Repsox

Manufactured by Selleck Chemicals

Sourced in United States, China

RepSox is a small molecule inhibitor that selectively targets the transforming growth factor beta (TGF-β) signaling pathway. It functions by inhibiting the phosphorylation of SMAD2 and SMAD3, which are key mediators of the TGF-β signaling cascade.

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Lab products found in correlation

Brain derived neurotrophic factor (bdnf), by R&D Systems (5 mentions) Dmem f12, by Thermo Fisher Scientific (5 mentions) N2 and b27 supplements, by Thermo Fisher Scientific (4 mentions) Glucose, by Merck Group (4 mentions) Minimum essential medium (mem), by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) Chir99021, by Selleck Chemicals (2 mentions) Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions) Ly2157299, by Selleck Chemicals (2 mentions) Fibroblast growth factor (fgf), by R&D Systems (2 mentions) Nanodrop one, by Thermo Fisher Scientific (2 mentions) Pf 03084014, by Selleck Chemicals (1 mentions) Human epidermal growth factor (hegf), by Merck Group (1 mentions) Basic fibroblast growth factor (bfgf), by R&D Systems (1 mentions) Isoproterenol, by Merck Group (1 mentions) Puromycin, by Selleck Chemicals (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Dmem f12 medium, by Thermo Fisher Scientific (1 mentions) H1299, by National Collection of Authenticated Cell Cultures (1 mentions) H1975, by National Collection of Authenticated Cell Cultures (1 mentions)

22 protocols using repsox

Hydrogel-Mediated Hair Cell Regeneration

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The key drug cocktail used for hair cell regeneration and protection in our study included CHIR (S1263), RepSox (S7223), and DAPT (S2215) from Selleck Chemicals, Houston, TX, USA. During hydrogel preparation (Section 2.1), these medications were added in PBS before crosslinking (the respective combinations for proliferation and differentiation were explained in Section 2.11). During hydrogel degradation in a physiological solution, the profile of drug release from the hydrogel was examined using dexamethasone as a model drug. The supernatant of the conductive hydrogel was collected and measured using a micro-UV-vis spectrometer (NanoDrop™ One, Thermo Scientific, Waltham, MA, USA) against a calibration curve to quantify the released drug at 1, 2, 4, and 6 days.

Tan F., Li X., Li X., Xu M., Shahzad K.A, & Hou L. (2024). GelMA/PEDOT:PSS Composite Conductive Hydrogel-Based Generation and Protection of Cochlear Hair Cells through Multiple Signaling Pathways. Biomolecules, 14(1), 95.

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Intestinal Small Molecule Treatments

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All small molecule information for intestinal treatment is listed in Supplemental Table 2. DBZ was from Apexbio Technology; RepSox and PF-03084014 were from Selleck Chemical and FBT10 from ForkheadBio Therapeutics. For STZ mice in vivo treatment, DBZ and RepSox were formulated in 1% DMSO, 0.5% methylcellulose, and 0.2% Tween-80 PBS solution, respectively. For NOD mice in vivo treatment, FBT10, PF-03084014, and RepSox were formulated together into N,N-dimethylacetamide/solutol HS 15/water = 5:10:85 (v/v/v) solution, pH4–5.

Du W., Wang J., Kuo T., Wang L., McKimpson W.M., Son J., Watanabe H., Kitamoto T., Lee Y., Creusot R.J., Ratner L.E., McCune K., Chen Y.W., Grubbs B.H., Thornton M.E., Fan J., Sultana N., Diaz B.S., Balasubramanian I., Gao N., Belvedere S, & Accili D. (2022). Pharmacological conversion of gut epithelial cells into insulin-producing cells lowers glycemia in diabetic animals. The Journal of Clinical Investigation, 132(24), e162720.

