Pd173074

Antibodies were obtained from the following commercial sources: PARP, caspase-3, caspase-8, caspase-9, phospho-Erk, and Erk (Cell Signaling Technology); p-c-Met, c-Met, and FGFR3 (Santa Cruz Biotechnology); pan-actin (Millipore); phospho-stat3-Ser⁷²⁷ and Stat3 (BD Biosciences). propidium iodide, MTT, z-VAD–FMK, PD98059, PD173074, and all of the other chemical reagents were obtained from Sigma. RPMI-1640 medium, Dulbecco's Modified Eagle Medium (DMEM), FBS, penicillin, streptomycin, and all other tissue culture regents were obtained from GIBCO/BRL Life Technologies. Sorafenib (purity ≥ 99%) was purchased from Biovision. Tivantinib (ARQ 197) was purchased from ChemieTek. For in vitro administration, Sorafenib, tivantinib or DE605 (structure and scheme of DE605 synthesis shown in supplemental Fig. S1) were dissolved in DMSO (Sigma) to a concentration of 10 mM and further diluted to appropriate final concentration in RPMI 1640 with 10% fetal bovine serum. DMSO in the final solution did not exceed 0.1% (v/v). For in vivo testing, Sorafenib or DE605 were dissolved in Cremophor EL/ethanol (50:50; Sigma Cremophor EL, 95% ethanol) at 4 × concentration. This 4 × solution was prepared fresh every 4 days. Final dosing concentration was prepared by diluting the 4 × solution to 1× with sterile water. The 1× solution was prepared just before it was given to the mice.

iPS cells were maintained routinely on MEFs with hES media (10 ng/mL FGF2). For the trophoblast differentiation, 2.4 × 10⁴ cells per square centimeter were seeded on Matrigel coated plates with conditioned hES medium by a monolayer of mitomycin-C treated MEF feeder cells (MEF-CM) containing FGF2 (10 ng/mL). On the next day, medium was changed to MEF-CM containing 4 ng/mL of FGF2. Next day, the medium was changed to BMP4 (10 ng/mL; RD Systems), the ALK4/5/7 inhibitor, A83-01 (1 μM; Tocris), and the FGF2-signaling inhibitor PD173074 (0.1 μM; Sigma) containing (BAP) hESC basal medium not conditioned with MEF feeder cells^{22 (link),27 (link),28 (link)}. Control cultures were grown in the presence of FGF2 and in the absence of BAP. The medium was replenished daily.

For the acute injury model induced by Aβ(25-35) and drug administration, siRNA was given (ICV) to C57BL/6J mice on day 1 and day 3, followed by ICV administration of Aβ(25-35) (10 nmol) and FGF21 (1 μg) on day 3. For the small molecule inhibitor group, the mice were preinjected (ICV) with PD173074 (25 μg) (Sigma-Aldrich, St. Louis, USA) prior to treatment with Aβ(25-35) and FGF21. The mice were sacrificed 4 days after the peptide injection for biochemical tests.
In vivo transfection experiments were performed by two ICV injections of 1 μL siRNA (Biomics Biotechnologies Co., Ltd., Nantong, China). Briefly, 40 μg siRNA was dissolved in 20 μL of endotoxin-free pure water and then mixed with Entranster^TMin vivo transfection reagent (Engreen Biosystem Co., Ltd., Beijing, China) at a ratio of 2:1. siRNA sequences: monocarboxylate transporter 2 (MCT2): sense 5'-CUGUCACAGUAUUCUUCAAdTdT-3', antisense 5'-UUGAAGAAUACUGUGACAGdTdT-3'; monocarboxylate transporter 4 (MCT4): sense 5'-GGAGCUUAUGCAUGAGUUUdTdT-3', antisense 5'-AAACUCAUGCAUAAGCUCCdTdT-3'; negative control: sense 5'-UUCUCCGAACGUGUCACGUdTdT-3', antisense 5'-ACGUGACACGUUCGGAGAAdTdT-3'.

FGFR3 antibody (B-9, C-15) and FGFR1/2/4 antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to TAK1, ERK, phospho-ERK, phospho-Tyrosine (4G10), p65 and p84 were from Millipore (Billerica, MA), as was normal rabbit IgG. Recombinant human FGF1 was obtained from R&D Systems (Minneapolis, MN) and PD173074 from Sigma (St. Louis, MO). Non-targeting and TAK1-specific siRNA (both ON-TARGETplus SMART-pool) were purchased from Dharmacon (Thermo Scientific). Human Collagen type IV was from Sigma.

For the systematic small-molecular inhibitor screen, PM organoids were generated and differentiated until day 3 (PSM) of our protocol. On day 3, media was replaced with fresh media containing combinations of small-molecule inhibitors targeting the FGF, WNT, BMP, and TGF-β signaling pathways at indicated concentrations. For targeting the WNT pathway, we used C59 (Tocris, Cat# 5148), XAV939 (Tocris, Cat# 3748), and CHIR99021 (Sigma-Aldrich, Cat# SML1046). For inhibiting the FGF pathway, we used PD173074 (Sigma-Aldrich, Cat# P2499). For inhibiting the BMP pathway, we used LDN193189 (Stemgent, Cat# 04-0074). For inhibition of the TGF-β pathway, we used A-83-01 (Tocris, Cat# 2939). Media was changed daily. We analyzed three replicates per condition in the primary screen and five replicates per condition in the secondary screen.