The shRUNX1 and LV-RUNX1 lentiviruses were obtained from Genechem (Shanghai, China). The siSUV39H1 siRNA was obtained from Genepharma (Shanghai, China). The LV-SUV39H1 plasmid was obtained from (Vigenebio, Jinan, China). The shRUNX1 target sequence, siSMAD3 sequence and siSUV39H1 sequence are provided in Table S6 . Flag-RUNX1RHD-del (del 135–167) and Flag-SMAD3MH1-del(del 57–94) were created by polymerase chain reaction with the insertion of a BglII restriction site to join the fragments. The mutated sequence was inserted into the PCMV-N-FLAG series vectors, which was made by IBSBIO(Shanghai, China), based on the website (http://asia.ensembl.org/ ). Mutated or wild-type promoters containing the putative target regions of BCL3, MGP and POSTN were synthesized and cloned into pGL4.10 [luc2] (Promega, Madison, WI, USA) vector sites and the pGL4.70 [hRluc] (Promega Madison, WI, USA) was used as a promoter-less control vector. The open reading frames (ORFs) of RUNX1, SMAD3, and SUV39H1 were cloned with a C-terminal Flag into pENTER (Vigenebio, Jinan, China). The siRNAs and plasmids were transfected into cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s protocol.
Penter
Manufactured by Charles River Laboratories
Sourced in China
The PENTER is a versatile laboratory equipment designed for precision cell and tissue processing. It functions as an automated tissue processing system, efficiently preparing samples for further analysis and study.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Dual luciferase assay kit, by Promega (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pgl4.70 hrluc, by Promega (1 mentions)
Pgl4.10 luc2, by Promega (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Penter plasmid, by Charles River Laboratories (1 mentions)
Quickchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Pcdnatm3.1, by Thermo Fisher Scientific (1 mentions)
Pcmv6 entry, by Thermo Fisher Scientific (1 mentions)
Psi lvru6gp vector, by GeneCopoeia (1 mentions)
Pgl3 basic luciferase reporter vector, by Promega (1 mentions)
Pcmv6 entry, by OriGene (1 mentions)
Lipofectamine ltx and plus, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Gv248 lentiviral vector, by Genechem (1 mentions)
E1751, by Promega (1 mentions)
22 protocols using penter
1
Lentiviral Transduction and Plasmid Transfection
2
Silencing PITX2 Gene Expression
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siRNAs against PITX2 (Santa Cruz Biotechnology, sc-44016) were used at 20 nM/well using 2 μL Lipofectamine RNAiMAX (Invitrogen) in the cells seeded in 6-well plates. The full-length cDNA of human PITX2 were PCR-amplified and cloned into the expression vector pENTER (Vigene Biosciences). shRNA and microRNA mimics were conducted and purchased from Vigene Biosciences. Transfection of plasmids and miRNA mimics was performed according to the Lipofectamine 3000 Reagent (Invitrogen) protocol. Totally 10 μg plasmid vectors were transfected into cells seeded in 6-well plates for each group. Nonsense shRNA (sh-nc) and negative control mimics were used as the respective controls. The sequences of shRNAs and microRNA mimics are shown in Additional file 3 : Table S3.
3
Hep3B Cell Luciferase Assay
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Hep3B cells were plated at 2 × 105 cells/well in 24-well tissue culture plates. PIK3R1-promoter-pGL3-basic plasmid (Kidan Bio Co. Ltd., Guangzhou, China), pRL-TK plasmid (Kidan Bio Co. Ltd), and FOXA1-pEnter plasmid (Vigene Bioscience Inc.) or pEnter (Vigene Bioscience Inc.) vector were co-transfected into Hep3B cells. After culturing for 48 h, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA).
4
Transient Transfection Luciferase Assay
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The pENTER and pENTER/RIPK4-overexpressing human RIPK4 plasmids were purchased from ViGene Biosciences (Shandong, China). For the luciferase reporter assay, 293 T and SiHa cells (1.5 × 105) seeded in 24-well plates were transiently co-transfected with the TOP/FOP-Flash reporter and pTK-RL plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 24 h post-transfection, luciferase activity was analyzed using the Dual Luciferase Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Renilla was used as a co-reporter vector to normalize transfection efficiency. All experiments were performed three times with triplicate samples.
