Su5402

Doxycycline (Dox) (Sigma, D9891) was added from 3 days post fertilization (dpf) to 7 dpf at a dose of 10 μg/ml to induce kras expression and at 30 μg/ml to induce Myc expression. SU5402 (Tocris, 3300), SU6668 (tocris 3335), IWR1 (Tocris, 3552), cardionogen 1 (sigma, SML0458), cyclopamine (Tocris, 1623) and GANT61 (Sigma, G9048) were first dissolved in dimethyl sulfoxide (DMSO) as stocks and used for larva exposure from 4 to 7 dpf. The working concentrations used in the experiments were 1 μM SU5402, 1 μM SU6668, 10 μM IWR1, 10 μM cardionogen 1, 10 μM cyclopamine and 1 μM GANT61. All of these small molecular inhibitors have been previously tested and validated in zebrafish models, such as SU540240 (link), SU666841 (link), IWRI25 (link), cardionogene 142 (link), cyclopamine43 (link) and GANT6144 (link). The dosages were selected based on the highest all-survival concentrations and/or our validation in previous experiments45 (link)46 (link).

For sensory neuron differentiation, a previously reported method was used with modifications [19 (link)]. iPSCs were maintained in Stem Fit medium containing 0.3 μM LDN193189 (Stemgent), 2 μM A83-01 (Wako), 6 μM CHIR99021, 2 μM RO4929097 (Cellagen Technology), 3 μM SU5402 (TOCRIS), and 0.3 μM retinoic acid for 8 days from the day after passage. The cells were dissociated into single cells with 0.5× TrypLE Select Solution and replated on plates coated with poly-l-ornithine and laminin at a density of approximately 0.5 × 10⁴ cells/cm² to induce differentiation into sensory neurons. After seeding, the sensory neurons were cultured with neuron medium consisting of KBM neural stem cell medium containing 2% B27, 10 ng/ml neurotrophin-3 (R&D Systems), 20 ng/ml NGF (R&D Systems), 20 ng/ml BDNF, and 20 ng/ml GDNF. The medium was changed every 2–3 days.

Embryos were treated with 0.3 μM DMH1 (EMD Chemicals) from 12 hpf to 20 hpf or 3 μM SU 5402 (Tocris Bioscience) from 50 hpf to 72 hpf in egg water. To ablate β-cells, Tg(ins:CFP-NTR)^s892 embryos were treated with freshly prepared 5 mM metronidazole (MTZ) (Sigma) from 84 hpf to 108 hpf in the dark, followed by 24–48 hours recovery. Before ablation, Tg(ins:Kaede)^jh6-expressing β-cells were converted from green to red by exposing them to UV light. Control embryos from the same batch were treated with DMSO in egg water. Tg(hsp:fhl1b; hsp:GFP)^gt3 and Tg(hsp70l:bmp2b)^f13 embryos were heat shocked at various stages by transferring them into egg water pre-warmed at 40°C and 37°C, respectively. After a 30-minute heat shock, embryos were returned into a 28°C incubator and harvested at various stages.

The following antibodies were used in this study: anti-histone H2AX (07-627; dilution 1:2000) and anti-pSer139-H2AX (05-636; dilution 1:2000) (Upstate, Billerica, MA, USA); anti-JAK1 (3332, dilution 1:1000) and anti-JNK1 (3708; dilution 1:1000) (Cell Signaling Technology, Danvers, MA, USA); anti-FGFR4 (sc-124; dilution 1:200), anti-pThr183/Tyr185-JNK (sc-6254; dilution 1:200), and anti-ATM (sc-135663, dilution 1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-MARK3 (1952-1; dilution 1:2000) and anti-RAF1 (1560-1; dilution 1:500) (Epitomics, CA, USA) and anti-β-actin (A3854; dilution 1:5000) (Sigma-Aldrich); anti-CDKN1B (AHZ0452, Invitrogen; dilution 1:200); PHKA1 (H00005255-A01, Abnova, Taiwan; dilution 1:500); and anti-PIK3CD (AP8020a, Abgent, CA, USA; dilution 1:500). To inhibit the JNK1 and ERK pathways, specific inhibitors from Calbiochem (Billerica, MA, USA) were used (JNK inhibitor 420119, JNK1 inhibitor negative control 420123, ERK inhibitor 328007 and ERK inhibitor negative control 328008). Stock solutions were prepared at 20 mM in DMSO, and the cells were treated with a final concentration of 25 µM of inhibitor for 2 h before irradiation. PD173074 (FGFR1 and FGFR3 inhibitor) and SU5402 (FGFR inhibitor) were purchased from Tocris and Calbiochem, respectively, and both inhibitors were used at a final concentration of 10 µM.