iMSCs were derived from the 1231A3 strain of iPSCs (reprogrammed using episomal vectors, kindly provided from Yamanaka laboratory). The iMSCs were induced using a previously described method.10 (link) Briefly, iPSCs were cultured on an iMatrix-511 (Nippi, Tokyo, Japan)-coated cell culture dish containing StemFit AK03N (Ajinomoto, Tokyo, Japan) for 4 d and then cultured in StemFit Basic03 (equivalent to AK03N without basic fibroblast growth factor, Ajinomoto) containing 10 μM SB431542 (Fujifilm Wako, Osaka, Japan) and 1 μM CHIR99021 (Axon Medchem, Reston, VA, USA) for 10 d to induce neural crest cell (NCC) differentiation. NCCs were selectively harvested using a cell sorter and CD271 as a marker, and the sorted cells were seeded into human fibronectin (Thermo Fisher Scientific, Waltham, MA, USA)-coated dish containing Basic03 supplemented with 10 μM SB431542, 20 ng/mL epidermal growth factor (Fujifilm Wako), and fibroblast growth factor 2 (Fujifilm Wako) and expanded. Subsequently, differentiation into iMSCs was induced using PRIME-XV MSC Expansion XSFM medium (Fujifilm Wako). BM-MSCs were purchased from Lonza (Lot: 19TL168853, 19TL191055; Basel, Switzerland).
Sb431542
Manufactured by Fujifilm
Sourced in Japan, United States
SB431542 is a small molecule inhibitor that selectively blocks the transforming growth factor-beta (TGF-β) signaling pathway. It functions by inhibiting the TGF-β type I receptor kinases ALK4, ALK5, and ALK7. This product is intended for laboratory research use.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Axon Medchem (10 mentions)
Y 27632, by Fujifilm (9 mentions)
Chir99021, by Fujifilm (8 mentions)
8 br camp, by Biolog (6 mentions)
Dexamethasone (dex), by Merck Group (6 mentions)
Ldn193189, by Fujifilm (6 mentions)
Heparin, by Nacalai Tesque (5 mentions)
Y 27632, by LC Laboratories (5 mentions)
Pneumacult ali, by STEMCELL (5 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
Pd0325901, by Fujifilm (4 mentions)
3 isobutyl 1 methylxanthine ibmx, by Fujifilm (4 mentions)
Epidermal growth factor (egf), by Fujifilm (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
N2 supplement, by Thermo Fisher Scientific (4 mentions)
Neurobasal medium, by Thermo Fisher Scientific (4 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Y 27632, by Merck Group (3 mentions)
Hydrocortisone, by Merck Group (3 mentions)
58 protocols using sb431542
1
Derivation of iMSCs from iPSCs
2
Generation of Induced Neural Crest Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human iPSCs 1231A3 reprogrammed with episomal vectors (kindly provided by Yamanaka Laboratory) were maintained as described previously (Nakagawa et al., 2014 (link)). For NCC induction, 3 × 104 cells/well were seeded in 6-well plates coated with iMatrix-511 (Nippi, Tokyo, Japan) in StemFit AK03N medium (Ajinomoto, Tokyo, Japan). After 4 days, the medium was replaced with NCC induction medium containing 10 μM SB431542 (FUJIFILM Wako Pure Chemical Corporation, Japan) and 1 μM CHIR99021 (Axon Medchem, Reston, VA, United States), as described previously (Kamiya et al., 2022 (link)). On day 10, CD271high positive cells were sorted and replated on fibronectin-coated plates at a density of 1 × 104 cells/cm2 in NCC expansion medium: Basic03 medium supplemented with 10 μM SB431542, 20 ng/ml EGF (FUJIFILM Wako Pure Chemical Corp.), and 20 ng/ml FGF2 (FUJIFILM Wako Pure Chemical Corp.). The medium was changed every 2–3 days. For cell passage, the cells were dissociated with Accutase (Innovative Cell Technologies, San Diego, CA, United States). iNCC stocks were prepared using STEM-CELLBANKER GMP grade (Takara Bio Inc., Kusatsu, Japan).
