Fig. 2

Successful pharmacological targeting of ES using iP300w. A Bar graphs show the dose-dependent inhibition of viability in ES cell lines (SKES1, A673, A4573, and TC71) compared to normal human myoblast cells (LHCN) following iP300w treatment for 48 hours. The ATP assay data are represented as mean ± SEM; p<0.05, by two-way ANOVA. Results are presented as fold difference compared to control (untreated group at 48 hours) (n=3). B SKES1 cell morphology after 48 hours of treatment with 1µM iP300w. Scale bar 100µm. C Immunofluorescence for Ki67 (red) expression in SKES1 cells indicates cell-cycle arrest after 48 hours of iP300w treatment (1 µM). DAPI (blue) was used to stain the nuclei. D Western blot analysis reveals for EWS::FLI1, P300, CBP, acH3K9, acH3K18 and acH3K27, in SKES1 and TC71 cell lines treated with 1 µM iP300w for 4 and 12 hours. E Bar graphs show RT-qPCR results illustrating unchanged EWS::FLI1 expression following P300/CBP inhibition, with a significant reduction in EWS::FLI1 target gene expression (NKX2.2 and CMYC) at 24 hours post-treatment. Data were normalized to B2M. The data represent the mean ± SEM, p< 0.05, by t-test. Results are presented as fold differences compared to the control (untreated group at 24 hours) (n = 6). F Gross morphology of tumors from treated (iP300w, 5.6 mg/kg daily) and control mice at the terminal point of the experiment (day 14). G The graphs display tumor volume and weight measurements, demonstrating significant reductions in tumor size and mass following treatment. Data are presented as mean ± SEM; ***p< 0.001, ****p< 0.0001, by t-test (control, n=6; treatment, n=7). H Western blot for acetylated H3K18 and H3K27 in tumor samples presented in “F”. I RT-qPCR analysis of EWS::FLI1, its target genes, senescence-related genes, and cell cycle genes in the xenografts from experiment “F”. Data were normalized to B2M and compared to the control group using a t-test. The data represent mean ± SEM; *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001, (control, n=6; treatment, n=7)