Fig. 2: SKP2 is regulated by a MYOD bound enhancer.

a Representative profile of ChIP-seq read densities of MYOD (green), H3K27ac (yellow) and PAX3-FOXO1 (red) at SKP2 locus on RD (FN-RMS) and RH4 (FP-RMS) cells. TSS, Transcription start site. b Pearson’s correlation analysis of MYOD1 versus SKP2 expression in FN-RMS (n = 58) and FP-RMS (n = 54) primary tumors. Line represents linear regression of data. Pearson’s correlation and p values are reported in the figure. P-value was calculated by a two-tailed Pearson correlation test. Error bands represent the 95% confidence interval for the Loess curve fit of the data. c, e Representative western blot (n = 3 independent experiments) of the indicated proteins on RD and JR1 FN-RMS cells transfected with either Scrambled (siSCR) or MYOD siRNA (siMYOD.1) at 24 h (h) and 48 h post-transfection. Vinculin is the loading control. d, f mRNA levels (RT-qPCR) of MYOD1 and SKP2 on cells treated as in (c, e) were normalized to GAPDH levels and expressed as fold increase over siSCR. n = 3 (RD, JR1, RH4) and n = 4 (RH30) independent experiments, data presented as mean values ± SD, Student’s two-tailed t-test. g Workflow schematic model of Chromosome Conformation Capture (3 C) assay. Created with BioRender.com. h (left) Representative electrophoretic run and (right) band intensity quantification of amplification products from 3 C assay on RD (FN-RMS) and RH4 (FP-RMS) cells. P1-P2, primers were designed on SKP2 promoter and intronic enhancer, respectively (see g). As loading control a region lacking restriction sites at SKP2 enhancer has been used. As 3 C positive control we used MYOD looping between SNAI2 distal enhancer and promoter as described in our previous work. A negative control was selected as reported in Supplementary Methods. n = 3 independent experiments, data presented as mean values ± SD, one-way ANOVA. Source data are provided as a Source Data file.