MCF-7 cells were purchased from American Type Culture Collection and cultured according to their instructions. The stable MCF-7 cell line expressing pcDNA3 or the two independent cell lines expressing pcDNA3-HER2Δ16 and referred to here as MCF-7/pcDNA, MCF-7/HER2Δ16H, and MCF-7/HER2Δ16M1, respectively, have been described elsewhere [14] (link). For stable suppression of EGFR to generate the pooled MCF-7/HER2Δ16/EGFRKD cell line, MCF-7/HER2Δ16H cells were transfected with the MISSION shRNA plasmid-DNA TRCN0000121329 targeting EGFR (Sigma) or a pLKO.1 (Sigma) negative control using Fugene6 (Roche). For stable suppression of miR-7 to generate the pooled MCF-7/miR-7KD cell line, MCF-7 cells were transfected with the miRZip-7 anti-miR-7 microRNA construct MZIP7-PA-1 (System Biosciences) or the pGreenPuro Scramble Hairpin Control construct MZIP000-PA-1 (System Bioscience) using Fugene6. For stable expression of miR-7 to generate the pooled MCF-7/HER2Δ16H/miR-7 cell line, MCF-7/HER2Δ16H cells were transfected with the miR-7 expression vector MI0000263 (Origene) using the NEON Transfection System exactly as described by the manufacturer (Invitrogen). At two days post-transfection all pooled cell lines were selected for two days in 1 µg/ml puromycin (Gibco) and then maintained at 0.2 µg/ml puromycin.
Mzip000 pa 1
Manufactured by System Biosciences
Sourced in United States, United Kingdom
The MZIP000-PA-1 is a lab equipment product from System Biosciences. It is a multi-channel pipette designed for accurate and precise liquid handling in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Pmirh146apa 2, by System Biosciences (18 mentions)
Pcdh cmv mcs ef1 copgfp cd511b 1, by System Biosciences (12 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Mzip27a pa 1, by System Biosciences (6 mentions)
Pmirh000 pa 1, by System Biosciences (6 mentions)
Mzip203 pa 1, by System Biosciences (6 mentions)
Effectene reagent, by Qiagen (6 mentions)
Rnaimax, by Thermo Fisher Scientific (6 mentions)
Puromycin, by Thermo Fisher Scientific (3 mentions)
Neon transfection system, by Thermo Fisher Scientific (3 mentions)
Plko 1, by Merck Group (3 mentions)
Mcf 7 cell, by American Type Culture Collection (3 mentions)
Miscript target protector, by Qiagen (3 mentions)
Si03650325, by Qiagen (3 mentions)
Hiperfect transfection reagent, by Qiagen (3 mentions)
Moflo astrios eq cell sorter, by Beckman Coulter (3 mentions)
Polybrene, by Merck Group (3 mentions)
Mzip 330 5p pa 1, by System Biosciences (3 mentions)
Retronectin, by Takara Bio (1 mentions)
Pcmv mir, by OriGene (1 mentions)
8 protocols using mzip000 pa 1
1
Generating Stable Cell Lines for HER2 and miR-7 Studies
2
Modulating miR-27a in Colorectal Cancer
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Synthetic miR-27a mimic (Syn-hsa-miR-27a), miR-27a inhibitor (anti-hsa-miR-27a) or the appropriate scrambled controls (AllStar or mirScript Inhibitor-Negative Control) were purchased from Qiagen (Hilden, Germany). The miR-27a-antisense (MZIP27a-PA-1), the pre-miR-27a expression constructs (PMIRH27a-onlyPA-1) and scrambled control miRNAs (MZIP000-PA-1; PMIRH000PA-1) plasmids (System Biosciences, Mountain View, CA, USA) were transfected in the different CRC cell lines. microRNA functional studies were performed by inhibiting miRNA-mRNA target interactions either with a custom-designed calreticulin-miScript Target Protector or a negative control miScript Target Protector (MTP0075035; Qiagen). Detection of no variations in unrelated proteins validated target protector specificity. A gene-specific package of three preselected siRNAs against calreticulin (Flexi Tube siRNA GS811) or a negative control siRNA (SI03650325) (Qiagen) was used in transient transfections. Functional assays, RNA and protein analyses were performed within 24/72 h from transfections. In each experiment, the extent of miR-27a silencing/overexpression was assessed by qRT-PCR. Plasmids and oligonucleotides were transfected using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA) and HiPerFect Transfection Reagent (Qiagen) or RNAi Max (Thermo Fisher), respectively, according to the manufacturers' recommendations.