5
Overexpression of NAT2 and VDR in Colon Cancer
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To construct the NAT2 plasmid, full-length NAT2 (NM_000015) was inserted into a vector (pLV-CMV-MCS-3FLAG-IRES-Puro) (Umine, Guangzhou, China) and then transfected into SW480 or LoVo cells by Lipofectamine™ 3000 Transfection Reagent (Invitrogen, Carlsbad, United States) according to the manufacturer’s instructions. The interference of endogenous NAT2 was achieved by siRNA (GenePharma, Shanghai, China). The NAT2-targeting siRNA and negative control sequences are listed in the Supplementary Table S1 . The VDR-overexpressing plasmid was constructed based on pEnter (Vigene, Jinan, China), and a full-length VDR (NM_000376) was inserted.
6
USP21 and GATA3 Overexpression and Knockdown
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The USP21 or GATA3 coding sequence was cloned into the pENTER plasmid (ViGene Biosciences Inc., Rockville, MD, United States) to overexpress USP21 or GATA3. The Flag or Myc labeled-empty plasmid vector (pENTER) was purchased from Vigene. The small interfering RNA (siRNA) targeting USP21, GATA3, or MAPK1 was designed and synthesized by RiboBio (Guangzhou, China). All transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, United States). For in vivo experiment, cells were seeded into a 6-well plate and cultured overnight, and an appropriate lentivirus-packing vector overexpressing USP21 synthesized by GenePharma (Shanghai, China) was added to the cells for infection.
7
PRDM1, STRA8, and Other Overexpression Plasmids
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The full-length cDNA of PRDM1 was purchased and cloned into pEGFP-C1. The mutant PRDM1 overexpression plasmids were generated by overlap extension PCR. The methods to construct plasmids used in the minigene splicing assay of STRA8 are described in detail below. To validate the function of exon2 deletion of STRA8, the full-length cDNA of STRA8 was PCR amplified from human transcriptome cDNA and cloned into p3 × FLAG-CMV7.1 as the WT plasmids, and the exon2 deletion mutant STRA8 overexpression plasmid was generated using overlap extension PCR.
The WT overexpression plasmids of BLM, HFM1, MCMDC2, MCM8, MCM9, MSH4 and RECQL4 cloned in pcDNA3.1-3 × FLAG-C were purchased from YOUBAO Biology. The WT overexpression plasmid NR5A1 in pENTER was purchased from Vigene Biosciences. All the mutant overexpression plasmids were generated through QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturerʼs protocol.
The WT overexpression plasmids of BLM, HFM1, MCMDC2, MCM8, MCM9, MSH4 and RECQL4 cloned in pcDNA3.1-3 × FLAG-C were purchased from YOUBAO Biology. The WT overexpression plasmid NR5A1 in pENTER was purchased from Vigene Biosciences. All the mutant overexpression plasmids were generated through QuickChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturerʼs protocol.
8
Cloning and Characterization of Sperm-Specific Proteins
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The full-length cDNA of AKAP3, TSSK4, ODF2, and QRICH2 was synthesized and separately cloned into pENTER (Vigene), pCMV6-Entry (Origene), pCMV6-Entry, and pcDNATM3.1(+) (Invitrogen) vectors containing a FLAG, Myc, and HA tag sequence. CABYR and ODF2 promoter fragments were amplified and cloned into the pGL3-Basic luciferase reporter vector (Promega). The shRNAs designed to interfere with QRICH2 expression and the negative control SuperSilencing shRNA (shNC) were synthesized and cloned into the psi-LVRU6GP vector by GeneCopoeia. The target sequences of QRICH2 shRNAs were 5′-GGGTTCACTTCCTTAACATCA-3′. The primers for promoter region amplification are listed in Supplementary Table 5 .
9
Generation of Lin28B Overexpressing Cells
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Cells were transfected with either pEnter or pEnter-Lin28B (Vigene Biosciences, China) using Lipofectamin 2000. After 72h incubation, puromycin was then added to the transfected cells for generating cells stably overexpressing Lin28B. Lin28B stable expression cell lines were identified by western blot, and then used for the experiments.
10
PCSK9 Overexpression in HepG2 Cells
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