To investigate whether the effects of EMD occur via regulation of the PI3K/Akt/VEGF pathway, the p-Akt level was evaluated via western blotting. EMD powder (30 mg/vial) was dissolved in 1 mL of acetate buffer (pH 5) and diluted to 100 μg/mL in D-MEM containing 10% FBS [27 (link)]. Cells were then incubated in D-MEM with 100 μg/mL EMD to analyze Akt phosphorylation and Vegf expression. In addition, the cells were treated with the Akt inhibitor wortmannin (Wako Pure Chemical Industries, Tokyo, Japan) at 100 nM 30 min before EMD treatment [28 (link)].
Wortmannin
Manufactured by Fujifilm
Sourced in Japan, United States
Wortmannin is a chemical compound produced by the fungus Penicillium wortmannii. It functions as a potent and selective inhibitor of phosphoinositide 3-kinase (PI3K), a key enzyme involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
3 methyladenine 3 ma, by Fujifilm (2 mentions)
Recombinant annexin 5, by BD (2 mentions)
Pma and lps e coli o55 b5, by Merck Group (2 mentions)
Pam3csk4 hydrochloride, by InvivoGen (2 mentions)
Recombinant human il 4 and il 13, by R&D Systems (2 mentions)
Endospecy es 50m set, by Seikagaku (2 mentions)
Tamoxifen, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Fujifilm (1 mentions)
Can get signal immunoreaction enhancer solution, by Toyobo (1 mentions)
Phenazine methosulfate pms, by Fujifilm (1 mentions)
Ribonuclease a rnase a, by Merck Group (1 mentions)
Fetal bovine serum fbs, by Nichirei Biosciences (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti collagen type 1, by Rockland Immunochemicals (1 mentions)
Anti total akt, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Pd98059, by Cayman Chemical (1 mentions)
α sma, by Agilent Technologies (1 mentions)
Anti total erk1, by Santa Cruz Biotechnology (1 mentions)
Fk506, by Merck Group (1 mentions)
11 protocols using wortmannin
1
Regulation of PI3K/Akt/VEGF by EMD
2
Sodium Arsenite and Tetrandrine Cytotoxicity Assay
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Sodium arsenite (NaAsO2, AsIII) (>99% purity) and tetrandrine (99.2% purity) were purchased from Tri Chemical Laboratories (Yamanashi, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively. Fetal bovine serum (FBS) was purchased from Nichirei Biosciences (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 medium, phenazine methosulfate (PMS), and dimethyl sulfoxide (DMSO) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Propidium iodide (PI), proteinase K, ribonuclease A (RNaseA), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)3.2-5-[(phenylamino)carbony]-2H-tetrazolium hydroxide (XTT), and tamoxifen were purchased from Sigma-Aldrich (St. Louis, MO, USA). MAPK inhibitors and their negative controls (JNK inhibitor SP600125 and its negative control SP600125NC; p38 MAPK inhibitor SB203580 and its negative control SB202474; ERK inhibitor PD98059) were purchased from Calbiochem (La Jolla, CA, USA). Autophagy inhibitors, 3-methyladenine (3-MA) and wortmannin, were purchased from Wako Pure Chemical Industries and Calbiochem, respectively. Can Get Signal® Immunoreaction Enhancer Solution was purchased from Toyobo Co., Ltd. (Osaka, Japan).
3
Endostatin Signaling Pathways in Wound Healing
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Reagent sources were as follows: Recombinant mouse endostatin (Sigma-Aldrich, St. Louis, MO, USA), PD98059 (Cayman, Ann Arbor, MI, USA), wortmannin (Wako, Osaka, Japan).
Antibodies sources were as follows: anti-phospho-Akt (Ser473), anti-total Akt, anti-phospho-ERK (Cell Signaling Technology, Beverly, MA, USA), anti-total-ERK1, anti-endostatin (Santa Cruz Biotech, Santa Cruz, CA, USA), anti-type I collagen (Rockland immunochemicals, Gilbertsville, PA, USA), anti-MMP-2 (Kyowa pharma chemical, Toyama, Japan), anti-total-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Wako), α-SMA (Dako, Glostrup, Denmark), anti-rabbit immunoglobulin G (IgG) horseradish peroxidase linked whole antibody, anti-mouse IgG horseradish peroxidase linked whole antibody (Amersham Bioscences, Buckinghamshire, UK).
Antibodies sources were as follows: anti-phospho-Akt (Ser473), anti-total Akt, anti-phospho-ERK (Cell Signaling Technology, Beverly, MA, USA), anti-total-ERK1, anti-endostatin (Santa Cruz Biotech, Santa Cruz, CA, USA), anti-type I collagen (Rockland immunochemicals, Gilbertsville, PA, USA), anti-MMP-2 (Kyowa pharma chemical, Toyama, Japan), anti-total-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Wako), α-SMA (Dako, Glostrup, Denmark), anti-rabbit immunoglobulin G (IgG) horseradish peroxidase linked whole antibody, anti-mouse IgG horseradish peroxidase linked whole antibody (Amersham Bioscences, Buckinghamshire, UK).
