Effectene

Manufactured by Qiagen

Sourced in Germany, United States, United Kingdom, Spain, Netherlands, Switzerland, France

Effectene is a transfection reagent developed by Qiagen. It is designed to efficiently deliver DNA, RNA, or other molecules into eukaryotic cells for various research applications.

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Lab products found in correlation

695 protocols using effectene

Hippo Pathway Regulation in S2 Cells

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S2 cells cultured at 25°C were transfected with Actin-Gal4, pIE1-4-myc-Hippo, pIE1-4-myc-Warts, pIE1-4-HA-Merlin and UAS-FLAG-His6-Rae1, pAc5.1-His6-FLAGx3-Rae1, pAc5.1-Rae1-V5-His using Cellfectin II (Invitrogen) or Effectene (Qiagen). HEK-293T, U87MG and HeLa cells were cultured in DMEM (Invitrogen) containing 10% FBS (Gemini) and 50 μg/mL penicillin/streptomycin (Gemini). Transfection with pCMV5-FLAG-Mst1, pCMV2-FLAG2 Lats1, pCMV-FLAG Yap2 S127A, pCMV-FLAG-Yap2 5SA, using Effectene (Qiagen) was performed according to the manufacturer's instructions.

Jahanshahi M., Hsiao K., Jenny A, & Pfleger C.M. (2016). The Hippo Pathway Targets Rae1 to Regulate Mitosis and Organ Size and to Feed Back to Regulate Upstream Components Merlin, Hippo, and Warts. PLoS Genetics, 12(8), e1006198.

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Stability and Ubiquitylation Assay for RpS6 Protein

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For the stability assay, pAc5.1/V5-HisA-RpS6 was transiently transfected into HA-PALLFL stable S2 cell line with Effectene (QIAGEN) according to the supplier’s instructions. The cells were then treated with CuSO4 at 24 hr post-transfection followed by MG132 (Sigma) at a concentration of 50 μM for 4h. For the ubiquitylation assay, pAc5.1/V5-HisA-RpS6 and pAc5.1/UB were transiently transfected into HA-PALLFL stable S2 cell line with Effectene (QIAGEN). PALL expression was induced with CuSO4 at 24 hr post-transfection. The cells were then treated with MG132 (Sigma) at a concentration of 50 μM for 4h, lysed and subjected to IPs and WBs as described above. The Ub (P4D1) mouse monoclonal Ab (Santa Cruz Biotechnology) was used at a 1:200 dilution.
In vivo phagocytosis assay, phagocytosis index quantification, RNA interference and S2 cell phagocytosis assays, and their statistical analyses were performed as in (Cuttell et al., 2008 (link); Silva et al., 2007 (link)).

Xiao H., Wang H., Silva E., Thompson J., Guillou A., Yates JR Jr I.I.I., Buchon N, & Franc N.C. (2014). The Pallbearer E3 Ligase Promotes Actin Remodeling via RAC in Efferocytosis by Degrading the Ribosomal Protein S6. Developmental cell, 32(1), 19-30.

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Piezo1 Knockdown in MDCK Cells

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Madin-Darby Canine Kidney (MDCK) cells (ATCC) were grown to confluence in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin and streptomycin. The cells were trypsinized and seeded in the wells and allowing to grow for 30 min in the incubator. The cells were then washed with media that removed unattached cells and grown for additional 2 h. Live cell imaging were conducted in a stage-top incubator (INUB-ZILCSD-F1-LU, Tokai Hit Co., Ltd., Japan), maintained at 37°C and 5% CO₂. No-phenol Red DMEM media with 10% fetal bovine serum (Gibco, TX) was used for fluorescence imaging to reduce the background illumination. Isotonic saline solution was used for experiments with GsMTx4 and Gd³⁺, as it was known that culture media reduced the efficiency of the peptide (Bae et al., 2011 (link)).
Piezo1 knockdown was performed using previously validated Piezo1 miRNA targeting Piezo1 and co-expressed with EGFP to verify transfection (Jetta et al., 2019 (link)). Cells were cultured to ∼60% confluence and transfected with plasmid DNA miRNA (0.4 µg) using Effectene (Qiagen, Valencia, CA) at 1:50 DNA to Effectene ratio. Cells were incubated in the transfection media for another 48 h prior to experiments. The transfection efficiency was ∼25%.

