Pcmvδr8

To generate the VSV-G-pseudotyped single-round luciferase-expressing HIV-based pseudoviruses HIV_wt, HIV_mut, or HIV_E152A, 293T cells were co-transfected with HIV-1 packaging vector pCMVΔR8.2 (Addgene plasmid #12263), pCMVΔR8.2_E212A/L213A [21 (link)], or pCMVΔR8.2_E152A [21 (link)], a vector for the expression of protein G from the vesicular stomatitis virus (VSV) pCMV-VSVG (Addgene plasmid #8454), and reporter plasmid pUCHR-inLuc-mR [38 (link)] using calcium-phosphate transfection. Forty-eight and seventy-two hours post-transfection supernatants were harvested, pseudoviruses were concentrated using centrifugation at 80,000× g for 1 h 30 min and resuspended in PBS. The level of p24 was assayed using the HIV-1 p24-antigen ELISA Kit (Vector Best, Novosibirsk, Russia).

The lentiviral packaging plasmid pCMV-ΔR8.2 (Addgene plasmid 12263) and pCAG-VSVG were obtained from Addgene (Plasmid #35616). The lentiviral vector pLentiCRISPR, which expresses CAS9 and gRNA, was obtained from Addgene as well. The gRNA that targets mouse miR-155 genomic sequence was subcloned into the lentiCRISPR vector by following the instruction. The gRNA sequence is 5′-TAGTGTTAATCGTAATTGTC-3′.

The plasmid pLV-mCherry (catalog no.36084, Addgene) was cotransfected with the packaging plasmids pCMV Δ R8.2 (catalog no.12263, Addgene) and pMD2.G (catalog no.12259, Addgene) into 293T cells to package lentivirus Lenti-mCherry. The plasmid pCDH-3xFLAG-GFP-puroR (catalog no.167463, Addgene) was inserted into cloned mouse polyomavirus middle T antigen transcripts before cotransfection with pMDLg/pRRE (catalog no.12251, Addgene) and pRSV-Rev (catalog no.12253, Addgene) in 293T cells to package Lenti-MT. The viral supernatant was harvested at 48 and 72 hours after transfection. After centrifuging at approximately 500g for 5 minutes to pellet any packaging cells and filtering through a 0.45 μm PES filter, the virus supernatant was used for the transduction of target cells. DMSO3-1-mCherry cells was selected using fluorescence-based screening, while melanoma-MT cells were selected using puromycin selection. The cells were subsequently screened to confirm the expression of the target protein.

To generate ROR1 shRNA lentiviral particles, HEK293T cells (ATCC) were co-transfected with the packaging plasmids pMD2.G (Addgene plasmid: 12259) and pCMVΔR8.2 (Addgene plasmid: 12263, both provided by Didier Trono, École Polytechnique Fédérale de Lausanne, Laboratory of Virology and Genetics, Lausanne, Switzerland) and either the pGIPZ non-silencing control (ns ctl) shRNA or shROR1 plasmid (Thermo Scientific, Schwerte, Germany) through calcium phosphate precipitation. While the ns ctl sequence is proprietary, the mature ROR1 targeting sequence is 5′-ATTTATAGGATCTGCCATG-3′. Virus-containing supernatants were concentrated using lentiviral enrichment reagent (MobiTech, Göttingen, Germany) and viral titers were calculated based upon the GFP expression of HEK293T transduced with serial dilutions of the shRNA of interest. MDA-MB-231 cells were finally transduced with a multiplicity of infection of 5.0. Cells were selected in medium with 2 μg/mL puromycin (Sigma, Munich, Germany).
For ROR2 overexpression, the plasmids pcDNA 3.1/Zeo(+) (Invitrogen, Paisley, UK) and pcDNA-hsROR2 were introduced into MCF-7 and SK-BR-3 cells using the Nanofectin transfection reagent (PAA, Cölbe, Germany). Stable expression was achieved by selecting for zeomycin (100 µg/ml) resistance.

Stable gene knockdown was performed using shRNAs directed against HSPB1 (the gene name for HSP27) as described previously [4 (link),5 (link)]. All reagents and TRCN numbers for shRNAs are listed in a previous publication [5 (link)]. Lipofectamine 2000 (Life technologies; #11668019, Carlsbad, CA, USA) was used as a liposome carrier to co-transfect of pCMV-ΔR 8.2 (Addgene; #8455, Watertown, MA, USA), pCMV-VSVG (Addgene; #8454, Watertown, MA, USA), and a lentiviral construct containing shRNA for HSPB1 (Sigma-Aldrich; St. Louis, MO, USA) into Lenti-X 293T cells [5 (link)]. Lentivirus-containing media was collected 48 h later and filtered through a 0.45 µm PVDF low protein-binding membrane filter (Celltreat, Pepperell, MA, USA). A2780CIS and PEO4 cells were incubated in the lentivirus-containing medium supplemented with 8 µg/mL polybrene (EMD Millipore; #TR-1003-G, Burlington, MA, USA) for 72 h at 37 °C with 5% CO₂. HSP27-knockdown cells (or scramble control cells) were then selected with 5 µg/mL puromycin (ThermoFisher; #A1113803, Waltham, MA, USA). Knockdown efficiency is shown in Figure S3A–C.

HEK293T cells were co-transfected with the lentiviral overexpressing or silencing plasmid, the envelope plasmid pMD2.G, and the packaging plasmid pCMVΔR8.2 (both Addgene, Watertown, MA, USA) using Fugene HD (Promega, E2311) and reduced medium. After 24 h, the medium was replaced with complete medium and viral particles were harvested. After an additional 24 h, they were used to transfect A549 cells using a final concentration of 0.8 mg/mL Polybrene (Merck, TR-1003-G) twice after 6 h. Puromycin (Gibco, A1113803) was used to select and culture transduced A549 cells. Lentiviral constructs for IRF9 overexpression (pLV IRF9) and knockdown (pLKO.1 shIRF9) as well as respective vector controls (pLV EV; pLKO.1 sh scr), as previously described [18 (link)], were generously gifted from George R. Stark’s lab (Cleveland Clinic Lerner Research Institute, OH, USA).