4
Phosphate Buffered PI Stimulation
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Stimulation by Pi was performed using 0.1 M Pi buffer, pH 7.4, containing Na2HPO4 and NaH2PO4. FGF2 was purchased from R&D Systems. U0126, wortmannin, and PD1703074 were purchased from Wako. FK506 was purchased from Sigma-Aldrich. Calcitriol [1,25(OH)2D3] was purchased from Carbosynth.
5
Insulin Signaling Pathway Investigation
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Insulin (human, recombinant), dexamethasone, 2-deoxyglucose (2-DG), and wortmannin were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The details of the other critical reagents are as follows: dorsomorphin (compound C) from Abcam (Cambridge, UK); oil red O from Cosmo Bio (Tokyo, Japan); primary antibodies against GLUT4 (rabbit, polyclonal, #NBP1-49533) from Novus Biologicals (Littleton, CO); and fluorescent antibody Alexa Fluor® 488 (donkey, polyclonal, #711-545-152) from Jackson ImmunoResearch (West Grove, PA). Details regarding reagents have been placed in the supplemental materials.
6
Preparation and Use of PI Stock Solutions
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All soluble analogues of PIs, such as PI(3,4)P2-diC8, were purchased from Echelon Biosciences and dissolved in water as a 0.2-mM concentration of stock solution. PI stock solutions were directly used for PI injection experiments and diluted into the bath solution for inside-out patch experiments. Insulin (Wako) was dissolved in 10 mM HCl solution and kept as a 1-mM stock solution. Thapsigargin (Sigma-Aldrich) and wortmannin (Wako) were dissolved in DMSO at 1 mM and 10 mM for stock solution, respectively.
7
Osteolineage Differentiation of hMSCs
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H89 (Cayman Chemical, Ann Arbor, MI, United States), the PKA inhibitor, and wortmannin (FUJIFILM Wako Pure Chemical Corp.), the PI3K inhibitor, were used. hMSCs were seeded at 10,000 cells/well on a 48-well Biocoat Collagen I plate and cultured in a growth medium for 3 days. Confluent hMSCs were cultured in the OM for 3 days and then AKDS001 (100 nM) and H89 (0, 5, 10, and 20 μM) or wortmannin (0, 20, 100, and 500 nM) were added to the medium. The medium was changed every 3–4 days. ALP activity was determined after 7 days of culture with AKDS001 or DMSO.
8
Evaluating Claspin's Role in Cell Cycle
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Three sets of 293T cells were transfected with siControl or siClaspin for 24 h in serum-free medium for 24 h. One set was harvested at 24 h after serum starvation. The remaining two sets were released into growth by addition of serum for 12 h or 24 h. Asynchronously growing 293T cells were also transfected with siControl or siClaspin for 24 h. Inhibitors or DMSO (control) were added during the last 12 h before the harvest. Inhibitors added and their concentrations were as follows: Wortmannin (FUJIFILM Wako Pure Chemical Corporation), 2 µM; PI-103 (Cayman Chemical Company), 2 µM; Flavopiridol (Selleck), 10 µM; UCN-01 (Cayman Chemical Company), 1 µM; VE-821(Sigma-Aldrich), 10 µM; GSK2334470 (Selleck), 5 µM; XL413(AdooQ Bioscience), 10 µM; Torkinib (Selleck), 5 µM; rapamycin (Medchemexpress), 2.5 µM and MIRIN (Sigma-Aldrich), 10 µM. Cells were observed under a microscope.
9
Synthesis and Validation of NK-4 Compound
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NK-4 was synthesized at Functional Dyes Unit, Hayashibara Co., Ltd. (Okayama Japan). A stock solution of 10 mM NK-4 was prepared with DMSO and stored frozen at − 80 °C. Endotoxin content in 100 μM NK-4 was below detection limits (0.00625 endotoxin units/ml), as determined according to a protocol listed in the Japanese Pharmacopoeia using an Endospecy ES-50 M set (Seikagaku Co., Tokyo, Japan). Final concentrations of DMSO at 0.05% or less did not affect the results of the experiments. PMA and LPS (E. coli O55:B5) were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Pam3CSK4 hydrochloride was purchased from InvivoGen (San Diego, CA). Poly(I:C) was purchased from Calbiochem (La Jolla, CA). Recombinant human IL-4 and IL-13 were purchased from R&D Systems (Minneapolis, MN). Recombinant annexin V was purchased from BD Biosciences (Franklin Lakes, NJ). Wortmannin was purchased from Wako Pure Chemical (Osaka, Japan). Human TNF-α, and monoclonal antibodies (mAb) for the human TNF-α ELISA were prepared and purified in our laboratories.
10
Cytotoxicity and Autophagy Induction by Hellebrigenin and Arenobufagin
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Hellebrigenin (Helle) (≥98% purity) and arenobufagin (Areno) (≥98% purity) were purchased from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). Fetal bovine serum (FBS) and HEPES were purchased from Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640 and DMEM medium, 3-methyladenine (3-MA) and wortmannin were obtained from Wako Pure Chemical Industries (Osaka, Japan). 25% glutaraldehyde solution were purchased from Kanto chemical CO., INC. (Tokyo, Japan). WST-1 and 1-Methoxy PMS were obtained from Dojindo Molecular Technologies, Inc. (Tokyo, Japan). Propidium iodide (PI), ribonuclease A (RNaseA), crystal violet (C.I. 42555) Certistain® were purchased from Merck KGaA (Sigma-Aldrich; Darmstadt, Germany).
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