Jetta D., Shireen T, & Hua S.Z. (2023). Epithelial cells sense local stiffness via Piezo1 mediated cytoskeletal reorganization. Frontiers in Cell and Developmental Biology, 11, 1198109.

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Targeted Silencing of Key Regulators

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Target-specific pools of four independent small interfering RNA (siRNA) species against human CD98hc (M-003542-02), GEF-H1 (M-009883-01), GEF-H1 3′-UTR (CTM-651415, CTM-651416), eIF4A1 (M-020178-01), eIF4A2 (M-013758-01), CPSF73 (M-006365-00), eEF1ε1 (M-015983-01), and non-targeting siRNA control pool #1 (D-001206-13) (siGENOME SMARTpool) were purchased from Dharmacon (Horizon Discovery). siRNA pools were transfected using Effectene (QIAGEN) at a final concentration of 50 nM for 48 hr unless otherwise stated before subsequent treatments. ARHGEF2/GEF-H1 ORF cDNA construct (OHu26696) and empty vector were purchased from GenScript, and transfected using Effectene (QIAGEN) following manufacturer protocols.

Ho J.J., Cunningham T.A., Manara P., Coughlin C.A., Arumov A., Roberts E.R., Osteen A., Kumar P., Bilbao D., Krieger J.R., Lee S, & Schatz J.H. (2021). Proteomics reveal cap-dependent translation inhibitors remodel the translation machinery and translatome. Cell Reports, 37(2), 109806.

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Generating Stable Prestin Expressing Cell Lines

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Sf9 cells (Invitrogen) were maintained in Sf-900 III SFM supplemented with 5% fetal bovine serum (Gibco) and 1X antibiotic antimycotic solution (Sigma). To generate stable Sf9 cells with WT gPrestin, Sf9 cells were transfected with pIZ-gPres-ceGFP using Effectene (Qiagen), and selected with 1 μg/μl zeocin (Invitrogen). A single clone was chosen to establish the stable cell line. The tetracycline-inducible WT-gPrestin stable HEK293 cell line (293-TRxST-gPrestin-YFP4TOmycHisC) was a generous gift from Drs. Santo-Sacchi and Navaratnam. Growth conditions and induction of the cell line were defined previously47 (link). For transient transfections of Sf9 and HEK293T cells with prestin constructs, Effectene (Qiagen) and jetPRIME (Polyplus) were used according to the manufacture’s instructions.

Takahashi S., Homma K., Zhou Y., Nishimura S., Duan C., Chen J., Ahmad A., Cheatham M.A, & Zheng J. (2016). Susceptibility of outer hair cells to cholesterol chelator 2-hydroxypropyl-β-cyclodextrine is prestin-dependent. Scientific Reports, 6, 21973.

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Cell Culture and Transfection Protocols

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Studies were performed using ATDC5 murine chondroprogenitor cells (RCB0565, RIKEN, Wako, Japan), NIH3T3 fibroblasts, MCF10A human mammary epithelial cells, and Human Embryonic Kidney (HEK) 293 cells. Cells were treated as indicated with TGFβ1 (5 ng/ml, HumanZyme, Chicago, IL), Y27632 (10 μM, Sigma-Aldrich, St. Louis, MO), and blebbistatin (10 μM, Cayman Chemical, Ann Arbor, MI).
For imaging experiments, glass-bottom imaging wells (Cellvis, Mountain View, CA ) were coated with collagen II (1 mg/ml in acetic acid diluted 1:100 in PBS), fibronectin (1 mg/ml diluted 1:100 in PBS), or poly-l-lysine (0.1 mg/ml). ATDC5, NIH3T3, and MCF10A cells were transfected using Nucleofection (Lonza, Basel, Switzerland) or Effectene (Qiagen, Valencia, CA), and then plated on to the imaging wells. For biochemical assays, 293 cells were plated in 100 mm cell-culture dishes and transfected using Effectene (Qiagen) at 80% confluency.

