Sciatic nerve explant culturing, which is an in vitro model of WD, was performed as described previously (Shin et al., 2013; Park et al., 2015; Li et al., 2021). Briefly, nine adult rats were anesthetized by intraperitoneal injection of 12 mg/mL tribromoethanol (180 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) and decapitated by guillotine. Sciatic nerve segments (0.5 cm in length) were isolated and incubated in the Dulbecco’s modified Eagle’s medium/F12 (Corning, New York, NY, USA) containing 3% fetal bovine serum (Corning), 3 mM forskolin (Sigma-Aldrich), 10 ng/mL heregulin (PeproTech, Rocky Hill, NJ, USA), and 100 mg/mL penicillin-streptomycin (Gibco, Grand Island, NY, USA). Paclitaxel (Sigma-Aldrich) or nocodazole (Sigma-Aldrich) was dissolved in anhydrous dimethyl sulfoxide (Sigma-Aldrich) to produce a 2 or 10 mg/mL stock solution, respectively, which was stored at –20°C until administration. Paclitaxel or nocodazole was added to the culture medium, and the nerve explants were collected after culturing for 5 or 8 days in vitro (div) for subsequent analysis. A vehicle-only control group was included.
Heregulin
Manufactured by Thermo Fisher Scientific
Sourced in United States
Heregulin is a recombinant protein that functions as a growth factor. It is used in cell culture applications to stimulate cell proliferation and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Forskolin, by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Corning (3 mentions)
Sb202190, by Merck Group (3 mentions)
N acetylcysteine, by Merck Group (3 mentions)
A83 01, by Bio-Techne (3 mentions)
Dulbecco s modified eagle s medium f12, by Corning (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Noggin, by Thermo Fisher Scientific (2 mentions)
Hepes, by Merck Group (2 mentions)
Fgf10, by Thermo Fisher Scientific (2 mentions)
R spondin3, by R&D Systems (2 mentions)
Gentamycin, by Euroclone (2 mentions)
Y 27632, by AbMole (2 mentions)
Amphotericin b, by Euroclone (2 mentions)
Pennicilin streptomycin, by Euroclone (2 mentions)
L glutamine, by Euroclone (2 mentions)
9 protocols using heregulin
1
Sciatic Nerve Explant Culturing for WD Study
2
Ascorbic Acid Effects on Nerve Explant Culture
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The in vitro cultures of nerve explants were performed according to previous publications (Shin et al., 2013; Park et al., 2015), with minor modifications. Briefly, after rats were anesthetized with tribromoethanol (180 mg/kg by intraperitoneal injection), the sciatic nerves were collected under aseptic conditions and cut into segments with lengths of 5 mm. Next, the nerve segments were explanted in a culture dish and incubated with basic medium, which consisted of Dulbecco’s modified Eagle’s medium/F12 (Corning, New York, NY, USA) containing 3% fetal bovine serum (Corning), 10 ng/mL heregulin (PeproTech, Rocky Hill, NJ, USA), 3 mM forskolin (Sigma-Aldrich), and 100 mg/mL penicillin-streptomycin (Gibco, Grand Island, NY, USA). The explant cultures were divided into the ascorbic acid group and the control group. In the ascorbic acid group, ascorbic acid was added to the basic culture medium with a final concentration of 200 μM. The same volume of solvent used for the ascorbic acid solution was added into the basic medium of the control group. After being cultured for 5 or 8 days in vitro (div), the explants were collected for immunohistochemistry, western blot assay, and transmission electron microscopy experiments.
3
Breast Cancer Organoid Culture Protocol
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The medium used for culturing the breast cancer organoid cultures contained the following ingredients: 50% conditioned medium from L-WRN cells (ATCC #CRL-3276) (Miyoshi and Stappenbeck, 2013 (link)) (containing Wnt3a, R-spondin 3, and Noggin), Heregulin 5 nmol/L (Peprotech, NJ, USA), fibroblast growth factor 7 (FGF7) 5 ng/ml (Peprotech), fibroblast growth factor 10 (FGF10) 20 ng/ml (Peprotech), epidermal growth factor (EGF) 5 ng/ml (Peprotech), A83-01 500 nmol/L (Tocris, Wiesbaden, Germany), Y27632 5 µmol/L (Hölzel), SB202190 (Sigma-Aldrich), Gibco B27 Supplement 2% (Thermo Fisher Scientific), N-acetyl-cysteine 1.25 mmol/L (Sigma-Aldrich), nicotinamide 5 mmol/L (Sigma-Aldrich), Primocin 50 µg/ml (InvivoGen, Toulouse, France), Gibco advDMEM/F12 50% (Thermo Fisher Scientific).
