We used transwell chambers (Corning, NY, USA) with a pore size of 8 μm to detect cell migration. In total, 24 h before the experiment, the medium was changed to a serum-free medium, and the cells were starved and placed in a 37°C, 5% CO2 incubator. The cells were trypsinized and centrifuged. SB203580 (MedChemExpress), Y27632 (MedChemExpress), and LY294002 (MedChemExpress) were diluted in DMEM containing 0.3% BSA at a concentration of 25 nM/ml for SB203580, 10 nM/ml for Y27632, and 10 nM/ml for LY294002. The concentration of the cell suspension was adjusted to 8.0 × 105 cells/ml added to the upper chamber of the Transwell. The intervention group was pretreated by adding the above signal pathway inhibitors in the upper chamber for 30 min, the control group was added with a basal medium in the lower chamber, and the experimental and intervention groups were added with VEGF (40 ng·ml−1) in the lower chamber. After 24 h of incubation at 37°C, the cells that had migrated to the lower surface were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30 min. Then, the migrating cells were photographed with an inverted microscope (×200 magnification) and counted using ImageJ software.
Y 27632
Manufactured by MedChemExpress
Sourced in United States
Y-27632 is a Rho-associated protein kinase (ROCK) inhibitor. It functions by selectively inhibiting the activity of ROCK, which is an important regulator of various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (5 mentions)
Matrigel, by Corning (4 mentions)
Chir99021, by MedChemExpress (4 mentions)
A83 01, by MedChemExpress (3 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (3 mentions)
N acetylcysteine, by Merck Group (3 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (3 mentions)
Ly294002, by MedChemExpress (2 mentions)
Sb203580, by MedChemExpress (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Relesr, by STEMCELL (2 mentions)
Mtesr1 medium, by STEMCELL (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sb431542, by MedChemExpress (2 mentions)
Accutase, by Biozym (2 mentions)
Y 27632, by Bio-Techne (2 mentions)
Mouse laminin, by Thermo Fisher Scientific (2 mentions)
Ara c, by Merck Group (2 mentions)
Normocin, by InvivoGen (2 mentions)
38 protocols using y 27632
1
Transwell Assay for Cell Migration
2
Organoid Culture from Human Intestinal Epithelial Cells
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Matrigel (CORNING, 354230, Corning, NY, USA) was placed at 4 °C overnight, and pipette tips, injectors and collection tubes were all pre-chilled in an ice bath. 104 cells were resuspended with 25 μL Matrigel, and 40 μL cell suspension was added into one well of 24-well plates, followed by solidification at 37 °C in a 5% CO2 incubator for 10 min. Thereafter, 500 μL 37 °C complete DMEM/F12 medium was added into the well, which was supplemented with 50% L-WNT3A, 20% R-spondin 1, 10% Noggin-Fc, 1 × B27 (Invitrogen, 17504-044, USA), 1 × N2 (Invitrogen, 15502-048, USA), 1 mM N-acetylcysteine (Sigma-Aldrich, A0737, St. Louis, MO, USA), 50 ng/mL EGF (Invitrogen, PMG8044, USA), 10 mM nicotinamide (Sigma-Aldrich, N0636, USA), 500 nM A-83-01 (Tocris, 2939, Shanghai, China), 10 μM SB202190 (Sigma-Aldrich, S7067, USA), 10 μM Y-27632 (MedChemExpress, HY-10071, China) and 10 nM Gastrin I (Sigma-Aldrich, 05-23-2301, USA). The above complete medium with growth factors was named CMGF+. Culture was refreshed using CMGF+ medium every other day until the HIEs were ready to be passaged.
3
Molecular Mechanisms of Fibrosis Inhibition
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Sodium hydrogen sulfide (NaHS), PPG (a CSE inhibitor) and 7-Azido-4-Methylcoumarin (AzMC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). SKF38393 (a DR1 agonist) was obtained from Abcam (Cambridge, MA, USA). Y-27632 (a ROCK inhibitor) was obtained from MedChemExpress (Shanghai, China). The primary antibodies for anti-CSE, Cyclin D1, proliferating cell nuclear antigen (PCNA), p21 Cip/W AF -1 , collagen I (Col-1), collagen III (Col-3), matrix metalloproteinase 9 (MMP-9), osteopontin (OPN) and α-smooth muscle actin (α-SMA) were purchased from Proteintech (Wuhan, China). The anti-p-RhoA, t-RhoA, p-ROCK1, t-ROCK1 were from Affinity Biosciences (Cincinnati, OH, USA). The anti-DR1 antibody was from GeneTex (Irvine, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG antibody, the Cell Counting Kit-8 (CCK-8) and anti-β-actin were obtained from Boster Bioengineering Limited Company (Wuhan, China). The EdU Cell Proliferation Assay Kit were obtained from Ribobio (Guangzhou, China). Fluo-4 AM were obtained from Beyotime Biotechnology (Shanghai, China). Enhanced ECL Chemiluminescent Substrate Kit was obtained from Yeasen Biotechnology (Shanghai, China). All other chemicals were from Solarbio (Beijing, China) or Beyotime Biotechnology.
4
Microglial Migration Assay via Transwell
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Primary microglia were subjected to invasive migration assay by Transwell chamber (Costar, 3415). Cell suspensions without serum mixed with Y-27,632 (5 µM, MedChemExpress, HY-10,071) and inoculated in the upper chamber at a concentration of 1 × 105 cells per chamber. The control serum-free medium was mixed with an equal amount of 0.1% DMSO. The lower chambers were supplemented with medium containing 10% FBS. After 24 h, microglia suspended in the small chamber were removed with cotton swabs and then fixed with 4% formaldehyde for 20 min. Finally, microglia were dyed using 0.1% crystal violet for a duration of 15 min and then rinsed thrice with PBS.
