Cyclopamine

The following chemicals were used: blebbistatin (FUJIFILM Wako Pure Chemical Corporation, #021-17041), cyclopamine (FUJIFILM Wako Pure Chemical Corporation, #038-19311), SU5402 (FUJIFILM Wako Pure Chemical Corporation, #197-16731), and PD0325901 (FUJIFILM Wako Pure Chemical Corporation, #162-25291).

U251MG cells (1 × 10⁵) were plated on 6-cm dishes, cultured at 37°C for 24 h, and then transfected with scrambled or Bbf2h7 small interfering RNA (siRNA) using Lipofectamine 2000 according to the manufacturer’s protocol. For Bbf2h7 knockdown, the following sequences were used: 5′-GGAAGAAGGUAGAGGUUCU-3′ (siBBF2H7-1) and 5′-CCAGAAACCUGCUGAUCUA-3′ (siBBF2H7-2). U251MG cells were incubated at 37°C for 24 h after transfection, and then the culture medium was replaced. Cell numbers were counted at the indicated days after transfection. siRNAs were purchased from Bioneer (Daejeon, South Korea).
Additionally, after transfection with scrambled or Bbf2h7 siRNAs, Bbf2h7-knockdown U251MG cells were exposed to conditioned medium collected from HEK293T cells transfected with an empty vector (Mock) or BBF2H7 C-terminus (C-Sup.), or to C-Sup. in which BBF2H7 C-terminus was pulled down by immunoprecipitation using an anti-BBF2H7 C-terminus antibody (C-Sup. + Ab.). Cell numbers were counted at the indicated days after transfection. For the cell proliferation assay, we also used a cell counting kit (WST-8 assay, DOJINDO) according to the manufacturer’s protocol.
To confirm activation of Hh signaling by BBF2H7 C-terminus, U251MG cells were treated with recombinant Shh (R&D Systems), cyclopamine (WAKO) or purmorphamine (WAKO).

To induce differentiation into the neuroectoderm, ESCs were cultured for 4 days in the MT-CDM medium in the presence of 10 µM SB431542 (SB; Wako), 1 µM PD0325901 (PD; Wako), and 1 µM dorsomorphin (DM; Wako). The resulting cells were transferred to non-adherent conditions for 7 days in the MT-fCF medium (MT-fCFA medium without activin) in the presence of 10 µM SB and were then plated back on a poly-D-lysine/laminin-coated plate for 32–39 days in Neurobasal medium (Invitrogen) supplemented with B-27 Supplement Minus Vitamin A (Invitrogen) and 1 µM cyclopamine (Wako) to promote the generation of cortical neurons. To induce mesendodermal differentiation, ESCs were cultured in the MT-CDM medium in the presence of 10 ng/ml activin and 1 µM DM for 4 days