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Kc7f2

Manufactured by Selleck Chemicals
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KC7F2 is a laboratory equipment product. It is a specialized instrument used for scientific applications. The core function of KC7F2 is to perform specific tasks in a laboratory setting.

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Lab products found in correlation

Pfkfb3 small interfering rnas sirnas, by Horizon Discovery (1 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) Immobilon p pvdf, by Merck Group (1 mentions) A2103, by Thermo Fisher Scientific (1 mentions) Luminata forte western hrp substrate, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Everolimus, by Selleck Chemicals (1 mentions) Lapatinib, by Selleck Chemicals (1 mentions) Doxycycline, by Merck Group (1 mentions) U73122, by Bio-Techne (1 mentions) C188 9, by Selleck Chemicals (1 mentions) Chir99021, by Selleck Chemicals (1 mentions) Sc75741, by Selleck Chemicals (1 mentions) Ly294002, by Selleck Chemicals (1 mentions) Pd98059, by Selleck Chemicals (1 mentions) T0157, by Merck Group (1 mentions) Human tnf α, by Merck Group (1 mentions) Rat tnf α, by Merck Group (1 mentions) I3275, by Merck Group (1 mentions)

15 protocols using kc7f2

1

Silencing Pfkfb3 in INS-1 cells

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Pfkfb3 small interfering RNAs (siRNAs; L-095107-02-0005) were purchased from Dharmacon, Lafayette, CO, USA. INS-1 832/13 cells were plated at 0.7×106 cells per well in a 6-well plate. Twenty-four hours later, cells were transfected with siRNAs at 75 nmol/l using Lipofectamine RNAiMAX (Invitrogen) and cultured for 48 h before collection. The cytokine mix was added to cells for the last 24 h of culture.
For inhibition of HIF1α we used HIF1α translation and transactivation inhibitor KC7F2 (S7946; Selleckchem, Houston, TX, USA), which was screened for efficacy and toxicity (see ESM Fig. 1). We used concentrations that inhibited HIF1α without toxicity.
Nomoto H., Pei L., Montemurro C., Rosenberger M., Furterer A., Coppola G., Nadel B., Pellegrini M., Gurlo T., Butler P.C, & Tudzarova S. (2019). Activation of the HIF1α/PFKFB3 stress response pathway in beta cells in type 1 diabetes. Diabetologia, 63(1), 149-161.
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2

Western Blot Analysis for Protein Detection

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Western blot analysis was performed as described previously [28 (link)]. Briefly, U251 MG cells were treated with test compounds or inhibitor (KC7F2, Selleckchem, Houston, TX, USA). Western blot samples were obtained by scraping 106 cells from 6-well plates in 250 µL of hot 1× Laemmli buffer. Then 20 µL samples were run on a 4–15% SDS-PAGE and blotted onto Immobilon P PVDF (polyvinylidene fluoride) membrane (0.45 µm pore size, Millipore, (Budapest, Hungary). After blocking with 0.5% nonfat dry milk powder in PBS containing 0.1 % Tween 20, membranes were labeled 1:700 with HF1 alpha antibody (PA1-16601, ThermoFisher, (Waltham, MA, USA). As secondary antibodies goat anti-rabbit whole IgG antibodies were used (111-035-003, Jackson ImmunoResearch, (Cambridgeshire, UK)
Proteins were visualized using the ECL system (Luminata Forte Western HRP substrate, Millipore, Budapest, Hungary, # WBLUF0100).
Quantification was carried out using ImageJ software by referring the relative density of HF1 alpha bands to the untreated sample relative density. Data are shown as adjusted relative density by referring the relative intensity of HF1 alpha bands to the corresponding loading control intensities (Actin: Sigma-Aldrich, A2103 or GAPDH: Invitrogen, PA1-987).
Hackler L J.r., Gyuris M., Huzián O., Alföldi R., Szebeni G.J., Madácsi R., Knapp L., Kanizsai I, & Puskás L.G. (2019). Enantioselective Synthesis of 8-Hydroxyquinoline Derivative, Q134 as a Hypoxic Adaptation Inducing Agent. Molecules, 24(23), 4269.
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3

