293FT cells was collected and used to infect mESCs on two subsequent days. Each day mESCs were incubated in a mix of virus-containing and standard mESC medium (1:1) for 4 hours before changing the medium to standard mESC medium alone. Cells were then washed with cold PBS and collected for analyses.