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Induced Somatic Granulosa Cell Generation from Human Dermal Fibroblasts

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HDFs between passages five and nine were used for iSGCs generation. The viral particles for iSGCs generation were produced after transfection of 293FT cells with a single pMX retroviral vector encoding EDA (pLV-hef1a-EDA-GFP) together with packaging plasmid psPAX2 and envelope plasmid pMD2.G. Lipofectamine™ 2000 was used for further transfection according to the manufacturer’s instructions. puromycin with a final concentration of 0.4 µg/ml was added at 48 h post-transfection to obtain stably transfected cells, and puromycin selection was continued for 15 d. HDFs transfected with EDA were cultured in SGM in the presence or absence of the following small molecules and proteins: Repsox (10 μmol/L, Selleck, USA), CHIR99021 (10 μmol/L, Selleck, USA), isoproterenol (5 μmol/L, Sigma-Aldrich, USA), RA (10 μmol/L, Sigma-Aldrich, USA) and BMP4 (20 ng/ml, R&D, USA). SGM consisted of DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1 × B27 (17,504,044, Gibco), 1 × Glutamax™ (Gibco), human epidermal growth factor (50 ng/ml, Sigma-Aldrich), basic fibroblast growth factor (20 ng/ml, R&D), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Ji S.F., Zhou L.X., Sun Z.F., Xiang J.B., Cui S.Y., Li Y., Chen H.T., Liu Y.Q., Gao H.H., Fu X.B, & Sun X.Y. (2022). Small molecules facilitate single factor-mediated sweat gland cell reprogramming. Military Medical Research, 9, 13.

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Modulating NCAPG expression in LUAD cells

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The human bronchial epithelial and LUAD cell lines—BEAS-2B, and PC9, A549, H827, H1299, and H1975—were purchased from the cell bank of the Chinese Academy of Sciences in Shanghai, China; they were then cultured in RMPI-1640 medium with 10% fetal bovine serum and incubated at 37 °C with 5% CO₂. Cell lines were authenticated to be free of mycoplasma contamination. PC9 and A549 cells were infected with lentiviruses harboring NCAPG overexpression. NCAPG-targeting short hairpin RNA (shRNA) was synthesized by Hanheng Biology Co., Ltd. (Shanghai, China). The target sequences of shRNA were as follows: sh-NCAPG 1#: 5′-GCTGTCAGAAAGCTGGCTTAT-3′ and sh-NCAPG 2#: 5′-GCTGAAACATTGCAGAAATGT-3′. RepSox was purchased from Selleck (Shanghai, China).

Wu Y., Lin Y., Pan J., Tu X., Xu Y., Li H, & Chen Y. (2021). NCAPG promotes the progression of lung adenocarcinoma via the TGF-β signaling pathway. Cancer Cell International, 21, 443.

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Induced Pluripotent Stem Cell Protocol

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Expanded MEFs that had been cultured in H-DMEM for 48 h (confluency 90%) were then transferred into a chemical conditional medium and maintained for 6 days. The chemical conditional medium was composed of KnockOut Serum Replacer (KSR) medium (Life Technologies), 15% knockout serum replacement, 1% nonessential amino acid (Invitrogen), 1% GlutaMax (Gibico), 1% sodium pyruvate (Gibico), 0.1 mM β-mercaptoethanol (Millipore), and 1,000 U/mL leukemia inhibitory factor (PeproTech). In primary screening, chemical cocktail VCR (valproic acid, 500 μM, Sigma; CHIR-98014, 3 μM Selleck; Repsox (E616542), 1 μM, Selleck) were added in KSR medium. In the optimized induction approach, VCRTc (VCR plus TTNPB, 3 μM, Selleck; celecoxib, 5 μM, Selleck) were added in KSR medium. Cells were cultured at 37°C under 5% O₂ (hypoxia) and 5% CO₂ in an O₂/CO₂ incubator (MCO-5M, Sanyo). Medium was changed every 3 days.

Chen Y., Wu B., Lin J., Yu D., Du X., Sheng Z., Yu Y., An C., Zhang X., Li Q., Zhu S., Sun H., Zhang X., Zhang S., Zhou J., Bunpetch V., El-Hashash A., Ji J, & Ouyang H. (2020). High-Resolution Dissection of Chemical Reprogramming from Mouse Embryonic Fibroblasts into Fibrocartilaginous Cells. Stem Cell Reports, 14(3), 478-492.

