Plpcx

The human B⁰AT1 cDNA sequence was inserted in the multiple cloning site of pIRES2-EGFP (catalog no. 6029-1, Life Technologies, Zug, Switzerland), upstream of the internal ribosomal entry site and the EGFP reporter gene. Human TMEM27 cDNA sequence was then subcloned in the above mentioned vector in place of EGFP sequence. The resulting bicistronic construct containing B⁰AT1 and TMEM27 upstream and downstream of IRES, respectively, was then excised from the plasmid and inserted in the retroviral vector pLPCX (Clontech, Saint-Germain-en-Laye, France). EGFP was introduced in pLPCX as previously described [28] . Production of supernatants containing the pseudoviruses and subsequent transduction of MDCK target cells was performed as previously described [9] . The first subcultivation after transduction was defined as passage 1. Stable MDCK cell lines were selected and maintained in standard growth DMEM containing 2 µg/mL puromycin (Sigma-Aldrich). Human TMEM27 cDNA sequence was subcloned as a PCR fragment flanked by SmaI and XhoI restriction sites into the Eco47III and XhoI sites of pLenti6-EGFP (Life Technologies), thus yielding to pLenti6-TMEM27 vector. Lentiviral production was performed according to the protocol described elsewhere [29] . Infected MDCK cells were selected with 6 µg/mL blasticidin S (Life Technologies).

Plasmids encoding green fluorescent protein (GFP) reporter HIV-1, SIVmac251, EIAV, FIV, and MLV viral vectors have been described previously [25 (link)]. HIV-1 CA, IN, NC, and RT mutations were generated with site-directed mutagenesis of the HIV-1_NL43-based pHP-dI-N/A packaging plasmid [59 (link)] (AIDS Research and Reference Reagent Program [ARRRP]). WT and mutant plasmids were cotransfected with the pHI-Luc transfer vector [25 (link)]. Plasmid pLPCX was obtained from Clontech.
DNA sequence encoding human MxB (accession number NM_002463.1) was engineered with a C-terminal HA-tag into the pLPCX-based MLV transduction vector. Mutations were generated using site-directed mutagenesis. The ΔLoop4 mutation was generated by replacing nucleotides corresponding to MxB residues 584 to 615 with those encoding the GAGAG peptide. Stalk Loop1 (residues 440 to 448) and Loop2 (residues 488–497) were replaced with the sequence GSGGSG. The Δ25 mutation was generated by deleting sequences encoding Met1 to Glu25. The NLS Δ25 mutant was created by inserting the SV40 large T antigen NLS sequence (PKKKRKV) subsequent to the initial Met but preceding Asn27. The NES mutant was generated by adding the NES-derived sequence from cAMP-dependent protein kinase inhibitor alpha (LALKLAGLDI) subsequent to the initial Met but preceding Ser2.

For SK-MEL-37 and WM1366 Gal-3 shRNA knockdown, cells were transduced with a lentivirus containing a LGALS3 shRNA sequence (OpenBiosystems, pLKO-shGAL-3 Cat. TRC0000029305) and then subjected to cell selection by puromycin (1 μg/mL) (Gibco). The shRNA scramble sequence was used as a transduction control. Gal-3 knockdown was evaluated by western blot in different passages. The plasmidial vector pLPCX (Clontech) tandem-tagged LC3 construct mCherry-eGFP-LC3 was used as previously described in Grasso et al. (2016) [72 (link)]. For gal-3 siRNA knockdown assays, WM1366 cells were transfected using a mix containing Lipofectamine ^® RNAiMAX (Invitrogen), opti-MEM medium (Gibco, Invitrogen) and 20 μM of gal-3 siRNA (5′UUU CCU GAU UAG UGC UCC ACC CGC CGC-3′; 5′-GGG GGG UGG AGC ACU AAU CAG GAA A-3′) or 20 μM of scramble siRNA (IDT, Coralville, IA). NGM cells were electroporated (1200 V, 10 ms, 3 p) using Neon transfection system (Invitrogen) and 20 μM of gal-3 siRNA or 20 μM of scramble siRNA (IDT, Coralville, IA).

The Flag-Omomyc insert was excised from pCbsFlag-Omomyc plasmid with BamHI and HindIII restriction enzymes and inserted into pLNCX2 (Clontech, Saint-Germain-en-Laye France) using BglII-HindIII restriction sites. Flag-Myc was excised from the pCbsFlag-Myc vector and inserted in pLPCX (Clontech) in BamHI-ClaI restriction sites. The PRTM5 coding sequence was obtained from a human cDNA library by PCR amplification, and inserted in pCS2 vector50 (link). pRFP-C-RS plasmid expressing a COPR5 specific shRNA sequence (shCOPR5) and the relative scrambled control were purchased by OriGene (Origene, Rockville, MD, U.S.A). Transfections were performed by using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Mouse Fermt1 cDNA was cloned into pRetroQ-EGFP-C1 (Clontech) using the restriction sites BglII and XbaI for transient transfection. Mouse Fermt2 cDNA was cloned into pRetroQ-EGFP-C1 (Clontech) using the restriction sites XhoI and BamHI for retrovirus-mediated expression. The phospho-mimetic and phospho-inhibitory mutants were generated using a QuikChange Site-Directed Mutagenesis kit according to the manufacturer’s protocol (Agilent). For retrovirus-mediated expression of YPet–talin and paxillin–GFP, the mouse YPet–talin or paxillin–GFP cassette was cloned into pLPCX (Clontech) using the restriction sites XhoI and NotI. The plasmid pLenti.PGK.LifeAct-Ruby.W was a gift from R. Lansford (Addgene, plasmid 51009; http://n2t.net/addgene:51009; RRID: Addgene51009). The plasmid LV-CFP was a gift from E. Fuchs (Addgene, plasmid 25998; http://n2t.net/addgene:25998; RRID: Addgene25998). On-target Plus smart pool siRNA and non-targeting siRNA control were purchased from Dharmacon. The detailed sequences of siRNA are listed in Supplementary Table 8.