P3000 enhancer reagent

HEK 293T or eHAP TKO cells were seeded in 24-well plates and transfected with pCAGGS plasmids encoding Tky05 PB1 577E, PA 556R, PB2 and NP (0.05 μg each), alongside the viral reporter plasmid pPolI-firefly luciferase^{66 (link)} and ANP32B-encoding or empty pcDNA plasmids as indicated, using Lipofectamine™ 3000 (ThermoFisher) transfection reagent, with 2 μl P3000™ Enhancer Reagent and 3 μl Lipofectamine™ 3000 Reagent per μg plasmid DNA. Twenty-four hours after transfection cells were lysed in 60 μl Reporter Lysis Buffer for 30 min at room temperature with gentle shaking. Bioluminescence was then measured on a FLUOstar Omega plate reader (BMG Labtech), using the Luciferase Assay System (Promega).

For transfection with DNA plasmids, MDA-MB-231 cells were seeded in 6-well plates (Greiner Bio-One, CELLSTAR, 657-160) and grown to a confluence of 50–70%. Prior to transfection, the culture medium in each well was replaced with 2 ml of fresh culture medium. Cells were then treated with 245 μl Opti-MEM (Gibco, 31985062) containing 1 μg of the DNA plasmid, Lipofectamine 3000 (Invitrogen, L3000-015; 3 μl per well) and P3000 Enhancer Reagent (Invitrogen, #L3000-015; 2 μl per well). Medium was replaced with fresh culture medium after 4–6 h, and experiments performed after 24 h. The Src Y527F plasmid was pBK-CMV containing the chicken Src sequence with the Y527F mutation that renders the resulting Src protein constitutively active. The plasmid was kindly provided by Marie Kveiborg, BRIC, Copenhagen, Denmark.

MDA-MB-231 or MCF10A cells were seeded in 6-well plates (Greiner Bio-One, CELLSTAR, 657-160) and grown to a confluence of 50–70%. Prior to transfection, the culture medium in each well was replaced with 2 ml of fresh culture medium. Cells were then treated with 245 µl Opti-MEM (Gibco, 31985062) containing 2.5 µg of the relevant DNA plasmid (for dual expression, 1.25 µg of each plasmid), 7.5 µl Lipofectamine 3000 (Invitrogen, L3000-015) and 5 µl P3000 Enhancer Reagent (Invitrogen, L3000-015). After 4–6 h, the medium was replaced with fresh culture medium. Experiments were performed 24 h after transfection. The average transfection efficacy was 8.6% for MCT4 and 14.2% for CD147, estimated from immunofluorescence analysis (n=4). Plasmid constructs used were empty pcDNA3.1 (+) (vector control), CD147 in pcDNA3.1 (+) (GenScript, OHu27639) and rat MCT4 (rMCT4) in pcDNA3.1(+). The CD147 plasmid was a kind gift from Marie Kveiborg, BRIC, University of Copenhagen, Denmark. The rMCT4 sequence in a Xenopus vector (pGHJ-rMCT4) was kindly provided by Holger M. Becker, University of Kaiserslautern, Germany. rMCT4 was subcloned from pGHJ to pcDNA3.1 (+) using restriction enzyme double digestion and sequenced before use.

For transient protein expression, Lipofectamine 3000 (Invitrogen, P/N 100022052) and P3000 enhancer reagent (Invitrogen, P/N100022058) were used according to the manufacturer’s instructions. Cells were transfected with plasmids 24 h prior to experiments. Transient siRNA transfections were performed using Lipofectamine RNAiMAX reagent (Invitrogen, P/N 56532) according to the manufacturer’s instructions. SORLA-targeting siRNAs were ON-TARGETplus obtained from Dharmacon—siSORLA #1 (J-004722-08), siSORLA #2 (J-004722-06), siSORLA #3 (J-004722-07), siSORLA #4 (J-004722-05). For rescue experiments, siRNA against the 3′UTR end of SORLA was obtained from Qiagen (siSORLA 3′UTR, SI05039888). HER2-targeting siRNAs were ON-TARGETplus obtained from Dharmacon (siHER2 #2, J-003126-17; siHER2 #4, J-003126-20). For controls, Allstars negative control (Qiagen, Cat. No. 1027281) was used. SiRNA concentrations used ranged between 20 and 40 nM and cells were transfected with siRNAs 48 h prior to experiments.
The corresponding oligonucleotide sequences can be found in Supplementary Table 2.

For transient overexpression, cells were transfected 24 h before experiments using Lipofectamine 3000 (Invitrogen, P/N 100022052) and P3000 enhancer reagent (Invitrogen, P/N100022058) according to the manufacturer’s instructions. For interference assays, cells were transfected 72 h before experiments using Lipofectamine RNAiMAX reagent (Invitrogen, P/N 56532) according to the manufacturer’s instructions. SORL1-targeting siRNAs were obtained from Dharmacon – siSORL1 #3 (J-004722-07, (5’CCGAAGAGCUUGACUACUU3’)), siSORLA #4 (J-004722-05, (5’CCACGUGUCUGCCCAAUUA3’)). Allstars (Qiagen, 1027281) was used as a negative control. All siRNAs were used at a final concentration of 20 nM.

The pooled-sgRNA library (Mouse Brie kinome pooled library) targeting murine kinome was a gift from John Doench and David Root [18 (link)] (RRID: Addgene_75316, Addgene, Cambridge, MA, USA) and modified for the inclusion of pancreatic cancer-related genes by our lab (supplemental data). The resulting library contained 3548 sgRNAs for 924 murine genes. The lentivirus was produced as described previously [19 (link)]. Briefly, three T175 flasks of HEK293TN cells were plated at 70% confluence and incubated overnight. For each flask, 13.8 μg of pooled-sgRNA library, 9.2 μg of pMDLg/pRRE (Cat# 12251, Addgene), 4.6 μg of pRSV-REV (Cat# 12253, Addgene), 4.6 μg of pMD2.G (Cat# 12259, Addgene), 64.4 μL P3000 Enhancer Reagent, and 129 μL of Lipofectamine™ 3000 diluted in OptiMEM (Cat# 31985070, Gibco) were mixed and added to the HEK293TN cells. pMDLg/pRRE (Addgene, RRID: Addgene_12251), pRSV-REV (Addgene, RRID: Addgene_12253), and pMD2.G (Addgene, RRID: Addgene_12259) were gifts from Didier Trono [20 (link)]. The medium was changed 6 h post transfection and the virus was collected by filtering through a 0.45 μm strainer 24 h later.