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Inducible hESC line for hematopoiesis

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The establishment of P18 (CDKN2C) inducible hESC lines (referred as P18/hESCs) was described in “Supplemental materials and methods”. D0-induced P18/hESCs co-cultured with AGM-S3 cells were treated without induction or with DOX from D0, D2, D4, D6, D8, D10, D12, or with DOX and 0.33 μM RepSox (Selleck Inc, dissolved in DMSO) from D0, as previously described^{1 (link)}. An equal volume of DMSO was added to control samples. Treated co-cultures were subjected to cell-cycle analysis at D4 and flow cytometry using 7-AAD and anti-CD34/CD43 antibodies (D8) or 7-AAD and anti-CD34/CD43/CD45, CD71/GPA, or CD34/CD43/GPA/CD41a antibodies (D14). Untreated co-cultures were used as negative controls. The detail information of flow cytometry was described in “Supplemental materials and methods”.

Yi D., Zhu L., Liu Y., Zeng J., Chang J., Sun W., Teng J., Zhang Y., Dong Y., Pan X., Chen Y., Zhou Y., Lai M., Zhou Q., Liu J., Chen B, & Ma F. (2021). The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-β and NF-κB signaling. Scientific Reports, 11, 24014.

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Reprogramming Fibroblasts to Induced Motor Neurons

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Reprogramming was performed in 96-well plates (8 × 10³ cells/well) that were sequentially coated with gelatin (0.1%, 1 hour) and laminin (2–4 hours) at room temperature. To enable efficient expression of the transgenic reprogramming factors, iPSCs were cultured in fibroblast medium (DMEM + 10% FBS) for at least 48 hours and either used directly for retroviral transduction or passaged before transduction for each experiment. 7 iMN factors were added in 100–200 μl fibroblast medium per 96-well well with 5 μg/ml polybrene. For iMNs, cultures were transduced with lentivirus encoding the Hb9::RFP reporter 48 hours after transduction with transcription factor-encoding retroviruses. On day 5, primary mouse cortical glial cells from P1 ICR pups (male and female) were added to the transduced cultures in glia medium containing MEM (Life Technologies), 10% donor equine serum (HyClone), 20% glucose (Sigma-Aldrich), and 1% penicillin/streptomycin. On day 6, cultures were switched to N3 medium containing DMEM/F12 (Life Technologies), 2% FBS, 1% penicillin/streptomycin, N2 and B27 supplements (Life Technologies), 7.5 μM RepSox (Selleck), and 10 ng/ml each of GDNF, BDNF, and CNTF (R&D). The iMN neuron cultures were maintained in N3 medium, changed every other day, unless otherwise noted.

Kramer N.J., Haney M.S., Morgens D.W., Jovičić A., Couthouis J., Li A., Ousey J., Ma R., Bieri G., Tsui C.K., Shi Y., Hertz N.T., Tessier-Lavigne M., Ichida J.K., Bassik M.C, & Gitler A.D. (2018). CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9orf72 dipeptide repeat protein toxicity. Nature genetics, 50(4), 603-612.

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Endothelial Cell Differentiation Protocol

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SCAP (3×10⁵) were seeded on 0.1% gelatin-coated 6-well plates in α-MEM and incubated at 37°C in a humidified atmosphere with 5% CO₂. At 40–50% cell confluence, the culture medium was changed to EGM-2 (Lonza, Walkersville, MD, USA), plus 50 ng/mL VEGF (rhVEGF165, Peprotech, NJ, USA), 20 ng/mL BMP-4 (R&D Systems, Minneapolis, MN, USA) and the chemical cocktail 0.5 mM VPA (Sigma-Aldrich, St. Louis, MO, USA), 3 μM CHIR99021 (Stemgent, Beltsville, MD, USA), 1 μM Repsox (Selleck, Houston, TX, USA), 10 μM Forskolin (Caymen, Ann Arbor, MI, USA), 5 μM Y-27632 (Sigma-Aldrich) for a 4-day induction, and then BMP-4 was replaced by 100 μM 8-Br-3,5-cAMP (Sigma-Aldrich) in the next 4 days (Appendix Table 1). The medium was changed every 2 days. The obtained cells are passaged and cultured in a maintenance medium composed of EGM-2 and 0.5 mM VPA, 3 μM CHIR99021, 1 μM Repsox, 10 μM Forskolin and 5 μM Y-27632.

