MC38 colon adenocarcinoma cancer cells (4 × 105 cells) were injected subcutaneously into 8–12-week-old mice in 100 µL of phosphate buffered saline (PBS). Then, 200 µg of anti-PD-L1 mAb (clone MIH5) were injected intraperitoneally at days 6, 8, and 11 after inoculation. LY364947 was purchased from Selleckchem (Huston, TX, USA) and dissolved in dimethylsulfoxide (DMSO) to make final concentration of 20 mg/mL. Then, 10 mg/kg of LY364947 were injected intraperitoneally at days 6, 8, and 11 and once every three days after cancer cell inoculation. The KPC1 pancreatic cancer cell line was generated from KrasLSL-G12D/+, Trp53LSL-R172H/+, Pdx1-Cre (KPC) mice and was a gift from Thorsten Hagemann (Queen Mary University of London). The tumor cells (1 × 105 cells) were injected subcutaneously into 8–12-week-old mice in 100 µL of PBS. At days 9, 11, and 14 post tumor inoculation, mice were injected intraperitoneally with 200 µg of anti-PD-L1 mAb (clone MIH5). For the LY364947 or combination group, mice received 10 mg/kg of LY364947 (intraperitoneally) at day 9 and once every day post tumor inoculation. All tumors were measured twice weekly using calipers. Mice were sacrificed when tumors reached a size of 100 mm2 to avoid unnecessary suffering. Both cell lines were mycoplasma and mouse antibody production (MAP)-tested before the start of tumor studies.
Ly364947
Manufactured by Selleck Chemicals
Sourced in United States
LY364947 is a laboratory chemical product manufactured by Selleck Chemicals. It is a small molecule compound used for scientific research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ly2157299, by Selleck Chemicals (2 mentions)
Csii ef dhb venus, by Addgene (2 mentions)
Plifeact mruby lentiviral vector, by Addgene (2 mentions)
Rp00452, by ABclonal (2 mentions)
Phiv h2b mrfp, by Addgene (2 mentions)
Leptomycin b lmb, by Beyotime (1 mentions)
Sw1088, by American Type Culture Collection (1 mentions)
Sw1783, by American Type Culture Collection (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Tgfbr1 kinase inhibitor, by Selleck Chemicals (1 mentions)
Raw264, by American Type Culture Collection (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Oe nc, by Genechem (1 mentions)
Sri 011381, by Selleck Chemicals (1 mentions)
Millicell standing cell culture inserts, by Merck Group (1 mentions)
Dm4000b, by Leica (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Sb431542, by Bio-Techne (1 mentions)
Sb525334, by Selleck Chemicals (1 mentions)
22 protocols using ly364947
1
Anti-PD-L1 and TGF-β Blockade in Murine Cancer Models
2
Inhibition of TGF-β Signaling in AML
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TGF-β signalling inhibitor LY364947 (Selleckchem, TX, USA) was used to treat OCI-AML3 cells with different concentrations (0, 20, 30, 40, 50 μM) for 3 h. For inhibition of cytoplasmic PML levels, leptomycin B (LMB; Beyotime, Shanghai, China) was used to treat OCI-AML3 cells with 20 nM for 6 h. The treated cells were harvested for qRT-PCR and western blot assay.
3
Glioma Tissue and Cell Line Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All experiments described in the present study were performed after obtaining informed consent and were in accordance with an Institutional Review Board protocol approved by the Partners Human Research Committee at Dalian Medical University (Dalian, China). The human normal brain tissue samples and glioma tissue samples were provided by the Department of Neurosurgery at the Second Affiliated Hospital of Dalian Medical University (Dalian, China) between 2010 and 2012. The human glioma cell lines, U87, T98G, SW1088 and SW1783, were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The human glioma cell lines, U251 (#09063001) and U373 (#08061901) were obtained from Sigma (St. Louis, MO, USA). The cells were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) (both from Invitrogen, Carlsbad, CA, USA). Mouse monoclonal JARID1B (ab198884), p-Smad2 (ab53100), Smad2 (ab119907), transforming growth factor-β1 (TGF-β1; ab92486), Oct4 (ab181557), nestin (ab11306) and Bmi-1 (ab85688) antibodies were purchased from Abcam (Cambridge, MA, USA). Mouse monoclonal β-actin antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). LY364947, an inhibitor of TGF-βRI, was obtained from Selleckchem (Houston, TX, USA).