Therapeutic Compound KC7F2 Administration

Cited 1 time
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KC7F2 was purchased from Selleck Chemicals (Houston, TX, #S7946), dissolved by dimethyl sulfoxide (Sigma, St Louis, MO). For in vitro experiments, mother solution was freshly dilated to 10 µM with culture medium (dimethyl sulfoxide <0.1%), which did not affect the viability of HUVEC in preliminary experiments. For in vivo experiments, working solution was freshly made using PBS solution at a final concentration of 0.5% dimethyl sulfoxide, and intraperitoneally injected into mice (10 mg/kg/d) from postnatal day 12 (P12) to P17, which was referred to a previous study.25 (link) The doses for the rats (7 mg/kg/d, from P14 to P18) can be converted from the mouse dose according to the body surface area formula.
Tang X., Cui K., Lu X., Wu P., Yu S., Yang B., Xu Y, & Liang X. (2022). A Novel Hypoxia-inducible Factor 1α Inhibitor KC7F2 Attenuates Oxygen-induced Retinal Neovascularization. Investigative Ophthalmology & Visual Science, 63(6), 13.
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4

Modulating Oncogenic HER2 Signaling

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MCF7/NeuT cells were seeded onto a 6-well plate and allowed to attach overnight. On the next day, cells were treated with different concentrations of LY294002 (Selleckchem), PD98059 (Selleckchem), U73122 (Tocris), everolimus (Selleckchem) or CHIR-99021 (Selleckchem). For induction of the oncogenic rat HER2 variant, NeuT in the MCF7 cells, doxycycline (Sigma) was added with a final concentration of 1 µg/ml per well to one half of the experiment setup one hour after the inhibitor addition [16 (link)]. Medium was changed every three days, and cells were harvested after three and seven days, for RNA and protein expression analyses as described above.
For the EDI3 expression studies in endogenous HER2 expressing cell lines, SKBR3 and HCC1954 cells were seeded onto a 6-well plate and allowed to attach overnight. On the next day, cells were treated with different concentrations of lapatinib, everolimus, CHIR-99021, compound 3i (666–15), C188-9, KC7F2, SC75741, 10074-G5 or SR18662 (all from Selleckchem). After one, two and three days (as well as 4 days for lapatinib), cells were harvested at approximately 80% confluency for RNA and protein expression analyses as described above. Medium was changed after two days.
Keller M., Rohlf K., Glotzbach A., Leonhardt G., Lüke S., Derksen K., Demirci Ö., Göçener D., AlWahsh M., Lambert J., Lindskog C., Schmidt M., Brenner W., Baumann M., Zent E., Zischinsky M.L., Hellwig B., Madjar K., Rahnenführer J., Overbeck N., Reinders J., Cadenas C., Hengstler J.G., Edlund K, & Marchan R. (2023). Inhibiting the glycerophosphodiesterase EDI3 in ER-HER2+ breast cancer cells resistant to HER2-targeted therapy reduces viability and tumour growth. Journal of Experimental & Clinical Cancer Research : CR, 42, 25.
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5

Cytokine and HIF-1α Inhibitor Study

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The chemicals used in this study included: rat IL-1β (IL024; Millipore, Burlington, MA, USA); human IL-1β (I9401, Sigma); rat TNF-α (T5944, Sigma); human TNF-α (T0157, Sigma); rat IFN-γ (I3275, Sigma); human IFN-γ (I17001, Sigma); and the HIF-1α inhibitor KC7F2 (S7946, Selleckchem).
Nomoto H., Pei L., Montemurro C., Rosenberger M., Furterer A., Coppola G., Nadel B., Pellegrini M., Gurlo T., Butler P.C, & Tudzarova S. (2019). Activation of the HIF1α/PFKFB3 stress response pathway in beta cells in type 1 diabetes. Diabetologia, 63(1), 149-161.
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6

Immune Cell Profiling and Targeting

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The following antibodies and reagents were used: anti-GAPDH (sc-365062, Santa Cruz Biotechnology) for immunoblot; anti-FOXA1 (Abcam, ab23738) for immunoblot and ChIP; anti-HIF1A (Abcam, ab2185) for immunoblot and ChIP; anti-CA IX (H-11) (sc-365900, Santa Cruz Biotechnology) for IHC; anti-PE/Cy7-CD163 (#326513, Biolegend) and anti-APC-CD206 (#321109, Biolegend) for flow cytometry; CCL2 neutralizing antibody (#551226, BD Biosciences, USA); Cobalt(II) chloride hexahydrate (Sigma, C8661); KC7F2 (Selleck Chemicals).
Wang X., Brea L., Lu X., Gritsina G., Park S.H., Xie W., Zhao J.C, & Yu J. (2022). FOXA1 inhibits hypoxia programs through transcriptional repression of HIF1A. Oncogene.
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7

