One day before the administration of the compounds, half of the culture medium was replaced with the stimulating medium composed of Ham’s F-12 (FUJIFILM Wako, Osaka, Japan), N2 supplement (Thermo Fisher Scientific, Waltham, MA, USA), and 1% penicillin-streptomycin. The next day, all of the medium was replaced with stimulating medium. Five compounds, namely, SB431542 (18176–54, 5 μM, Nacalai, Kyoto, Japan), LDN193189 (6053–10, 1 μM, R&D Systems, Minneapolis, MN, USA), CHIR99021 (13122, 1.5 μM, Cayman, Ann Arbor, MI, USA), DAPT (208255-80-5, 5 μM, Sigma-Aldrich), and Y-27632 (18188–04, 0.5 μM, Nacalai), were added to the stimulating medium. The medium was changed every two days and the compounds were added at the same time.
Sb431542
Manufactured by Nacalai Tesque
Sourced in Japan
SB431542 is a potent and selective inhibitor of transforming growth factor-beta (TGF-β) type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. It effectively blocks TGF-β signaling by inhibiting the phosphorylation of SMAD2 and SMAD3.
Automatically generated - may contain errors
2 protocols using sb431542
1
Directed Differentiation of Cells
2
Intravitreal Injection of Small Molecules
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One day before administration of the compounds, half of the culture medium was replaced with the stimulating medium composed of Ham's F-12 (FUJIFILM Wako, Osaka, Japan), N2 supplement (Thermo, Waltham, MA, USA), and 0.1% penicillin-streptomycin. The next day, all of the medium was replaced with stimulating medium. Five compounds, namely, SB431542 (5 μM, Nacalai, Kyoto, Japan), LDN193189 (1 μM, R&D Systems, Minneapolis, MN, USA), CHIR99021 (1.5 μM Cayman, Ann Arbor, MI, USA), DAPT (5 μM, Sigma-Aldrich), and Y-27632 (0.5 μM, Nacalai) were added to the SM. The medium was changed every two days and the compounds were added at the same time.
Intravitreal injection. After anesthesia, the compounds were injected into the vitreous of 6week-old W T, ROSA-td-Tomato, and 2-and 4-week-old rd10 mice under topical mydriasis.
The final concentrations of SB431542, LDN193189, CHIR99021, and DAPT were 0.1 μM, 0.02 μM, 0.03, and 0.1 μM, respectively. Micro syringes with 33-gauge needles (Hamilton, Reno, NV, USA) were used. After anesthesia, we injected intravitreally of 1 μl pAAV.GFAP.Cre.WPRE.hGH, with a concentration was 7 x 10 13 vg/μL, to 2-week-old ROSAtd-To m a t o mice. PAAV.GFAP.Cre.WPRE.hGH was a gift from James M. Wilson (Addgene plasmid # 105550 ; http://n2t.net/addgene:105550 ; RRID:Addgene_105550).
Intravitreal injection. After anesthesia, the compounds were injected into the vitreous of 6week-old W T, ROSA-td-Tomato, and 2-and 4-week-old rd10 mice under topical mydriasis.
The final concentrations of SB431542, LDN193189, CHIR99021, and DAPT were 0.1 μM, 0.02 μM, 0.03, and 0.1 μM, respectively. Micro syringes with 33-gauge needles (Hamilton, Reno, NV, USA) were used. After anesthesia, we injected intravitreally of 1 μl pAAV.GFAP.Cre.WPRE.hGH, with a concentration was 7 x 10 13 vg/μL, to 2-week-old ROSAtd-To m a t o mice. PAAV.GFAP.Cre.WPRE.hGH was a gift from James M. Wilson (Addgene plasmid # 105550 ; http://n2t.net/addgene:105550 ; RRID:Addgene_105550).
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