4
Evaluating TGF-β Receptor Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For treatment with LY2157299 (20 μM) and LY364947 (1 μM) (TGFBR1 kinase inhibitor, Selleckchem, Houston, TX), 300,000 H1650-M3 cells were plated in a 6 cm2 plate. Inhibitor was added the next day and the mixture was incubated for 3–5 days for LY2157299 and 2–3 days for LY364947. The cells were lysed with TRIzol and processed for RNA preparation. To determine IC50 values for various drugs (17-AAG (this drug is not mentioned in main text), cisplatin, doxorubicin, etoposide, erlotinib, epitaxol and tunicamycin), the cells were plated in 96-well plates at 2,000 cells/well. The next day, individual drugs were added to the wells at the indicated concentrations and the mixture was incubated for 5 days. The plates were then washed once with PBS, fixed with 3.7% formaldehyde and stained with crystal violet. Each stained well was destained in 50–100 µl of 10% acetic acid and the absorbance was read in a spectrophotometer at 590 nm.
5
Simulating Ischemia-Reperfusion in Cardiomyocytes and Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cardiomyocytes (H9c2) and macrophage cells (RAW 264.7) were obtained from American Type Culture Collection. Cells were cultured in RPMI-1640 medium (Hyclone; Cytiva) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin. The cells were cultured in a 37˚C humidified atmosphere with 5% CO2 and glucose-free balanced salt solution in the presence of 95% N2 for 6 h to mimic hypoxia and hypoglycemic conditions. Subsequently, these cells were cultured in RPMI-1640 medium in 95% air and 5% CO2 at 37˚C for 12, 24 and 48 h. This process was performed to simulate ischemia-reperfusion in vitro (20 (link)). LY364947 (Selleck Chemicals) is a receptor kinase I inhibitor that was added into the medium to inhibit TGF-β function. lncRNA ATB overexpression plasmid (Oe-ATB) and its negative control (Oe-NC) were synthesized by Shanghai GeneChem Co., Ltd. and transfected into H9c2 cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.
6
Scratch-Wound Healing Assay: Bleomycin Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The scratch-wound healing assay was performed to examine the effects of bleomycin on collective migration and recolonization in a 2-dimensional environment. AF cells with or without TGFβR1 siRNA knockdown and BMSCs were cultured to 100% confluence. Under sterile conditions, a linear scratch line was made straight down the center of the cell monolayer using the tip of a sterilized 200-μl micropipette tip. Cell media were carefully aspirated to remove cellular debris and floating cells and then replaced with fresh serum-free DMEM/F12 or MEMα without or with bleomycin (5 or 10 μg/ml) ± TGFβR1 inhibitor (LY364947, 10 μM; Selleck, USA). Phase-contrast images were captured of the initial scratch wound for reference and designated time 0. Further images were captured at 24 and 48 h, and the distance between the leading cell edges at each time point was measured using the ImageJ software (National Institutes of Health, USA).
