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R spondin3

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R-spondin3 is a recombinant human protein that is a member of the R-spondin family of secreted proteins. R-spondin3 functions as an agonist of the Wnt/β-catenin signaling pathway.

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Lab products found in correlation

Noggin, by Thermo Fisher Scientific (3 mentions) Sb202190, by Merck Group (3 mentions) A83 01, by Bio-Techne (3 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions) N acetylcysteine, by Merck Group (3 mentions) Dmem f12, by Thermo Fisher Scientific (3 mentions) Mouse anti β actin, by Proteintech (2 mentions) Wnt3a, by R&D Systems (2 mentions) Gentamycin, by Euroclone (2 mentions) Heregulin, by Thermo Fisher Scientific (2 mentions) Y 27632, by AbMole (2 mentions) Amphotericin b, by Euroclone (2 mentions) Pennicilin streptomycin, by Euroclone (2 mentions) Hepes, by Merck Group (2 mentions) L glutamine, by Euroclone (2 mentions) Fgf10, by Thermo Fisher Scientific (2 mentions) Nicotinamide, by Merck Group (2 mentions) B27 supplement, by Thermo Fisher Scientific (2 mentions) Advanced dmem f12, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)

6 protocols using r spondin3

1

PDO Establishment from Surgical Specimens

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The study for in vitro/ex vivo MM model preparation was previously approved by the Ethics Committee of the IRCCS Istituto Tumori Giovanni Paolo II (Prot. 737/CE) and written informed consent was obtained from all the patients enrolled in the study. PDOs were obtained from the surgical specimen of a RES enrolled in our Institutional Ethical Committee‐approved protocol (Prot. 737/CE) and conducted in accordance with the international standards of good clinical practice. Informed consent was signed by the patient. The tumor tissue was processed as previously described [16 ] and cultured in Advanced DMEM F12 (GibcoTM, USA) supplemented with 10% (v/v) FBS (GibcoTM, USA), B27 (GibcoTM, USA), R-spondin3 (R&D System, USA), Glutamax (GibcoTM, USA), Noggin (PeproTech, USA) and antibiotics and maintained in ultra-low attachment tissue culture plates.
Serratì S., Di Fonte R., Porcelli L., De Summa S., De Risi I., Fucci L., Ruggieri E., Marvulli T.M., Strippoli S., Fasano R., Rafaschieri T., Guida G., Guida M, & Azzariti A. (2023). Circulating extracellular vesicles are monitoring biomarkers of anti-PD1 response and enhancer of tumor progression and immunosuppression in metastatic melanoma. Journal of Experimental & Clinical Cancer Research : CR, 42, 251.
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2

Investigating Molecular Pathways in Oncogenesis

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Rabbit anti-PCNA and CyclinD1 were purchased from Abcam (Cambridge, MA). Rabbit anti-HIF-1α, mouse anti-β-actin and GAPDH were obtained from Proteintech (Chicago, US). In situ cell death detection POD kit (Cat. No. 11684817910) were from Roche (Penzberg, Germany). RNATrip and BCA protein quantitative assay kit were purchase from Applied Gene (Beijing, China). Lgr4 shRNA, β-catenin shRNA and NC control vectors were obtained from Vigene Biosciences (Shandong, China). RIPA lysis buffer was from Beyotime Biotechnology (Shanghai, China). Neofect™ DNA transfection re-agent was purchased from Neofect (Beijing, China). DMOG was obtained from Cayman Chemical (Michigan, USA). R-spondin3 and Wnt3a were purchased from R&D Systems (Minneapolis, MN, USA).
Liu S., Yin Y., Yu R., Li Y, & Zhang W. (2018). R-spondin3-LGR4 signaling protects hepatocytes against DMOG-induced hypoxia/reoxygenation injury through activating β-catenin. Biochemical and biophysical research communications, 499(1), 59-65.
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3

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.
Mazzucchelli S., Piccotti F., Allevi R., Truffi M., Sorrentino L., Russo L., Agozzino M., Signati L., Bonizzi A., Villani L, & Corsi F. (2019). Establishment and Morphological Characterization of Patient-Derived Organoids from Breast Cancer. Biological Procedures Online, 21, 12.
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4

Lipid Metabolism Regulation Protocol

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Compound C, oleic acid (OA), palmitic acid (PA) and collagenase IV were purchased from Sigma Aldrich (St. Louis, MO, USA). AICAR was purchased from MCE (NJ, USA). R-spondin1 and R-spondin3 were purchased from R&D system (Minneapolis, MN). Rabbit anti-LGR4 was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-AMPKα, rabbit anti-pAMPKα (Thr 172) and rabbit anti-β-catenin were obtained from Cell Signaling Technology (Beverly, MA). Rabbit anti-SREBP2, Rabbit anti-AMPKα1, Rabbit anti-AMPKα2 were from Abcam (Cambridge, MA). Mouse anti-β-actin and mouse anti-laminB1 were purchased from Proteintech (Chicago, USA). RNAtrip was obtained from Applygen (Beijing, China). Reverse transcription (RT) system was from Invitrogen Inc. (Carlsbad, CA). Nuclear protein extraction kit was purchased from BestBio (Shanghai, China). Cholesterol assay kit and triglyceride assay kit were from Biosino Bio-technology & Science Inc. (Beijing, China). Neofect DNA transfection reagent was purchase from Neofect Biotech (Beijing, China).
Liu S., Gao Y., Zhang L., Yin Y, & Zhang W. (2020). Rspo1/Rspo3-LGR4 signaling inhibits hepatic cholesterol synthesis through AMPKα-SREBP2 pathway. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(11), 14946-14959.
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5

Colorectal Tumor Organoid Culture

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Colorectal tumor tissues were obtained from The Sixth Affiliated Hospital of Sun Yat‐sen University with informed consent and the study was approved by the ethical committee (SYSU‐IACUC‐2020‐000570). Fresh tumor tissues were dissociated into single‐cell suspensions with the Tumor Dissociation Kit (Miltenyi Biotec, 130‐095‐929). CRC organoids were cultured in CRC organoid medium.[57] The composition of CRC organoid medium was: 14 mL advanced DMEM/F12 medium (Life Technologies) with 1% GlutaMax I 100x (Life Technologies), 1% Hepes (Life Technologies), 1% Penicillin Streptomycin 100x (Thermofisher), 0.1% Primocin (Invivogen), 2% B27 supplement 50x (Life Technologies), 3.5 µg R‐Spondin 3 (R&D), 2.8 µg EGF (Peprotech), 2.95 µg A83‐01 (Tocris), 1.4 µg Wnt3A (R&D), 46.4 µg SB202190 (Sigma), 2.24 mg N‐acetylcysteine (Sigma), 0.168 mg nicotinamide (Sigma), and 1% N2 supplement 100x (Life Technologies).
Yu Z., Deng P., Chen Y., Liu S., Chen J., Yang Z., Chen J., Fan X., Wang P., Cai Z., Wang Y., Hu P., Lin D., Xiao R., Zou Y., Huang Y., Yu Q., Lan P., Tan J, & Wu X. (2021). Inhibition of the PLK1‐Coupled Cell Cycle Machinery Overcomes Resistance to Oxaliplatin in Colorectal Cancer. Advanced Science, 8(23), 2100759.
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6

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.
Liu Y., Zhou Y., Ma X, & Chen L. (2021). Inhibition Lysosomal Degradation of Clusterin by Protein Kinase D3 Promotes Triple‐Negative Breast Cancer Tumor Growth. Advanced Science, 8(4), 2003205.
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