Pcdna3.1 flag

GFP-, DsRed-, Flag-, and GST-tagged
PAK4 were constructed by polymerase chain reaction (PCR) and sub-cloned into pEGFP-C1, DsRed, pcDNA3.1-Flag (Invitrogen), and pGEX-5X-1/2 (GE Healthcare, Bethesda, USA) vectors, respectively. The full length CORO1C was amplified by PCR using cDNA derived from SGC-7901 cells as a template and cloned into pEGFP-C1, DsRed, pcDNA3.1-Flag and pGEX-4T-2 vectors. CORO1C deletion constructs were obtained by PCR and cloned into pGEX-4T-2. The site-directed mutagenesis (PAK4S99A or PAK4S99D) was generated from PAK4 WT using the QuikChange kit (Stratagene, La Jolla, USA) according to the manufacturer′s instructions. eYFP-RCC2 was a kind gift from Prof. Sheng Xiao of Harvard Medical School (Boston, USA).

Hoechst 33342 and Pitstop 2 were purchased from Sigma-Aldrich. Pitstop 2 were dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer’s instructions. The lipophilic dyes DiO and DiD were purchased from Biotium. The fluorescent dyes Alexa Fluor 647 and Alexa Fluor 488 phalloidin were purchased from Invitrogen. anti-β-tubulin was purchased from Abcam (USA). peroxidase-conjugated affinipure goat anti-rabbit IgG were purchased from proteintech (USA).
Using the primers listed in Table 1, the full-length CLCs were constructed in vectors including pcDNA3.1-flag, pEGFP-N3, and pmDsRed-C1 (Invitrogen). Site-directed mutants, including EaCLCa-W119R and EaCLCb-W122R, were all subcloned into the pEGFP-N3, pmDsRed-C1 and pcDNA3.1-flag vectors using specific primers (Table 1) and the Fast Mutagenesis Kit V2 (Vazyme). Tryptophan (W)119 of CLCa and W122 of CLCb were both replaced with arginine(R). In addition, the pEGFP-Rab5 vector was maintained in our laboratory. The constructed plasmids were confirmed by sequencing.

To design specific small interfering RNA (siRNA) targeting GPI and gp78 (named siGPI and sigp78), several sequences from the human GPI gene were selected using BLOCK-iT™ RNAi Designer (Life Technologies, Carlsbad, CA, USA). The target sequences for GPI siRNA (siGPI: 5′-CCATACGGAAGGGTCTGCATCACAATT-3′), gp78 siRNA (sigp78: 5′-GTCG GCACAAGAACTATCTTT-3′) and negative control siRNA (si-NC: 5′-TTCTCCGAA-CGTGTCACGT-3′) were synthesized by Genepharma Inc (Shanghai, China). SiRNA transfection was performed using Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA). According to the sequence of GPI cDNA (NM_001184722.1), we designed the primers 5′-CGGAATTCCA TGGTAGCTCTCTGCAGCCT-3′ (forward) and 5′-CCCTCGAGGTTATTGGACTCTGG CCTCG C-3′ (reverse), and obtained the full length of GPI cDNA by PCR. GPI cDNA was inserted into pcDNA3.1-Flag (Invitrogen) vector and plasmid DNA transfection was performed using Lipofectamine -LTX (Invitrogen). PCI-Neo-gp78/JM20 (plasmid 13303) was purchased from Addgen (Teddington, UK).

pcDNA-EGFP-PAK6 wild-type (WT)/K436A (KA, kinase-dead) were gifts from Dr. G. M. Bockoh (The Scripps Research Institute). Flag/GST-tagged PAK6 and PAK1,4,5 were constructed by polymerase chain reaction (PCR) and subcloned into pcDNA3.1-Flag (Invitrogen) and pGEX-5X-1/2 (GE Healthcare) vectors, respectively. Flag-tagged SIRT4 was purchase from GeneChem (Shanghai, China). HisA-ANT2 WT was constructed by PCR and subcloned into pcDNA3.1/Myc-HisA (Invitrogen) vectors, respectively. The site-directed mutagenesis (ANT2 T107A mutant, ANT2 K105R mutant and ANT2 T107A/K105R mutant) was generated from ANT2 WT using the QuikChange kit (Stratagene), according to the manufacturer's instructions. The myc-ubiquitin constructs were previously generated in our laboratory.

MARVELD1 ORF region was cloned into the vector pcDNA3.1/flag (Invitrogen, Carlsbad). The cells were transfected with the MARVELD1 construct using Lipofectamine 2000 (11668-019, Invitrogen) according to the manufacturer's protocol. The negative control and MARVELD1 over-expressing lentiviruses/flags were purchased from Invitrogen. Next, lentiviruses infected 5 × 10⁵ cells in a 6-well plate with 4-6 µg/mL polybrene (107689, Sigma). After 24 h, the infected cells were subjected to selection with 4 µg/ml puromycin (540411, Calbiochem) and cultured for 3 days. Stable overexpression cell lines were identified using qRT-PCR and western blot assays.

To determine EcRab5c function in vitro, the full-length EcRab5c was cloned into pEGFP-C1 and pcDNA3.1-Flag (Invitrogen, United States) plasmids, using PCR primers (Supplementary Table 1), and verified by sequencing. The EcRab5c glutamine codon (Q) at position 80, and serine codon (S) at position 35 were mutated to leucine (L) and asparagine (N), respectively, using the Fast Mutagenesis Kit V2 (Vazyme, China) to create the mutant plasmids, pEGFP-EcRab5c-Q80L (CA EcRab5c) and pEGFP-EcRab5c-S35N (DN EcRab5c). The pEGFP-EcRab7 and pEGFP-LC3 plasmids were maintained in our laboratory.

cDNAs of HBx, FoxP3 and E2F1 were amplified and sub-cloned into pcDNA3.1-Flag (Invitrogen, Carlsbad, CA, USA). 3′-UTR fragment of wild-type (WT) CDH2 and mutant (MUT) CDH2 were cloned into the dual-luciferase expression vector pmirGLO (Invitrogen, Carlsbad, CA, USA). The promoter region of WT and MUT of miR-187-5p (WT-miR-187-5p-Luc and MUT-miR-187-5p-Luc), WT and MUT of FoxP3 (WT-FoxP3-Luc and MUT-FoxP3-Luc) were cloned into the luciferase vector pGL3-Promoter (Addgene, Watertown, MA, USA). All cloning primers have been shown in Supplementary Table 1.