5
Neuroprotective Effects of Y-27,632
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Mice were administered Y-27,632 (10 mg/kg, MedChemExpress, HY-10,071) through intraperitoneal injection on a daily basis from 4 h post-injury until the time of sacrifice. Y-27,632 was diluted in a solution of 10% DMSO, 40% polyethylene glycol 300, 5% polysorbate 80, and 45% saline, according to the manufacturer’s instructions. An equal volume of drug solvent was used as the control.
6
Prostate Contractility Assay with Y-27632
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Fresh prostate specimens obtained from patients (or brain-dead men) were cut into vertical strips of the same length, approximately 1 × 0.5 × 0.5 cm, and installed lengthwise in a 10 mL organ bath (Multi-Myograph Model 810MS; Danish Myo Technology, DEN). The myograph was connected in line to a PowerLab 4/30 Data Acquisition System (ADInstruments, USA) and in turn to a Dual-Core processor Pentium computer for real-time monitoring of physiological force. The prostate strips were equilibrated at least 1 h in Krebs buffer at 37 °C with continuous bubbling of 95% O2 and 5% CO2. The buffer was composed of (in mM): NaCl 110, KCl 4.8, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and dextrose 11. During the organ bath experiments, the Krebs buffer was changed every 10 min. During the process of equilibration, the tension of the strips was continuously adjusted to 2000 mg. After equilibration, prostate strips were contracted with phenylephrine (PE, Grandpharma, CHN) (10−5M). They then were completely washed out and pre-incubated with 10 μM Y-27632 (MedChemExpress, CHN) for 15 min. Then, they were contracted with the same dose of PE. The inhibitory effect of Y-27632 was calculated.
7
Cytoskeletal Inhibitor Effects on Spheroid Viability
8
Probing Vasodilation Mechanisms
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To elucidate the role of the endothelium, K+ channel, and PI3K/Akt/Rho-kinase pathways in GPS-mediated vasodilation, the aortic rings with intact endothelium were preincubated with nitric oxide synthase (NOS) inhibitor (100 μM L-NAME), cyclooxygenase (COX) inhibitor (100 μM indomethacin), and soluble guanylyl cyclase (sGC) inhibitor (100 μM methylene blue), respectively, for 30 min, while the aortic rings without endothelium were preincubated with different K+ channel blockers of tetraethylammonium chloride (TEA, 10 mM), 4-aminopyridine (4-AP, 1 mM), BaCl2 (1 mM), glibenclamide (0.01 mM), L-type Ca2+ channel inhibitor (100 μM verapamil), PI3K/Akt inhibitor (10 μM LY294002), and Rho-kinase inhibitor (10 μM Y27632) for 30 min (all inhibitors were obtained from MedChem Express, NJ, USA). After achieving a plateau of PE-induced contracted tension, GPS was added in a cumulative manner (20, 80, and 160 μΜ) for 20 min, and the concentration-response curves were recorded.
9
Single-cell transcriptomics of colon samples
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PBL or colon single-cell suspensions from each patient were pooled with equivalent number of live cells and resuspended at 1–2.5 × 103 cells/µl in 0.04%BSA/PBS, with the addition of 10 µM Y-27632 (MedChem Express) for colon samples. Samples from unique individuals were pooled, and samples from the proximal (Right,R) or distal (Left,L) colon of the same individual were placed in separate pools, so each sample could later be uniquely identified using demuxlet21 (link). For the primary biopsy UC and VDZ analysis, the two pools were loaded into four wells each of a Chromium Single Cell 3′ v2 Reagent Kit (10X Genomics), with a total of 8 wells (Supplementary Table 2 ). 1 × 106 cells of both single-cell colon suspension pools were stained with a custom TotalSeq-A panel, (BioLegend) (Supplementary Table 3 ) according to the manufacturer’s instructions and loaded into two wells. For all experiments, 60,000 cells were loaded per well and processed for single-cell encapsulation and cDNA library generation using the Chromium Single Cell 3′ v2 Reagent Kits (10X Genomics). TotalSeq-A library generation was performed according to manufacturer’s instructions (BioLegend). Libraries were sequenced on an Illumina NovaSeq6000 to obtain 25,000 reads per cell for the gene expression libraries and 10,000 reads per cell for the TotalSeq libraries.
10
Liver Progenitor Cell Generation Protocol
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The generation of liver progenitor cells were basically according to previously published protocol17 (link). Concisely, isolated primary mouse hepatocytes were seeded on plates coated with collagen type I (Sigma) at 2 × 104 cells/cm2. The cells were cultured in DMEM supplemented with 10% FBS for 6 h and then the medium changed to SHM medium (DMEM/F12 containing 2.4 g/L NaHCO3 and L-glutamine (Gibco) supplemented with 5 mM HEPES (Solarbio), 30 mg/L L-proline (Alfa Aesar), 0.05% BSA (Solarbio), 10 ng/ml epidermal growth factor (PeproTech), insulin-transferrin-serine (ITS) (Sigma-Aldrich), 10−7 M dexamethasone (Dex) (Sigma-Aldrich), 10 mM nicotinamide (Solarbio), 1 mM ascorbic acid-2 phosphate (Wako) and 1× antibiotic/antimycotic solution (Solarbio)) supplemented with YAC (10 mM Y-27632 (Medchemexpress), 0.5 mM A-83-01 (Medchemexpress), 3 mM CHIR99021 (Medchemexpress)). Cells were cultured for 14 days to generate liver progenitor cells and the medium was changed every other day thereafter.
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