Human PC3 and DU145 cells were obtained from ATCC (Manassas, VA). Cells were maintained in DMEM high glucose medium (Hyclone, Logan, UT) with 10% FBS (Atlanta Biologicals, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified incubator at 37°C and 5% CO2, and routinely passaged when 80–90% confluent. Antibodies for pβ-catenin, β-catenin, E-cadherin, and N-cadherin were purchased from Cell Signaling (Danvers, MA). Anti-TGFβ-RII was purchased from Abcam (Cambridge, MA). Anti-β-actin was purchased from Sigma (St. Louis, MO). Compound inhibitors such as TCBN, ICG001, IWR-1, LY2109761, SB431542 and SB415286 were purchased from Selleckchem (Houston, TX).
Iwr 1
Manufactured by Selleck Chemicals
Sourced in United States
IWR-1 is a laboratory instrument designed for the analysis and detection of various compounds. It utilizes infrared spectroscopy technology to provide analytical data on the chemical composition of samples. The core function of IWR-1 is to accurately identify and quantify the presence of specific molecular structures within a given sample.
Automatically generated - may contain errors
Lab products found in correlation
Chir99021, by Selleck Chemicals (13 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Activin a, by Thermo Fisher Scientific (3 mentions)
Sb431542, by Selleck Chemicals (2 mentions)
Glis1, by Addgene (2 mentions)
Pmxs oct4, by Addgene (2 mentions)
Nanog, by Addgene (2 mentions)
Lin28a, by Addgene (2 mentions)
Matrigel, by Corning (2 mentions)
Rpmi b27, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Thermo Fisher Scientific (2 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (2 mentions)
Mtesr1 media, by STEMCELL (2 mentions)
B27 without insulin, by Thermo Fisher Scientific (2 mentions)
Rpmi b27 without d glucose, by Thermo Fisher Scientific (2 mentions)
Accutase, by Merck Group (2 mentions)
Y 27632, by Selleck Chemicals (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
27 protocols using iwr 1
1
Prostate Cancer Cell Line Maintenance
2
Generation of Polycistronic Reprogramming Vectors
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The DOT1L inhibitor EPZ004777 (iDOT1L) was purchased from AOBIOUS Inc. (Gloucester, MA, USA). WNT inhibitor IWR1 was purchased from Selleckchem (Houston, TX, USA). The constructs pMXs-OCT4, NANOG, LIN28A and GLIS1 were purchased from Addgene (Cambridge, MA, USA). Construction of the polycistronic vector pMXs-KLF4, MYC and SOX2 (KMS) was described in our previous study (Wang et al., 2017 (link)). To clone the pMXs-GNL or NL polycistronic vector, the coding sequences for human NANOG, LIN28A and GLIS1 were PCR-amplified from the above-mentioned Addgene constructs. The amplified DNA sequences for each gene were then inserted into linearized pMXs vectors (Cell Biolabs, San Diego, CA, USA) using an In-Fusion kit (Clontech Inc., Mountain View, CA, USA). 2A sequences (Carey et al., 2009 (link); Ryan and Drew, 1994 (link); Ryan et al., 1991 (link)) were inserted between each gene.
3
Cardiomyocyte Differentiation from Pluripotent Stem Cells
4
CCK-8 Assay for Cell Proliferation
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CCK-8 assay (DOJINDO) was performed to assess cell proliferation. CFP1-knockdown or control A549 cells (1 × 103 cells per well) were plated in 96 well plates for preincubation. Before each CCK8 test, the old medium was removed and 90 μL culture medium supplemented with 10 μL CCK-8 was added to each well, and plates were incubated in the incubator (37 °C, 5%CO2) for 2 h. The OD values were measured at 450 nm with a microplate reader. The same treatment was applied to CFP1-knockdown or control H1975 cells. In the WNT and TGF-β pathway inhibitors treatment assay, CFP1-overexpression or control H1975 cells were plated in 96 well plates (1 × 103 cells per well). SB431542 (50 μM, Selleck) and IWR-1 (50 μM, Selleck) were diluted in culture medium and added to plates. CCK8 assay was performed at 0, 24, 48, 72, and 96 h respectively. Differences in OD value were analyzed by Student’s t-test at different time points.
5
Cardiac Differentiation of Human ESCs
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Human ESC lines (H1) were maintained on Matrigel-coated plates (BD Biosciences) in Essential 8 Medium (STEMCELL Technologies) and ROCK inhibitor (Selleck). The medium was changed every day. For cardiac differentiation, human ESC lines (H1) at ~ 100% confluence were incubated with a differentiation medium comprising RPMI 1640 medium (Gibco) and B27 supplement minus insulin (Invitrogen). On day 0, CHIR99021 (Selleck), a selective glycogen synthase kinase 3β inhibitor, was added to the differentiation medium (3 μM final). On day 3, the Wnt antagonist, IWR-1 (Selleck), was added to the differentiation medium (5 μM final). On day 5, the medium was removed and replaced with a differentiation basal medium without any inhibitors. On day 8, the cells were incubated with a medium consisting of RPMI 1640 medium and B27 supplement plus insulin (Invitrogen). The medium was changed every 3 days for a desired time of culture.
