Picropodophyllin ppp

Manufactured by Bio-Techne

Sourced in United Kingdom, United States

Picropodophyllin (PPP) is a chemical compound used in laboratory research. It functions as a selective inhibitor of the insulin-like growth factor 1 receptor (IGF-1R). This receptor is involved in various cellular processes, including cell growth and survival. PPP can be utilized in experimental settings to study the role of IGF-1R in biological systems.

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5 protocols using picropodophyllin ppp

Osteogenic Differentiation Assay

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First, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), calcein, alizarin red, gelatin, donkey serum, 4′6-diamidino-2-phenylindole (DAPI), and β-glycerophosphate (GP) were purchased from Sigma (St. Louis, MO, USA). Minimum essential medium alpha modification (α-MEM), fetal bovine serum (FBS), and antibiotic mixtures were obtained from WelGENE (Gyeongsan, Gyeongsangbuk-do, Republic of Korea). Antibodies against GSK-3β (sc-81462), phospho-GSK-3β at Ser9 (sc-37800), RUNX2 (sc-101145) and peroxidase-labeled anti-mouse immunoglobulins (sc-516102) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-labeled anti-rabbit immunoglobulins (KO211708) were obtained from KOMA BIOTECH (Seoul, Republic of Korea). Picropodophyllin (PPP) and DOI hydrochloride (DOI) were obtained from Tocris (Bristol, UK). Alexa Fluor^® 488 goat anti-rabbit secondary antibody was purchased from Abcam (Cambridge, UK). Dako faramount aqueous mounting medium was purchased from Dako (Carpinteria, CA, USA). Commercial FO (product name: FO100) was kindly supplied by Marine Bioprocess Co. (Busan, Republic of Korea). FO contains approximately 60% protein, 30% carbohydrate, and 3% lipid, as previously described [17 (link)]. All other chemicals were purchased from Sigma.

Molagoda I.M., Jayasingha J.A., Choi Y.H., Park E.K., Jeon Y.J., Lee B.J, & Kim G.Y. (2020). Fermented Oyster Extract Promotes Insulin-Like Growth Factor-1-Mediated Osteogenesis and Growth Rate. Marine Drugs, 18(9), 472.

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Isolation and Culture of Rat DRG Explants

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Embryonic rat dorsal root ganglia (DRGs) were isolated as previously described by Greaves et al. (34 (link)). Whole-ganglion explants were plated onto poly-D-lysine- and Matrigel-coated wells (BD Biosciences) in 48-well plates and incubated in DRG medium (high-glucose DMEM supplemented with 0.01% penicillin-streptomycin, 10% fetal calf serum). Positive controls were supplemented with nerve growth factor (NGF) (Bio-Rad, Hercules, CA, USA) plus recombinant IGF-1 (R&D Systems, Minneapolis, MN, USA) (2–200 ng/ml) ± 125–500 nM Picropodophyllin (PPP; Tocris Bioscience, Bristol, United Kingdom). Some DRGs were incubated in PF or macrophage-conditioned medium diluted 1:1 in DRG medium ± 500 nM PPP. Images of explants were captured using an Axiovert microscope (Carl Zeiss, Oberkochen, Germany), an Axiovision camera, and software.

Forster R., Sarginson A., Velichkova A., Hogg C., Dorning A., Horne A.W., Saunders P.T, & Greaves E. (2019). Macrophage-derived insulin-like growth factor-1 is a key neurotrophic and nerve-sensitizing factor in pain associated with endometriosis. The FASEB Journal, 33(10), 11210-11222.

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Primary Myoblast Isolation and Culture

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Adult primary myoblasts were obtained from hind limb muscles of 10- to 20-week-old C57BL/6J mice. Briefly, dissected muscle was digested with collagenase type I (Worthington, USA) for approximately 1 h. A single cell suspension was obtained after filtering the samples through a 70 μm mesh (Falcon). Cells were plated on collagen-coated dishes and cultured for the indicated times in F12-C supplemented with 15% horse serum and 1 nM FGF-2, at 37°C and 5% CO₂ (Olguin and Olwin, 2004 (link); Olguin et al., 2007 (link)). Differentiation was induced by incubating the cultures in F12-C supplemented with 2% horse serum. When required, the inhibitor picropodophyllin (PPP) (Tocris Bioscience, USA; 1 µM) was added for 1 h before treatment with Ang-(1-7).

