Fig. S6

BIO alters the differentiation status of zebrafish ERM tumor cells. (A–C) Transplanted ERMS tumor-bearing larval zebrafish were treated with DMSO and BIO (300 nM) for 5 d. MF20 staining was performed on cryosections of treated tumors to assess the extent of terminal differentiation. (A) DMSO-treated tumor. (B) BIO-treated tumor. Three independent tumors from each treatment group were analyzed. Average percentage of MF20-positive cells ±SD are noted. (Scale bar, 20 μM.) (C) Real-time RT-PCR assessing expression of myogenic markers in DMSO- and BIO-treated fish (n = 3). Error bar indicates SD across experimental triplicate. *P < 0.05 in comparison with the DMSO-treatment group. Myosin heavy chain-6 (mhc). (D and E) Immunohistochemistry for Phospho-H3. The number of Phospho- H3-positive cells was quantified in three independent fields at 400× magnification. The values (with SEM) indicate average of three tumors analyzed for each treatment group. (F–O) EdU analysis. Larval fish engrafted with tumors expressing myf5:GFP and mylz2:mCherry were treated with DMSO or BIO and pulsed with EdU for immunofluorescence (IF) analysis. GFP (F and G), mCherry (H and I), DAPI (J and K), and EdU (L andM). (N and O) Merge image of all four channels. Yellow arrowheads denote representative myf5:GFP⁺/mylz2:mCherry cells that have EdU incorporation. (Scale bar, 50 μM.) (P) Analysis of apoptosis by Annexin V staining after multiparameter FACS. (Q) Quantitation of caspase-3 immunohistochemical staining. Average of number of positive cells in three high-power (400× magnification) fields is shown for three independent tumors treated with either DMSO or BIO. Each error bar indicates SD. NS, not significant.