Tianpure midi plasmid kit

Cis-platinum (DDP) was obtained from the first affiliated hospital of Xiamen University. MDR1 (ABCB1, Cat# sc-55510) and β-actin (Cat# sc-47778) antibodies were purchased from Santa Cruz Biotechnology. Pygo2 (Cat# 11555-1-AP) antibody was obtained from Proteintech and TIAN pure Midi Plasmid Kit (Cat# DP107) from TIANGEN. TurboFect Transfection Reagent (Cat# R0531) was from Thermo Scientific. E.coli pLL3.7-hPygo2-KD (knock-down) and pGL3.3-control were a gift from Xiamen University School of Life Sciences.

The BZLF-1 gene fragment (GenBank: MK_540470) of EBV was synthesized using primers (Forward: 5′-CCTGGTCATCCTTTGCCA-3′; Reverse: 5′-TGCTTCGTTATAGCCGTAGT-3′) and inserted into the pMD18-T vector (TaKaRa D1010A, Takara Bio, Dalian, China). Then, the gene sequence accuracy was verified by sequencing (Supplementary Material). The recombinant plasmids were extracted using a TIAN Pure Midi Plasmid Kit from TIANGEN BIOTECH (Beijing, China) Co., Ltd., and the plasmid concentration was measured using a NanoDrop microvolume spectrophotometer from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The DNA copy number of the recombinant EBV plasmid was calculated using the following equation:
Then, the DNA was serially diluted 10-fold from 1.0 × 10¹⁰ to 1.0 × 10⁰ copies/µL.

The heterologous protein expression vector pSVrPA/CaMVbar constructed in our laboratory was used [19 (link)]. The vector was maintained and amplified in E. coli Top10 strain. Plasmid DNA was isolated from E. coli with a TIANpure Midi Plasmid Kit (Tiangen, Beijing, China).

We isolated total RNA from 100 denuded GV-stage oocytes using an Arcturus PicoPure RNA Isolation Kit (Applied Biosystems), and cDNA was generated using a QuantiTect Purification Kit (Qiagen, Düsseldorf, Germany). For Rac1 plasmid construction, PCR products were purified and cloned into the linearized pCS2⁺ vector with six Myc tags using the Uniclone one-step seamless cloning kit (Genesand Biotech Co.,Ltd, Cat# SC612). The plasmid was propagated using Trans1-T1 (Transgen Biotech, Beijing, China) as the host strain and purified with a TIANpure Midi Plasmid Kit (DP107, TIANGEN Biotech, Beijing), followed by sequencing to confirm the open reading frame. For the synthesis of cRNA, the Rac1 plasmids were linearized with XbaI. Capped cRNAs were achieved using in vitro transcription with SP6 mMESSAGE mMACHINE (Ambion, CA, USA) according to the manufacturer’s instructions. Synthesized RNA was aliquoted and stored at − 80 °C (the related primer sequences can be found in Supporting Information Table S1).

PCR primers were designed by primerselect 7.0 (DNASTAR Inc, Madison, WI) and synthesized (Sangon Biotech, Shanghai, China). rs72755295 and rs4149909 surrounding regions (~1.5 kb) were amplified with Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA) and primers shown in Table S1. Thermocycling conditions for routine PCR was as follows: 98 ℃ for 30 s; 35 cycles of 98 ℃ for 10 s, 68 ℃ for 30 s, 72 ℃ for 45 s, and finally 72 ℃ for 2 min. The PCR product and pGL3-promoter vector (Promega, Madison, WI) were digested by MluI and XhoI (NEB), purified by GeneJET Gel Extraction Kit (Thermo Fisher Scientific) and ligated by T4 DNA ligase (NEB) according to the manufacturer’s manual. The recombinant plasmids were transformed into E.coli DH5α competent cells (Takara, Dalian, China), cultured, and then extracted by TIANpure Midi Plasmid Kit (Tiangen Biotech, Beijing, China). After sequencing, the plasmids with corresponding alleles were generated by Q5 Site-Directed Mutagenesis Kit (NEB) and primers in Table S1. Before transfection, all plasmids were sequenced to rule out artificial mutations and verify the haplotype orientation.

Hep3B, HepG2, and BxPC-3 cells were purchased from Tongpai Biotechnology Co., Ltd. (Shanghai, China). Huh7, MCF-7, MDA-MB-231, and LO2 cells were purchased from Hunan Fenghui Biotechnology Co., Ltd (Changsha, Hunan, China). Hep3B, Huh7, HepG2, LO2, and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, C12430500BT) supplemented with 10% fetal bovine serum (FBS, Gibco, 10,099-141C), penicillin (Shanghai Yuanye Bio-Technology Co., Ltd, B25911), and streptomycin (Sangon Biotech, A610494-0050). MCF-7 and BxPC-3 cells were cultured in RPMI Medium 1640 (Gibco, C11875500BT) with 10% FBS and penicillin/streptomycin. To avoid mycoplasma contamination, cells were treated with prophylactic plasmocin (InvivoGen, ant-mpp), according to the manufacturer’s instructions. For inhibitor treatments, cells were pretreated for 20 min with A66 (6 μM) and 30 min with TGX221 (4 μM) or cytochalasin D (10 μM). Lipofectamine 2000 (11,668,019; Thermo Fisher) was used for transfection according to the manufacturer’s protocol. Plasmid PH-Btk-GFP was purified using the TIANpure Midi Plasmid Kit (TIANGEN #DP107-02).