Rab20-knockdown RAW264.7 macrophages were produced using a MISSION shRNA system (Sigma-Aldrich, USA) as described previously (Pei et al., 2012 (link)). Cells stably transfected with Rab20 shRNA oligo 2643 (5′-CCTTTACAAGAAGATCCTGA-3′) or empty control shRNA plasmid pLKO.1 were seeded 2 d before experiment in T25 flasks. Then cells were washed twice with PBS and starved in serum-free DMEM for 5 h at 37°C. For EGF uptake, cells were washed twice with ice-cold PBS and incubated for 1 h on ice in uptake medium (2% BSA and 20 mM HEPES, pH 7.5, in serum-free DMEM) containing 2.5 μg/ml recombinant EGF (BioLegend). After incubation, cells were washed three times with ice-cold PBS to remove unbound ligands. PBS was replaced by uptake medium, and flasks were transferred to a 37°C incubator and incubated for the indicated time periods (10, 30, and 60 min). Then cells were washed with PBS, scraped, and processed for Western blot.
Epidermal growth factor (egf)
Manufactured by BioLegend
Sourced in United States
EGF (Epidermal Growth Factor) is a protein that stimulates cell growth and differentiation. It plays a key role in the regulation of cell survival, proliferation, and migration. EGF is a widely used reagent in cell culture applications to support the growth and maintenance of various cell types.
Automatically generated - may contain errors
Lab products found in correlation
Basic fibroblast growth factor (bfgf), by BioLegend (2 mentions)
Recombinant human noggin, by Thermo Fisher Scientific (2 mentions)
Matrigel, by BD (2 mentions)
A83 01, by Bio-Techne (2 mentions)
Organoid growth medium, by STEMCELL (2 mentions)
Gentle cell dissociation reagents, by STEMCELL (2 mentions)
Y 27632, by STEMCELL (2 mentions)
R spondin 1, by R&D Systems (2 mentions)
Dmem f12, by Corning (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Insulin, by Merck Group (2 mentions)
Mission shrna system, by Merck Group (1 mentions)
Mtt solution, by Merck Group (1 mentions)
Vascular endothelial growth factor (vegf), by Koma Biotech (1 mentions)
Egm 2, by Lonza (1 mentions)
Recombinant tnf α, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Fitc rat anti mouse cd8, by BD (1 mentions)
Pe anti mouse f4 80 antibody, by BioLegend (1 mentions)
Advanced dmem f12, by Thermo Fisher Scientific (1 mentions)
11 protocols using epidermal growth factor (egf)
1
Rab20 Knockdown Impacts EGF Uptake
2
HUVEC Proliferation Assay with VEGF and EGF
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Cell proliferation was determined by MTT assay. Briefly, HUVECs were seeded into a gelatin-coated 24-well plate at 3.2 × 104 cells/well and incubated at 37 °C in 5% EC growth medium (EGM-2, Lonza) overnight. After attachment, the cells were cultured with M199 (HyClone) supplemented with 1% FBS. Next, the cells were stimulated with VEGF (Koma Biotech, 20 ng/mL) and EGF (BioLegend, 20 ng/mL) for 24 h. The cells were then incubated for 4 h at 37 °C with MTT solution (0.1 mg/mL, Sigma) for evaluation of cell proliferation. After the 4-h incubation period, the MTT solution was removed, and a 50% dimethyl sulfoxide/ethanol solution (Sigma-Aldrich) was added (200 μl/well) to solubilize formazan crystals. The absorbance was then detected at a wavelength of 540 nm, and cell proliferation was calculated as a percentage of the control.
3
Investigating IL-33/ST2 Signaling Pathway
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Recombinant TNFα was obtained from PeproTech (Rocky Hill, NJ, USA), EGF was purchased from Biolegend Inc (San Diego, CA, USA) and PGE2 was obtained from Cayman Company (Ann Arbor, MI, USA). LPS was purchased from Invivogen (San Diego, CA, USA). Recombinant IL-33 was obtained from ProSpec (East Brunswick, NJ, USA). The primary antibodies used were as follows: polyclonal anti-human anti-ST2L antibody (cat # NBP1-85251) Novus, (Littleton, CO, USA), anti-mouse anti-ST2 antibody (cat#ab25877) Abcam (Cambridge, UK), anti-human anti-IL-33 antibody (Nessy-1) (cat#ALX-804-840) Enzo Life Sciences (Farmingdale, NY, USA). APC/Cy7 anti-mouse Ly-6G/Ly-6C antibody and PE anti-mouse F4/80 antibody were obtained from Biolegend Inc. FITC rat anti-mouse CD8 was purchased from BD Biosciences (Franklin Lakes, NJ, USA).