Hypoxia Preconditioning of MSCs

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MSCs were plated on 6-well plates and transfected using Lipofectamine 2000 (#11668019, Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. After 48 h, the cells were collected for further study. The in vitro preconditioning hypoxia model was established by flooding the chamber with 95% N2 and CO2 as described previously. The HIF-1α inhibitor KC7F2 (#S7946, Selleck Chemicals, Houston, TX, USA) was used before some experiments.
Wei H., Xu Y., Chen Q., Chen H., Zhu X, & Li Y. (2020). Mesenchymal stem cell-derived exosomal miR-223 regulates neuronal cell apoptosis. Cell Death & Disease, 11(4), 290.
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8

Investigating Hif1α Inhibition in EAU

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Mice were daily injected intraperitoneally with vehicle or an Hif1α inhibitor, KC7F2 (10 mg/kg/d; Selleck Chemicals, Houston, TX, USA)31 ,32 for 2 weeks after immunization. CDLN cells were isolated from the EAU group and these cells were treated with IRBP1–20 alone or IRBP1–20 + KC7F2 (10 µM)31 ,33 (link),34 (link) at 37°C for 72 hours before flow cytometry.
Zhu L., Li H., Wang R., Li Z., Zhao S., Peng X, & Su W. (2023). Identification of Hif1α as a Potential Participant in Autoimmune Uveitis Pathogenesis Using Single-Cell Transcriptome Analysis. Investigative Ophthalmology & Visual Science, 64(5), 24.
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9

Visualizing HIF-1α in Hypoxic OvCa Cells

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OvCa cells (OVCAR-3, SW 626, and TOV-112D) were seeded in 48-well plates overnight and treated with CoCl2 (150 μM), to mimic hypoxia, and with KC7F2 (40 μM, Selleckchem, USA), an inhibitor of HIF-mediated transcription, for 12 h at 37 °C and under 5% CO2. Next, cells were fixed with 4% paraformaldehyde and permeabilized with 0.05% saponin for 10 min. Cells were washed and stained with and HIF-1α primary antibody (Invitrogen, USA) at 4 °C overnight, then incubated with FITC conjugated fluorescent secondary antibody (R&D System, USA) for 1 h at room temperature. Cells were washed with PBS to remove unbound antibody. To visualize the F-actin cytoskeleton, cells were stained with Phalloidin TM Red 594 solution (1:40) (BioLegend, USA) for 20 min at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 300 nM, Invitrogen, USA) for 5 min. Images were captured by a fluorescent microscope with the appropriate filter for FITC/GFP, RFP, or DAPI with an EVOS FL microscope (Thermo Scientific, USA).
Singh S.K., Mishra M.K, & Singh R. (2019). Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells. Journal of Ovarian Research, 12, 42.
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10

EPC Colony-Forming Capacity Modulation

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For colony-forming unit (CFU) assays, EPCs derived from bone marrow cells were put in a T25 culture flask with M199 culture medium supplemented with 2% FBS. EPCs were pretreated with 500 μM DMOG alone or with 500 μM DMOG and the HIF-1α inhibitor KC7F2 (40 μM) (S7946, Selleck, Houston, TX, USA) for 24 h before MPS administration. The control group was cultured in only M199 medium supplemented with 2% FBS containing equal amounts of DMSO before MPS administration. The CFUs of the EPCs were counted on day 7. A colony of >50 cells was marked as one unit.
Shao W., Li Z., Wang B., Gong S., Wang P., Song B., Chen Z, & Feng Y. (2023). Dimethyloxalylglycine Attenuates Steroid-Associated Endothelial Progenitor Cell Impairment and Osteonecrosis of the Femoral Head by Regulating the HIF-1α Signaling Pathway. Biomedicines, 11(4), 992.
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