3
Downregulation and Overexpression of miRNAs in HaCaT Cells
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To down-regulate miR-203 in HaCaT cells, we used an antisense miR-203 vector (MZIP203-PA-1) and a miRZip scrambled hairpin control vector (MZIP000-PA-1) from System Biosciences (SBI; CA, USA). HaCaT cells were transfected with the pZip control and pZip-203 vectors using the Effectene reagent (Qiagen; MD, USA). Three days after the transfection, transfected cells were enriched by sorting based on the copGFP reporter, followed by puromycin selection. Two pooled miR-203 knockdown cell clones (Zip203-1 and Zip203-2) were generated via independent transfection followed by the selection procedures described above.
We used an miR-146a-overexpressing vector (PMIRH146aPA-2) and a scrambled control hairpin in pCDH-CMV-MCS-EF1-copGFP (CD511B-1) from SBI to generate stable miR-146a-overexpressing and control cell clones, respectively, using a similar protocol as describe above.
We used an miR-146a-overexpressing vector (PMIRH146aPA-2) and a scrambled control hairpin in pCDH-CMV-MCS-EF1-copGFP (CD511B-1) from SBI to generate stable miR-146a-overexpressing and control cell clones, respectively, using a similar protocol as describe above.
4
Downregulation and Overexpression of miRNAs in HaCaT Cells
5
Lentiviral Transduction for miR-27a Modulation
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Lentiviral constructs overexpressing (Cat# PMIRH27a-onlyPA-1, System Biosciences, Mountain View, CA, USA), or functionally knocking-down miR-27a (Cat# MZIP27a-PA-1, System Biosciences), along with the corresponding controls (Cat# PMIRH000-PA-1 and MZIP000-PA-1, System Biosciences) were transduced and packaged in 293T cells. Stable cell lines (HCT116, SW480 or HT29) overexpressing or silencing miR-27a or the corresponding controls were generated via lentiviral transduction, in the presence of polybrene (8μg/ml) (Sigma-Aldrich, S.Louis, MO, USA), and selected with puromycin or by flow cytometry sorting for GFP positive populations (the MoFlo Astrios EQ, Cell Sorter, Beckman Coulter, Indianapolis, IN, USA).
6
Modulating miR-330 Expression In Vitro
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A miRNA-suppressing miR-ZIP plasmid was used for in vitro miR-330-5p suppression (catalogue number MZIP-330-5p-PA-1, System Biosciences, California, USA) as previously described [20 (link)]. Cells were transfected with the miR-ZIP plasmid or a scrambled non-targeting vector control MIRZIP-VC plasmid (catalogue number MZIP000-PA-1, System Biosciences) using Lipofectamine 2000 (Invitrogen, ThermoFisher Scientific, UK). The single clone (SC) cell line was derived from an individual clone that was selected after assessing GFP expression using fluorescent microscopy. The SC cell line had high levels of GFP expression indicating high expression of the miRZIP-330-5p plasmid. The miRZIP-VC SC was derived from an individual clone that was selected after assessing GFP expression using fluorescent microscopy. The heterogeneous clonal (HC) cell line was derived from a mixed population of stable clones. The miRZIP-VC HC was derived from a mixed population of stable clones. The miR-330 precursor plasmid was used for in vitro miR-330-3p/5p overexpression (catalogue number PMIRH330PA-1, System Biosciences). The miR-VC (catalogue number CD511B-1, System Biosciences) vector control plasmid was used as a control. Transient overexpression of miR-330-3p and miR-330-5p was confirmed via qPCR analysis, as previously described [20 (link)].
7
Silencing miR-424 via Lentiviral Transduction
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To silence miR-424 the GFP tagged lentiviral vectors that express antisense hsa-miR-424 (MZIP424-PA-1) and corresponding scramble control lentiviral vectors (MZIP000-PA-1) were purchased from SBI (System Biosciences). Transfection of HEK293T packaging cell line (Forster et al., 2014) was performed using calcium-phosphate protocol and infection of target cells was done as previously described (Platzer et al., 2004) . In general, lentiviral supernatant was added to CD34 + cells in the RetroNectin (Takara) precoated 24-well suspension plate according to manufacturer protocol. Infections were repeated 2-3 times and on day 5 cells were replated into lineage-specific cytokine mix and further differentiated for 5-7 days as previously described. Taqman qPCR of GFP + FACS sorted cells at 4 day of differentiation under lineage-specific conditions revealed 50%-70% decrease in miR-424 levels (Figure S1C).
8
Stable miR-31 Overexpression in Cell Lines
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Transfections were performed using Lipofectamine 2000 reagent from Invitrogen according to the manufacturer’s instructions. miR-31 overexpression plasmid (MI0000089) and vector control (PCMVMIR) were purchased from Origene and stably transfected into the NCI-H2452 cell line under 500 μg/mL G418 (Thermo Fisher Scientific) selection for ∼21 days. Zip-miR-31 plasmid (MZIP31-PA-1) and Zip vector control (MZIP000-PA-1) were purchased from SBI and stably transfected into the P31 cell line under 3 μg/mL puromycin (Sigma Aldrich) selection for 10 days.
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