4
Culturing Mouse Neural Stem Cells
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NSCs were harvested from embryonic C57BL/6J mouse brain (E14) as previously described [14 (link)]. Briefly, cells were resuspended in a DMEM/F12 medium containing B27 (2%, Gibco, USA), EGF (20 ng/ml, BioLegend, USA), bFGF (20 ng/ml, BioLegend, USA), and ITSS (10 μg/ml, Roche, Switzerland) and cultured in a cell incubator. The medium was changed every 3 d, and cells were passaged when the neurospheres grew to 50-100 μm diameter. Cells were plated on poly-D-lysine and laminin precoated coverslips for further experiments.
5
Murine Breast Cancer Cells and Myoepithelial Cell Culture
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Sorted myoepithelial cells were centrifuged and resuspended in 1:20 Matrigel (cat. 354234; Corning) and cultured in advanced-DMEM/F12 (cat. 12634010; Life Technologies) supplemented with 10 ng/ml EGF (cat. 585506; Biolegend), 20 ng/ml bFGF (cat. 710304; Biolegend), 4 μg/ml heparin (cat. H3149-10KU; Sigma-Aldrich), 5% newborn calf serum (cat. SH3011803; HyClone), and 5 μM Y-27632.
AT-3 cells, a murine breast cancer cell line derived from MMTV-PyMT tumors in the C57Bl/6 background, were cultured at 7% CO2 in DMEM high glucose (cat. MT-10-013-CV; Corning) supplemented with 10% FBS premium-select, penicillin–streptomycin (cat. MT30002CI; Corning), 15 mM HEPES (cat. 15630080; Life Technologies), 2 mMl -glutamine (cat. SH3003401; HyClone), NEAA (cat. SH3023801; HyClone), 1 mM sodium pyruvate (cat. 13-115E; Lonza Walkersville), and 1:250,000 2-mercaptoethanol (cat. M6250-100ML; Sigma Aldrich).
AT-3 cells, a murine breast cancer cell line derived from MMTV-PyMT tumors in the C57Bl/6 background, were cultured at 7% CO2 in DMEM high glucose (cat. MT-10-013-CV; Corning) supplemented with 10% FBS premium-select, penicillin–streptomycin (cat. MT30002CI; Corning), 15 mM HEPES (cat. 15630080; Life Technologies), 2 mM
6
Assessing EGFR and IAP Signaling
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LCL161 and birinapant were purchased from ApexBio. The following antibodies were used; EGFR (PA1-1110—Thermo fisher), XIAP (MAB822—R&D Systems), cIAP1 (was a kind gift from John Silke—WEHI Melbourne Australia), LC3b (nb100–2220—Novus), LAMP2 (ab13524—Abcam). EGF was purchased from Biolegend (catalogue No. 713108). Alexafluor 647 Dextran MW10,000 (catalogue No. D-22914), Lysotracker Red DND-99 (catalogue No. L7528) and Mitotracker Green (catalogue No. M7514) were purchased from Thermo Fisher Scientific. Concanamycin A (catalogue No. Cay-11050-25) was purchased from Cayman Chemical.
7
Colonic Organoid Culture and Minocycline Treatment
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The University of Florida Institutional Review Board approved the protocol for this human study (no. 201903360). We received the consent of 16 subjects with high systolic blood pressure (SBP) (156 ± 4 mmHg, high blood pressure, HBP) and 22 subjects with normal BP (110 ± 3 mmHg, normal blood pressure, RBP) for the study. Details of clinical characteristics of these subjects were described in our previous publication [3 (link)]. Within 30 min of the collection of biopsies by colonoscopy, human colonic crypts were isolated from the biopsies (~2 × 4 mm) of colon descending aspects in these subjects with gentle cell dissociation reagents (STEMCELL Technologies) including 2 mM EDTA for 90 min at 4 °C. The crypts were grown and maintained as 3D-spheroid cultures in Matrigel (BD Biosciences) containing organoid growth medium (STEMCELL Technologies) with recombinant human Noggin [Pepro-Tech] and EGF [BioLegend], recombinant human IGF-1, FGF-basic [FGF-2; BioLegend] and R-spondin1 [R&D], Y-27632 [STEMCELL Technologies], and A83-01 [Tocris], as described previously [3 (link)]. The human colonic organoids were cultured for 8 days, then treated with 1 mM minocycline for 24 h in the following experiments.