Yi B., Ding T., Jiang S., Gong T., Chopra H., Sha O., Dissanayaka W.L., Ge S, & Zhang C. (2021). Conversion of stem cells from apical papilla into endothelial cells by small molecules and growth factors. Stem Cell Research & Therapy, 12, 266.

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Direct Reprogramming of Induced Neurons

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Reprogramming was performed in 96-well plates (8 × 10³ cells/well) or 13mm plastic coverslips (3.2 × 10⁴ cells/coverslip) that were sequentially coated with gelatin (0.1%, 1 hour) and laminin (2–4 hours) at room temperature. To enable efficient expression of the transgenic reprogramming factors, iPSCs were cultured in fibroblast medium (DMEM + 10% FBS) for at least 48 hours and either used directly for retroviral transduction or passaged before transduction for each experiment. 7 iMN factors or 5 iDA factors were added in 100–200 µl fibroblast medium per 96-well well with 5 μg/ml polybrene. For iMNs, cultures were transduced with lentivirus encoding the Hb9::RFP reporter 48 hours after transduction with transcription factor-encoding retroviruses. On day 5, primary mouse cortical glial cells from P1 ICR pups (male and female) were added to the transduced cultures in glia medium containing MEM (Life Technologies), 10% donor equine serum (HyClone), 20% glucose (Sigma-Aldrich), and 1% penicillin/streptomycin. On day 6, cultures were switched to N3 medium containing DMEM/F12 (Life Technologies), 2% FBS, 1% penicillin/streptomycin, N2 and B27 supplements (Life Technologies), 7.5 µM RepSox (Selleck), and 10 ng/ml each of GDNF, BDNF, and CNTF (R&D). The iMN and iDA neuron cultures were maintained in N3 medium, changed every other day, unless otherwise noted.

Shi Y., Lin S., Staats K.A., Li Y., Chang W.H., Hung S.T., Hendricks E., Linares G.R., Wang Y., Son E.Y., Wen X., Kisler K., Wilkinson B., Menendez L., Sugawara T., Woolwine P., Huang M., Cowan M.J., Ge B., Koutsodendris N., Sandor K.P., Komberg J., Vangoor V.R., Senthilkumar K., Hennes V., Seah C., Nelson A.R., Cheng T.Y., Lee S.J., August P.R., Chen J.A., Wisniewski N., Victor H.S., Belgard T.G., Zhang A., Coba M., Grunseich C., Ward M.E., van den Berg L.H., Pasterkamp R.J., Trotti D., Zlokovic B.V, & Ichida J.K. (2018). Haploinsufficiency Leads to Neurodegeneration in C9ORF72 ALS/FTD Human Induced Motor Neurons. Nature medicine, 24(3), 313-325.

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Intrathecal Repsox Injection in Rats

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Repsox was synthesized by Selleck Chemicals (Shanghai, China). After inducing anesthesia with sodium pentobarbital (50mg/kg), the rats were placed in a prone position. A 40 U insulin syringe was used to administer Repsox (10 µL at a concentration of 10 µg/µL in dimethyl sulfoxide [DMSO]) to rats in the experimental group; those in the control group received 10 µL of DMSO alone. The needle was into the gap between vertebrae L4–L5 until the characteristic tail flick was observed; drug was then injected slowly while making certain that another tail flick was observed while the injection was taking place. The tip of the needle was removed and the wound was disinfected with iodine before returning the rat to a clean cage; wounds were monitored carefully. The treatment time points are as indicated in the individual Figure Legends.

Peng C., Chen X.T., Xu H., Chen L.P, & Shen W. (2020). Role of the CXCR4/ALK5/Smad3 Signaling Pathway in Cancer-Induced Bone Pain. Journal of Pain Research, 13, 2567-2576.

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