Rys J.P., DuFort C.C., Monteiro D.A., Baird M.A., Oses-Prieto J.A., Chand S., Burlingame A.L., Davidson M.W, & Alliston T.N. (2015). Discrete spatial organization of TGFβ receptors couples receptor multimerization and signaling to cellular tension. eLife, 4, e09300.

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Melanoma Cell Cytoskeletal Dynamics

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To study the effects of different gene knockdowns on focal adhesion and cytoskeletal dynamics, we co-transfected WM266-4 melanoma cells with different combinations of plasmid reporters and siRNAs. Cells were seeded on a 6-well plate, 24h prior to transfection at a density of 1 × 10⁶ cells/well. Effectene (Qiagen) transfection mixtures contained 1 μg of total plasmid, 2.56 μL of siRNA, 97μL of EC buffer, 8 μL of enhancer and 5 μL of Effectene – a total volume of 113 μL of transfection mixture. Prior to the addition of transfection mixture, 1.6 mL of fresh complete DMEM was added per well. A final concentration of 25 μM of siRNA was used. Cells were incubated for 48h (unless otherwise indicated).

Bousgouni V., Inge O., Robertson D., Jones I., Clatworthy I, & Bakal C. (2022). ARHGEF9 regulates melanoma morphogenesis in environments with diverse geometry and elasticity by promoting filopodial-driven adhesion. iScience, 25(8), 104795.

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Stable CXCR4 and ACKR3 Cell Lines

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The mammalian cell expression vector pcDNA3.1Zeo (Thermo Fisher Scientific, Waltham, MA, USA) containing CXCR4 was transfected into CHO WT cells as described previously [74 (link)]. ACKR3 cDNA from the pCMV-XL5/ACKR3 vector (OriGene, Rockville, MD, USA) was cut and cloned into a pcDNA3 vector and transfected into CHO WT and CHO-CXCR4 cells using Effectene (Qiagen, Hilden, Germany). Stable CHO-ACKR3 cell lines were obtained by growing transfected cells at low density for 24 h followed by the addition of 800 μg/mL G418 for selection over 3 weeks. Clones were picked and expanded before ACKR3 expression assessment and a single cell dilution of the highest expressing colonies was carried out. Transient CHO-CXCR4-ACKR3 transfected cells were created using Effectene (Qiagen, Hilden, Germany) 24 h prior to the assay.

del Molino del Barrio I., Wilkins G.C., Meeson A., Ali S, & Kirby J.A. (2018). Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12. International Journal of Molecular Sciences, 19(11), 3592.

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Targeted siRNA-mediated knockdown

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Establishing Stable S2 Cell Lines

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S2 cells (ATCC CRL-1963) were maintained in Schneider’s medium (Life Technologies) supplemented with 10% FBS (HyClone Standard FBS, heat-inactivated) at 25°C. Cells were plated to 1.2×10⁶/ml and allowed to adhere before transfection with Effectene (Qiagen) at ratios of 400ng DNA/3.2ul enhancer/8ul Effectene/ml of cells). CuSO₄ was added to 100uM-200uM to the medium at transfection, and cells/supernatants harvested between 60 and 70 hours post-transfection.
The actin 5C promoter was introduced in the place of the metallothionein promoter of pMT-PURO for constitutive expression (“pACT-PURO”) when indicated for 6xHis+mCh3xFLAG. Stable lines were generated by linearization of plasmids with ScaI or NsiI, transfected using the protocol above, incubated for ~72 hours, and passaged 1:5 into fresh media with puromycin (Invivogen ant-pr-1) at 1 ug/ml. After 3–4 days, cells were passaged 1:5 again, and split 3–4 more times at intervals of 3–5 days before being taken off of antibiotic two passages before experiments.

Stepanik V., Sun J, & Stathopoulos A. (2020). FGF Pyramus has a transmembrane domain and cell-autonomous function in polarity. Current biology : CB, 30(16), 3141-3153.e5.

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