8
Spheroid Formation Assay for Drug Evaluation
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1.5 × 103 cells pre-treated with DMSO or IACS alone or in combination with cisplatin (cisplatin, 6 μM; IACS—010,759, 1 μM) for 3 h were plated in 24-well low attachment plates (Corning, 3473) in stem cell media [DMEM-F12 (Corning, #10-017-CV) with 100 U Penicillin–Streptomycin, 0.4% BSA, 10 ng/ml bFGF (Invitrogen, #13256-029), 20 ng/ml EGF (Biolegend, 585,506), 5 μg Insulin (Sigma, 19278)]. The plates were incubated for 14 days at 37 °C, 5% CO2. Fresh stem cell media was supplemented every 3 days and the spheroids were imaged at 14 days. The percent area of spheroids in each well was calculated using ImageJ for each biological replicate and the graph was plotted using all the three replicates.
9
Spheroid Formation Assay in Matrigel
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5 × 103 cells were mixed with Matrigel (Corning, Growth Factor Reduced, # 354230) and then platted in 24-well plates (Costar, #3738). The plate was incubated in a tissue culture incubator for 10 minutes to allow Matrigel to solidify followed by the addition of stem cell media [DMEM-F12 (Corning, #10-017-CV) with 100 U Penicillin–Streptomycin, 0.4% BSA, 10 ng/ml bFGF (Invitrogen, #13256-029), 20 ng/ml EGF (Biolegend, #585,506), 5 μg Insulin (Sigma, #19278)]. The plates were incubated for 20 days at 37°C, 5% CO2. Spheroids were manually quantified and the periphery of Matrigel were not included during quantification to reduce edge-effect bias. Images were taken using an EVOS FL Auto microscope (Life Technologies).
10
Isolation and Culture of Colonic Organoids
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Primary colonic crypts were isolated from descending aspects of the colons of REF and HBP subjects with gentle cell dissociation reagents (STEMCELL Technologies) including 2 mM EDTA for 90 min. They were grown and maintained as 3D-spheroid cultures in Matrigel (BD Biosciences) containing organoid growth medium (STEMCELL Technologies) with recombinant human Noggin [PeproTech] and EGF [BioLegend], recombinant human IGF-1, FGF-basic [FGF-2; BioLegend] and R-spondin1 [R&D], Y-27632 [STEMCELL Technologies], and A83-01 [Tocris], as described previously [17 (link),20 (link),55 (link)].
Colonic organoids were cultured for 8 days, then treated with 3.0 mM butyrate (Sigma-Aldrich, St. Louis, MO, USA) for 24 h in the following experiments. Selection of the butyrate dose was based on our published data [17 (link)], demonstrating that a 3.0 mM dose was optimal for gene expression without affecting organoid growth [20 (link)]. This is consistent with doses of butyrate in colonic epithelial cells (2 mM) in regulation of the assembly of tight junctions [56 (link)], in Caco-2 cells (10 mM) for apoptosis [57 (link)] and its levels in the gut, portal, hepatic and venous blood [58 (link)].
Colonic organoids were cultured for 8 days, then treated with 3.0 mM butyrate (Sigma-Aldrich, St. Louis, MO, USA) for 24 h in the following experiments. Selection of the butyrate dose was based on our published data [17 (link)], demonstrating that a 3.0 mM dose was optimal for gene expression without affecting organoid growth [20 (link)]. This is consistent with doses of butyrate in colonic epithelial cells (2 mM) in regulation of the assembly of tight junctions [56 (link)], in Caco-2 cells (10 mM) for apoptosis [57 (link)] and its levels in the gut, portal, hepatic and venous blood [58 (link)].
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