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Penter

Manufactured by WZ Biosciences
Sourced in China
About the product

The PENTER is a high-performance laboratory equipment designed for precise and efficient sample preparation. Its core function is to provide a controlled environment for the homogenization, disruption, and extraction of a wide range of biological samples, including tissues, cells, and microorganisms. The PENTER utilizes advanced technology to ensure consistent and reproducible results, making it a valuable tool for researchers and scientists in various fields of study.

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4 protocols using penter

1

Plasmid Overexpression Transfection Protocol

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We purchased the empty control plasmid (pENTER) and overexpression plasmid (pENTER-FGFR4) from WZ Biosciences Inc. (Shandong, China). We verified the efficiency of overexpression by qPCR. We list primers used to construct these plasmids in Supplementary Table S8. According to the manufacturer's protocol of Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific Inc., catalog No. L3000001), we gently mixed plasmid and Opti-MEM I (Invitrogen Inc., catalog No. 31985062) reduced serum medium and then incubated at room temperature for 5 min. Similarly, we mixed Lipo3000 and Opti-MEM I reduced serum medium and then incubated at room temperature for 5 min. Next, we gently mixed diluted plasmid and Lipo3000 and then incubated at room temperature for 20 min. Finally, we added the complex into the six-well plate, 2 mL per well. Then, we performed other experiments after culturing for another 24–48 h.
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2

Plasmid Transfection of Breast Cancer Cells

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The small interfering RNAs targeting MIDEAS-AS1, MATR3, NCALD and the scrambled oligonucleotides (NC; 20–50 nM) were purchased from GenePharma (Shanghai, China). Full sequence of MIDEAS-AS1, MATR3, NCALD complementary DNA (cDNA) was amplified by PCR and then, inserted into the vector pcDNA3.1-GFP (abbreviated as “pcDNA3.1”) or pEnter (WZ Biosciences, Jinan, China) to construct pcDNA3.1/MIDEAS-AS1, pcDNA3.1/NCALD, pEnter/MATR3 plasmids. Empty vector (pcDNA3.1, pEnter) was regarded as the control. Using Lipofectamine 2000 (Invitrogen, Texas, USA), all vectors were transfected into breast cancer cells. After 24 h of transfection, cells were collected for following experiments. The sequences that were used are shown in Additional file 1: Table S1.
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3

Transcriptional Regulation of NR1H3 by FOXC1

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Bioinformatic analysis (JASPAR database, http://jaspar.genereg.net/) was used to explore the potential binding sequence of transcriptional factors in relation to the genetic variants in this study. To verify whether rs11039149A>G influences the transcriptional activity of NR1H3, 200-bp NR1H3 promoter fragments containing rs11039149A or rs11039149G allele were amplified by PCR method using the following primers: forward 5′-GGCGGT​ACCCCT​CAG​CAG​CTT​GCC​TCC-3′, reverse 5′- GGCACG​CGTAGT​GGC​TGG​G CTGGGATCAGA-3′, then cloned into the pGL3-promoter luciferase vector (Promega, WI, USA) at KpnI and MluI restriction sites, and two plasmids were generated: pGL3-rs11039149A and pGL3-rs11039149G. Forkhead box C1 (FOXC1) gene coding sequence was amplified using the following primers: forward 5′-GCGCG​ATC​GCATG​CAG​GCG​CGC​TAC​TCC-3′, reverse 5′- GGCACGCGTCA AAA​CTT​GCT​ACA​GTC​GTA​G-3′, then cloned into eukaryotic expression vector pENTER (WZ Biosciences, Shandong, China) at AsisI and MluI restriction sites to generate the pENTER-FOXC1 plasmid. All plasmids were confirmed by DNA sequencing.
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4

Generating Mutant TP53 Plasmids

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To overexpress TP53, a plasmid expressing wild-type P53 (wtp53) was purchased from WZ Biosciences Inc (Shandong, China). The polymerase chain reaction (PCR) product was cloned into a pENTER (WZ Biosciences Inc., Shanghai, China) vector backbone. The cytomegalovirus (CMV)-TP53Y220C/R273H/H179L/R248Q/H193R plasmid was generated with a Quick Mutation™ Plus site-directed mutagenesis kit (D0208S, Beyotime Biotechnology, Shanghai, China) using CMV-wtp53 as a template. Products were validated by Sanger sequencing to authenticate the mutations and ensure the quality of the constructs. Plasmids were transfected into NCI-H1299 cells using Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The sequences of the mutant plasmids are listed in Table S1.
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