6
Efficient Endodermal Differentiation of H9 Cells
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H9 cells were cultured on gelatin-coated plates with a feeder layer of MEF for 5 days, then the cells were subjected for endodermal differentiation. At the first day, differentiation medium was X-VIVO 20 medium (Lonza) supplemented with 100 ng/ml Activin A (Peprotech), 50 ng/ml WNT3A (Peprotech) or 3 μM CHIR99021(Selleck), 10 μM ROCK inhibitor Y-27632 (Selleck). The following day, the medium was changed to X-VIVO 20 (Lonza) medium containing 100 ng/ml Activin A, 0.5% B27 (Gibco). At day 4 after the differentiation, endodermal cells were dissociated with TrypLE (Gibco), and reseeded on Matrigel (Corning)-coated plates with DMEM-F12 medium (Gibco)or EGM2 medium (Lonza), supplemented with 0.5% B27, 1 μM RA (Sigma), 10 μM SB431542 (MCE), 20 ng/ml BMP4 (Peprotech), 2.5μM IWP2 (Selleck) or 2.5μM IWR1 (Selleck). The medium was changed daily, and the differentiation lasted until day 9 for the induction of 3PPE.
7
Directed Differentiation of iPSCs into iCMs
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iPSCs (Coriell Institute for Medical Research, AICS-0048-039) were cultured in mTeSR1media (STEMCELL Technologies, 85850) on Matrigel (Gibco, A1413302)-coated plates. At 80% of confluency, iPSCs were differentiated into iCMs as previously described 26 (link). Briefly, the iPSCs were treated with 8μM CHIR-99021 (SelleckChem, S2924) in RPMI (Gibco, 11875093)-B27(no insulin) (Gibco, A1895601) from day 0–1. Media was changed on day 2 and the cells were treated with 5 μM IWR1 (SelleckChem, S7086) in RPMI-B27(no insulin) from day 3–4. Starting from day 7, RPMI-B27(with insulin) (Gibco, 17504044) media were given to iPSC-derived cardiomyocytes (iCMs). Two rounds of glucose starvation from day 12–15 and from day 20–23 were performed to eliminate non-cardiomyocyte cells. At day 30, iCMs were transferred to Matrigel-coated 24-well plates and maintained for further use. For co-culture of iCM and HCF, iCM cells were seeded at 2.5×104 cells per well in a Matrigel-coated 24-well plate on day 1, and HCF cells were seeded at 1.5×104 cells per well on day 2. Cells were cultured in RPMI-B27 (with insulin). Co-culture was used on day 3 for experiments and analysis.
8
Cardiac Differentiation from Human iPSCs
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Human iPSCs were maintained on Matrigel (BD Biosciences) coated plates in E8 medium (Life Technologies), which was changed daily33 (link). Prior to cardiac differentiation, hiPSCs were passaged once every four days with 0.5 mM EDTA at 37 °C for 5 minutes. After hiPSCs had achieved 95% confluence, cardiac differentiation was initiated by treating cells with 6 μM CHIR99021 (Selleck Chemicals) in RPMI + B27 without insulin for 48 hours (day 0 to day 2) according to a previously-published protocol23 (link). The medium was then changed to RPMI + B27 without insulin for 1 day, and 5 μM IWR1 (Selleck Chemicals) was added between days 3 to 5 of differentiation. From day 5 to 7, cells were returned to RPMI + B27 without insulin and without small molecules. Cells were then maintained on RPMI + B27 with insulin from day 7 onwards. Once spontaneous beating could be observed (usually between days 10 to 12 of differentiation), the medium was changed to RPMI without glucose + B27 with insulin for glucose starvation for 3 days to eliminate non-CMs in culture. This step typically resulted in greater than 90% CM purity. The surviving CMs were then passaged with trypLETM (Life Technologies) and replated for further studies.
9
Directed Differentiation of iPSCs to Cardiomyocytes
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Human pluripotent stem cells were differentiated into iCMs using an established differentiation procedure as previously described (Wanjare et al., 2017 (link)). Briefly, iCMs were generated using a directed differentiation method that involved incubating iPSCs with Wnt agonist CHIR 99021 (6 μM, Selleck), followed by Wnt antagonist IWR-1 (5 μM, Selleck) in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher), supplemented with insulin-free B27 (Invitrogen). After 7 days, the culture medium was replaced with RPMI 1640 supplemented with B27 and insulin (Invitrogen). To enrich the iCM populations, the differentiating cells were deprived of glucose after 9 days. The iCMs were used for experiments after 15 days of differentiation. Only dishes containing >80% spontaneously contracting iCMs were used for the described experiments.
10
Xeno-free Monkey PSC Culture Protocol
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Xeno-free monkey PSCs were cultured in a chemically defined medium under 20% O2 and 5% CO2 at 37°C. XF-PSC medium was prepared by including E8 medium supplemented with 1× Chemically Defined Lipid Concentrate (Gibco), 1× Glutamax (Gibco), 1.94 mg/L Glutathione (Sigma), 100 ng/mL of Nodal (MCE), 2 μmol/L IWR-1 (Selleck), and 10 ng/mL of Activin A (Peprotech). Prepared XF-PSC medium could be kept at 4°C for up to 1 week.
XF PSCs were cultured on Vitronectin XF (STEMCELL)—coated plate, which was diluted in Cell adhere dilution buffer (STEMCELL). The final concentration of Vitronectin XF was 50 μg/mL. For culturing XF PSCs in 24-well plates, 350 μL of diluted Vitronectin was added to one well, then the plates were incubated at 37°C for 1.5 h. Do not allow the culture surface to dry as the matrix will become inactivated. For the initial passaging and culturing, Y27632 (STEMCELL) or Clone R (STEMCELL) was needed.
XF PSCs were cultured on Vitronectin XF (STEMCELL)—coated plate, which was diluted in Cell adhere dilution buffer (STEMCELL). The final concentration of Vitronectin XF was 50 μg/mL. For culturing XF PSCs in 24-well plates, 350 μL of diluted Vitronectin was added to one well, then the plates were incubated at 37°C for 1.5 h. Do not allow the culture surface to dry as the matrix will become inactivated. For the initial passaging and culturing, Y27632 (STEMCELL) or Clone R (STEMCELL) was needed.
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