Fig. 2

Thermal proteome profiling of Napabucasin in zebrafish embryos. Embryos were treated with napabucasin (5 μM) or solvent control (0.2% DMSO). Alternatively, antisense STAT3-specific morpholino (MO stat3) was microinjected at the 1-cell stage. A, at 10.5 hpf, the embryos were fixed, and in situ hybridization was performed using a ntl-specific probe, which stains the notochord. B, the length of the notochord was determined in napabucasin treated and stat3-morpholino knockdown embryos compared with DMSO and nontreated E3 medium control. Significance was determined using a one-way ANOVA with Dunnetts multiple comparisons test, n = 26 to 66 embryos per condition; ∗∗∗p < 0.001. The global precipitation behavior of proteins in DMSO (C) or napabucasin treated (D) lysate is similar, in agreement with overall comparable melting points for proteins under these two conditions (E). The protocol captured a large dynamic range of proteins, ranging from high abundant proteins such as Vtg1, Ttnb, and Myhz1.2 (red) to low abundant proteins such as Sirt1, Cbx5 and Bbs7 (yellow). Additionally, Stat3 (orange) and all shifting Aldh proteins (green) are shown (F). hpf, hours postfertilization.