4
Optimized Stem Cell Culture Medium
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Hyclone DMEM-F/12 1:1 (Thermo Scientific #SH30023.01) supplemented with:
10 mM HEPES (Sigma-Aldrich; #H3885),
10 μg/mL Gentamycin (Euroclone #ECM0011B; 10 mg/mL),
2 mM L- Glutamine (Euroclone ECB3000D-20; 200 mM),
1% Pennicilin/streptomycin (Euroclone #ECB3001D, 100×),
2.5 μg/mL Amphotericin B (Euroclone #ECM00 09D; 250 μg/mL),
5 mM Nicotinammide (Sigma-Aldrich, #N0636, 250 mM).
1.25 mM N-acetylcysteine (Sigma-Aldrich, # A9165, 125 mM).
1× B27 supplement (Gibco, #17504–44, 50×).
250 ng/mL R-spondin 3 (R&D, #3500-RS/CF, 25 μg/mL)*.
5 nM Heregulin (Peprotech, #100–03, 7.14 μM)*.
5 ng/mL KGF (Peprotech, #100–19, 10 μg/mL)*.
20 ng/mL FGF10 (Peprotech, #100–26, 25 μg/mL)*.
5 ng/mL EGF (Peprotech, #AF-100-15, 5 μg/mL)*.
100 ng/mL Noggin (Peprotech, #120-10C, 20 μg/mL)*.
500 nM A83–01 (Tocris, #2939, 500 μM)*,**.
5 μM Y-27632 (Abmole,#M1817, 5 mM)*.
500 nM SB202190 (Sigma-Aldrich, #S7067, 500 μM)*.
* Add fresh the day of use.
** dissolve in DMSO.
10 mM HEPES (Sigma-Aldrich; #H3885),
10 μg/mL Gentamycin (Euroclone #ECM0011B; 10 mg/mL),
2 mM L- Glutamine (Euroclone ECB3000D-20; 200 mM),
1% Pennicilin/streptomycin (Euroclone #ECB3001D, 100×),
2.5 μg/mL Amphotericin B (Euroclone #ECM00 09D; 250 μg/mL),
5 mM Nicotinammide (Sigma-Aldrich, #N0636, 250 mM).
1.25 mM N-acetylcysteine (Sigma-Aldrich, # A9165, 125 mM).
1× B27 supplement (Gibco, #17504–44, 50×).
250 ng/mL R-spondin 3 (R&D, #3500-RS/CF, 25 μg/mL)*.
5 nM Heregulin (Peprotech, #100–03, 7.14 μM)*.
5 ng/mL KGF (Peprotech, #100–19, 10 μg/mL)*.
20 ng/mL FGF10 (Peprotech, #100–26, 25 μg/mL)*.
5 ng/mL EGF (Peprotech, #AF-100-15, 5 μg/mL)*.
100 ng/mL Noggin (Peprotech, #120-10C, 20 μg/mL)*.
500 nM A83–01 (Tocris, #2939, 500 μM)*,**.
5 μM Y-27632 (Abmole,#M1817, 5 mM)*.
500 nM SB202190 (Sigma-Aldrich, #S7067, 500 μM)*.
* Add fresh the day of use.
** dissolve in DMSO.
5
Neural Precursor Differentiation Protocol
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For induction, Neural precursors were seeded at a concentration of 5 × 103 cells/cm2 and were cultured with a standard medium supplemented with 35 ng/mL of all-trans-retinoic acid (Sigma-Aldrich®, St. Louis, MO, USA). After 72 h, the cells were washed with PBS (Sigma-Aldrich®, St. Louis, MO, USA), and the differentiation medium composed of DMEM/Ham-F12 (Sigma-Aldrich®, St. Louis, MO, USA), 5 ng/mL of PDGF-AA (Peprotech®, East Windsor, NJ, USA), 10 ng/mL of bFGF (Peprotech®, East Windsor, NJ, USA), 10µM of forskolin (Sigma-Aldrich®, St. Louis, MO, USA) and 25 ng/mL of heregulin (Peprotech®, East Windsor, USA) NJ, USA) was added [21 (link)].