3
Immunocytochemistry of ENPP2 in hTM Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunocytochemistry was performed as previously described [11 (link), 18 (link)]. Cells were grown in chamber slides. hTM cells were fixed in ice-cold 4% paraformaldehyde at 24 h after application of TGF-β2 or SB431542 (Fujifilm, Osaka, JAPAN). The primary antibody was anti-ENPP2 antibody [5H3] (1:1,000; Abcam, Cambridge, MA, USA). The corresponding Alexa Fluor 488 secondary antibody (1:1,000) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Images were acquired using a BX51 fluorescence microscope (Olympus, Tokyo, Japan).
4
Investigating TGF-β1 Signaling Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HFF-1 cells were seeded in 6-well plates with DMEM at 200,000 cells/well. Cells were treated with control (DMEM with 2% FBS-added vehicle) or 4 ng/mL TGF-β1 (R&D Systems) with or without each inhibitor, and the lysates were collected after 48 h for quantification by WB. The following inhibitors were used: LY364947 (123-05981, Fujifilm), SB431542 (dispensed from a Tocriscreen Kinase Inhibitor Toolbox [3514, Tocris Bioscience]), and T-5224 (S28966, Selleck).
5
Immunocytochemistry for Cell Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunocytochemistry was performed as previously described55 (link). The primary antibodies were Anti-Cytomegalovirus Antibody, clone 8B1.2 (1:2,000; Merck Millipore, Billerica, MA, USA), Anti-ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase family member 2) antibody [5H3] (1:1,000; Abcam, Cambridge, MA, USA), Anti-TGF-β1 antibody (1:100; Sigma-Aldrich), anti-αSMA (1:500; Sigma-Aldrich) and anti-fibronectin [IST-9] (1:400; Abcam). Alexa Fluor 488 and 594 secondary antibodies (1:1,000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). To assess the characteristic changes in hTM and SCE cells after exposure to the conditioned medium, we performed immunocytochemistry using rhodamine phalloidin (7:1,000, Thermo Fisher Scientific) and ZO-1 (1:100; Abcam), followed by Alexa Fluor 488 and 594 secondary antibodies (1:1000; Thermo Fisher Scientific). We further explored whether the changes induced by the conditioned medium could be suppressed by ROCK inhibitors Y27632 (Merck, Kenilworth, NJ, USA) and K115 (KOWA, Nagoya, Japan), as well as Ki16425 (Merck) and SB431542 (Fujifilm, Osaka, JAPAN).
6
Comparative Study of Human Uterine Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SKN, which is a human uLMS cell line, and MES-SA, which is human uterine sarcoma cell line, were used in this study. SKN was purchased from Health Science Research Resources Bank (HSRRB, Osaka, Japan), and MES-SA was purchased from American type culture collection (ATCC, Virginia, USA). SKN cells were cultured in Ham's F 12 (Sigma-Aldrich Japan K.K., Tokyo, Japan), and MES-SA cells were cultured in McCoy's 5a medium (ATCC). Both media were supplemented with 10% heat-incubated fetal bovine serum (FBS). The cells were seeded at a density of 5×104 cells/well in a six-well plate, and incubated at 37°C in a humidified 5% CO2 incubator for 5 days. The cells were trypsinized and counted by a cell counter (Vi-CELL XR; Beckman Coulter, Tokyo, Japan) at each time point, as reported previously (36 (link)).
For TGF-β treatment, cells were cultured in Ham's F12 and McCoy's 5a medium supplemented with 10% FBS containing 100 or 500 pg/ml TGF-β1 (Sigma-Aldrich Japan K.K.) for 24 h. In order to block TGF-β signaling, SB431542 (WAKO, Tokyo, Japan), which is a TGF-β type I receptor-selective blocker, was dissolved at a concentration of 10 µM in dimethylsulfoxide (DMSO). Cells were seeded at a density of 5×104 cells/well in a six-well plate and cultured for 24 h and then cultured with new medium supplemented with 10 µM SB431542 for more 48 h.
For TGF-β treatment, cells were cultured in Ham's F12 and McCoy's 5a medium supplemented with 10% FBS containing 100 or 500 pg/ml TGF-β1 (Sigma-Aldrich Japan K.K.) for 24 h. In order to block TGF-β signaling, SB431542 (WAKO, Tokyo, Japan), which is a TGF-β type I receptor-selective blocker, was dissolved at a concentration of 10 µM in dimethylsulfoxide (DMSO). Cells were seeded at a density of 5×104 cells/well in a six-well plate and cultured for 24 h and then cultured with new medium supplemented with 10 µM SB431542 for more 48 h.