Morales M.G., Abrigo J., Acuña M.J., Santos R.A., Bader M., Brandan E., Simon F., Olguin H., Cabrera D, & Cabello-Verrugio C. (2016). Angiotensin-(1-7) attenuates disuse skeletal muscle atrophy in mice via its receptor, Mas. Disease Models & Mechanisms, 9(4), 441-449.

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Effects of Arecoline, NAC, and PPP on Cellular Markers

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Arecoline was purchased from Sigma–Aldrich (St. Louis, MO, USA). N-acetyl cysteine (NAC) and (picropodophyllin) PPP were purchased from Tocris Bioscience (Bristol, UK). Monoclonal mouse anti-human antibodies against α-SMA (1A4), vimentin (9E7E7), and ZEB1 (416A7H10) and rabbit polyclonal anti-human antibody against COL1A1 (H-197) and IGF-1Rβ (C-20) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Rabbit polyclonal anti-human antibodies against p-IGF-1R^Tyr1161 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was purchased from GeneTex Inc. (Hsinchu City, Taiwan). A collagen solution from bovine skin was purchased from Sigma–Aldrich. Horseradish peroxidase–conjugated antimouse IgG or anti-rabbit IgG antibodies were purchased from PerkinElmer (Waltham, MA, USA). Alexa 488–conjugated antimouse IgG and Alexa 594–conjugated anti-rabbit IgG antibodies were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA).

Chang Y.C., Tsai C.H., Lai Y.L., Yu C.C., Chi W.Y., Li J.J, & Chang W.W. (2014). Arecoline-induced myofibroblast transdifferentiation from human buccal mucosal fibroblasts is mediated by ZEB1. Journal of Cellular and Molecular Medicine, 18(4), 698-708.

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Inhibitor Effects on Chromaffin and Neuroblast Cells

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Inhibitors were dissolved in Dimethylsufoxide (DMSO) at 2–5 mM, diluted to appropriate final concentrations, and added on day one of the culture period. The inhibitors that were used were the BET inhibitors JQ1 (Tocris Biotechne, Wiesbaden, Germany; 4499) and GSK1324726A (iBET 726) (Selleckchem Biozol, Eching, Germany), the CDK-7 inhibitors THZ1 (Medchem Express Biotrend, Köln, Germany) and YKL-5-125 (Selleckchem), the CDK12/13 inhibitor THZ 531 (Selleckchem), the IGF1-R inhibitor picropodophyllin (PPP) (Tocris 2956), the EZH2 inhibitor EPZ 6438 (Axon Medchem, Groningen, NL; 2227), the WNT inhibitor ICG001 (Axon Medchem 1766), and the ALK inhibitor Alectinib (Selleckchem S276).
Cell proliferation was analyzed for chromaffin cells and NESTIN-expressing cells after 6 days in culture and for neuroblasts after 3 days in culture by 4 h 5-ethynyl-2-deoxyuridine (EdU) labeling using the Click-iT EdU Alexa Fluor 594 imaging kit (Invitrogen Thermofisher, Dreieich, Germany) combined with staining for PHOX2B and NESTIN. The effects of the BET and CDK inhibitors on the chromaffin cells were also analyzed in the neuroblast-medium after a 3d culture period.

Sriha J., Louis-Brennetot C., Pierre-Eugène C., Baulande S., Raynal V., Kramdi A., Adameyko I., Ernsberger U., Deller T., Delattre O., Janoueix-Lerosey I, & Rohrer H. (2022). BET and CDK Inhibition Reveal Differences in the Proliferation Control of Sympathetic Ganglion Neuroblasts and Adrenal Chromaffin Cells. Cancers, 14(11), 2755.

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