7
Zebrafish TGFβ signaling modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The TGFβ type I receptor kinase antagonist LY364947 was sourced from Selleckchem or Sigma. A stock concentration of 10 mM was prepared in DMSO. Owing to minor batch-to-batch variation, the final working concentration was determined empirically for each batch, which was 10 µM or 20 µM based on the lowest concentration that would induce a robust phenotype. The TGFβ signaling agonist SRI-011381 was sourced from Selleckchem. A stock concentration of 100 mM was prepared in DMSO. The final working concentration was 500 µM, based on a dosing study to determine the highest non-lethal concentration of the drug. Healthy embryos without chorions were arrayed in clear multi-well plates on 2 dpf. Typically, 25 embryos were arrayed in 5 ml of E3 in 6-well plates or ten embryos were arrayed in 500 µl of E3 in 24-well plates. At the desired stage, a stock solution of LY364947, SRI-011381 or equivalent volumes of DMSO were added to each well to achieve the final working concentrations of LY364947 and SRI-011381. Plates were incubated at 28.5°C in a Ziploc bag containing a wet towel. For experiments taking longer than 24 h, fresh E3 and DMSO, LY364947 or SRI-011381 were added approximately every 24 h. Embryos were fixed and processed for immunostaining as described.
8
Multimodal Cellular Imaging Toolkit
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pHIV-H2B-mRFP was purchased from Addgene (#18982) for stable H2B-RFP expression, FG12-GFP was kindly provided by the Soengas lab (CNIO, Spain) (Olmeda et al., 2017 (link)) for stable GFP expression, CSII-EF-DHB-Venus (sensor for Cdk2 activity) was kindly provided by the Spencer lab (University of Colorado, USA) for cell cycle analysis, and a pLifeact-mRuby lentiviral vector was kindly provided by the Moreau and Saltel laboratories (DR2 INSERM, France) as in (Juin et al., 2014 (link)) for imaging actin dynamics. CFP-YFP RhoA biosensor (Pertz et al., 2006 (link)), a CFP-YFP Cdc42 biosensor (Hanna et al., 2014 (link)), and a CFP-YFP Rac1 biosensor (Miskolci et al., 2016 (link)) were kindly provided by the Hodgson lab (AECOM, USA). Recombinant Human TGF-beta 2/TGFβ2 Protein was purchased from AbClonal (RP00452) and diluted according to manufacturer’s instructions. LY364947 (Selleck Chemical, S2805), an ATP-competitive inhibitor of TGFβR-I was diluted according to manufacturer’s instructions.
9
Bleomycin Effects on 3D Cell Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effects of bleomycin on single-cell migration through a three-dimensional environment were examined using the transwell migration assay (Millicell Standing Cell Culture Inserts, 8-μm pore size; Merck-Millipore, Burlington, MA, USA). Briefly, AF cells with or without TGFβR1 siRNA knockdown and BMSCs were seeded into the upper chamber of the Millicell Standing Cell Culture Inserts at a density of 8 × 104 cells/well in serum-free DMEM/F12 or MEMα media without or with bleomycin (5 or 10 μg/ml) ± TGFβR1 inhibitor (LY364947, 10 μM; Selleck, USA). The inserts were then placed into 24-well plates filled with complete media (FBS as chemoattractant source) and cultured for 24 h. At the end of the experiment, the media in the inserts were discarded, and adherent cells were fixed in 4% paraformaldehyde (PFA) for 30 min and then stained with 0.2% crystal violet for 5 min. Cells adhering to the membrane inside the inserts (i.e., cells that have not migrated) were gently removed using a cotton-tipped applicator. Migrated cells on the other side of the inserts were imaged under a light microscope (Leica DM4000 B; Leica Microsystems) and staining intensity analyzed with the Image-Pro Plus 6.0 software to evaluate the ratio of integrated optical density (expressed as the IOD/area for each sample).
10
Modulating VMHc Neuron Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tg(vmhc:mCherry-NTR) larvae were treated with 5 mM of MTZ (Sigma) in E3 water at 72 hpf for 4 h as previously described (Zhang et al., 2013 (link)). After being washed, the larvae were then incubated in 2 μM of SIS3 (Selleck), 25 μM of SB431542 (Tocris), 20 μM of LY364947 (Selleck), or 0.2% dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific) as control from 76 to 124 hpf.
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!