7
Differentiation of Airway and Alveolar Epithelial Cells from iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The air–liquid interface culture of airway and alveolar epithelial cells (Fig. 4G ) was differentiated from human iPSC-derived lung progenitor cells as previously described1 (link)–4 (link),21 (link),62 (link),65 (link),66 (link). Briefly, alveolar progenitor cells were induced stepwise from human iPSCs according to a 21-day and four-step protocol65 (link). At day 21, alveolar progenitor cells were isolated with the specific surface antigen carboxypeptidase M and seeded onto the upper chamber of a 24-well Cell Culture Insert (Falcon, #353104), followed by 28-day and 7-day differentiation of airway and alveolar epithelial cells, respectively. Alveolar differentiation medium with dexamethasone (Sigma-Aldrich, Cat# D4902), KGF (PeproTech, Cat# 100-19), 8-Br-cAMP (Biolog, Cat# B007), 3-isobutyl 1-methylxanthine (IBMX) (FUJIFILM WAKO, Cat# 095-03413), CHIR99021 (Axon Medchem, Cat# 1386), and SB431542 (Fujifilm Wako, Cat# 198-16543) was used for the induction of alveolar epithelial cells. PneumaCult ALI (STEMCELL Technologies, Cat# ST-05001) with heparin (Nacalai Tesque, Cat# 17513-96), Y-27632 (LC Laboratories, Cat# Y-5301), and hydrocortisone (Sigma-Aldrich, Cat# H0135) was used for induction of airway epithelial cells.
8
Establishment and Characterization of Trophoblast Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TSmole cells were established as described previously (14 (link)). Briefly, CT cells were isolated from CHM tissues and cultured on plates coated with 5–10 µg/mL Col IV (Corning) using TS medium (DMEM/F12 [Wako] supplemented with 0.1 mM 2-mercaptoethanol [Wako], 0.2% FBS [Thermo Fisher Scientific], 0.5% Penicillin-Streptomycin [Thermo Fisher Scientific], 0.3% BSA [Wako], 1% ITS-X supplement [Wako], 1.5 μg/mL l -ascorbic acid [Wako], 50 ng/mL EGF [Wako], 2 μM CHIR99021 [Wako], 0.5 μM A83-01 [Wako], 1 μM SB431542 [Wako], 0.8 mM VPA [Wako], and 5 μM Y27632 [Wako]). TSbip #1, #2, and #3 were established in our previous study (14 (link)) and correspond to TSCT #1, #2, and #3, respectively. TSbip #4 was established in this study and used only for CNV analysis. Unless otherwise noted, we used TSmole and TSbip cells passaged 10–20 times for the analysis.
9
Investigating Molecular Mechanisms in Cellular Processes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chlorpyrifos (CPF), Y-27632, SB431542, and LDN193189 were obtained from Wako (Tokyo, Japan). Penicillin-streptomycin mixture (PS) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). U0126 was obtained from Enzo Life Sciences (Farmingdale, NY, USA). Poly-L-ornithine, 2-mercaptoethanol (2-ME), and carbonylcyanide m-chlorophenylhydrazone (CCCP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade and obtained from commercial sources.
10
Anti-tumor Effects of Nanaomycin K
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed cell proliferation assays using KK47 and T24 in the presence of nanaomycin K to determine anti-tumor bioactivity in vitro. Two thousand KK47 and T24 cells were seeded for 24 h and then divided into 3 groups and switched to media10 (link) with or without 5 ng/mL TGF-β (FUJIFILM Wako Pure Chemicals, Osaka, Japan), or 5 ng/mL TGF-β11 (link) and 36 ng/mL SB-431542 (FUJIFILM Wako Chemicals), a TGF-β receptor inhibitor. After switching media, two different concentrations of nanaomycin K (5 µg/mL, 50 µg/mL) or DMSO (0.05%, 0.5%) were added to the cultures. After incubation for 0, 24, 48, 72 and 96 h, cell proliferation was measured by using 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega Corporation, Madison, WI) according to the manufacturer’s instructions. All experiments were carried out in triplicate.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!