6
Breast Cancer Cell Line Maintenance and Stimulation
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The human breast cancer cell lines MDA-MB-231 (ATCC® HTB-26™), BT549 (ATCC® HTB122™), HCC1937 (ATCC® CRL 2336™), MDA-MB-468 (ATCC ® HTB-132™), (American Type Culture Collection, Manassas, VA), SUM149, and SUM159 (Asterand Bioscience, Detroit, MI now acquired by BioreclamationIVT, Westbury, NY) were authenticated using a panel of microsatellite markers. Cell lines were maintained at 37 °C in a humidified atmosphere of 5% CO2 as previously described in Turdo et al. [3 (link)]. For stimulation experiments, MDA-MB-231 cells were starved in serum-free medium for 24 h and then treated for 48 h with a pool of 5 WHFs at a final concentration of 5% as described [23 (link)] or with PDGF-BB, Mib1b, MCP1, IP10, Il1ra, Il1b, G-CSF, Il8, Il6, EGF, FGF, Heregulin, PDGF-AA, PDGF-AB (PeproTech, Rocky Hill, NJ) at 50 ng/mL. Cells were treated in indicated experiments with cycloheximide (1 μM) or UO126 (2 μM), both of which were dissolved in DMSO (maximum concentration 0.1%) (Sigma-Aldrich).
7
Isolation and Culture of Rat Sciatic Nerve Schwann Cells
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As described in our previous publication, 22 SCs were isolated from the sciatic nerves of SD rats at postnatal day 3. The nerve tissues were harvested and dissociated with 0.25% of trypsin-EDTA (Gibco) at 37°C for 30 minutes, and single cells suspended in DMEM/F12 (Hyclone) containing 10% FBS (Hyclone) were plated onto Poly-L-lysine-coated
dishes. After overnight incubation, the cultures were treated with cytosine arabinoside (10 mM, Sigma-Aldrich, St. Louis, MO, USA) for 48 hours to eliminate fibroblasts. Then, the cells were routinely cultured with SC medium [DMEM/F12 containing 3% FBS, 3 mM forskolin (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL heregulin (PeproTech), and 100 mg/mL penicillin-streptomycin (Gibco)] to expand the cells. The maintenance medium was changed every 3 days.
For ANXA1 treatment, cells were fed with media containing rat recombinant ANXA1 (rrAnxA1, RPE787Ra01, Cloud-Clone, US), ranging from 0.1 to 100 m/M, and incubated at 37°C for various durations of time as previously described. 20
dishes. After overnight incubation, the cultures were treated with cytosine arabinoside (10 mM, Sigma-Aldrich, St. Louis, MO, USA) for 48 hours to eliminate fibroblasts. Then, the cells were routinely cultured with SC medium [DMEM/F12 containing 3% FBS, 3 mM forskolin (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/mL heregulin (PeproTech), and 100 mg/mL penicillin-streptomycin (Gibco)] to expand the cells. The maintenance medium was changed every 3 days.
For ANXA1 treatment, cells were fed with media containing rat recombinant ANXA1 (rrAnxA1, RPE787Ra01, Cloud-Clone, US), ranging from 0.1 to 100 m/M, and incubated at 37°C for various durations of time as previously described. 20
8
Isolation and Expansion of Rat Spinal Nerve Schwann Cells
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As described in our previous publication (Wen et al., 2017 (link)), SCs were isolated from the spinal nerves of SD rats at postnatal day 3. The collected nerves were dissociated with 0.25% trypsin-EDTA (Gibco) at 37°C for 30 min, and single cells suspended in DMEM/F12 (Corning) containing 10% FBS (Corning) were plated onto PLL-coated Petri dishes. After overnight incubation, the cultures were treated with cytosine arabinoside (10 μM, Sigma-Aldrich, St. Louis, MO, USA) for 48 h to eliminate fibroblasts. Then, the cells were routinely cultured with SC medium [DMEM/F12 containing 3% FBS, 3 μM forskolin (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/ml heregulin (PeproTech) and 100 mg/ml penicillin-streptomycin (Gibco)] to expand the cells.
9
TNBC Organoid Culture and Drug Treatment
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Briefly, a TNBC tissue was digested by Collagenase I, then cultured in DMEM‐F/12 (Gibco) supplemented with 10 mm HEPES (Sigma‐Aldrich), 10 µg mL−1 Gentamycin (Euroclone), 2 mm L‐Glutamine (Euroclone), 1% Pennicilin/streptomycin (Euroclone), 2.5 µg mL−1 Amphotericin B (Euroclone), 5 mm Nicotinamide (Sigma), 1.25 mm N‐acetylcysteine (Sigma), 1 × B27 supplement (Gibco), 250 ng mL−1 R‐spondin 3 (R&D), 5 nm Heregulin (Peprotech), 5 ng mL−1 KGF (Peprotech), 20 ng mL−1 FGF10 (Peprotech), 5 ng mL−1 EGF (Peprotech), 100 ng mL−1 Noggin (Peprotech), 500 nm A83–01 (Tocris), 5 µm Y‐27632 (Abmole), 500 nm SB202190 (Sigma). The TNBC organoids were treated with CRT0066101 (1 µm ) and/or OGX‐011 (300 nm ) for cell growth assay.
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