Fig. 5: Overexpression of HSPB3 promotes the differentiation of fusion-negative rhabdomyosarcoma (FN-RMS) cells.

A Comparative analysis of HSPB3 fragments per kilo base per million mapped reads (FPKM) in FN-RMS cells (n = 3 cell lines), human skeletal muscle-proliferating myoblasts (HSMMs), and differentiated myotubes (HSMMtubes) (n = 3 biological replicates), ± s.e.m.. The analysis was performed using publicly available RNA-seq data: GSE52529 (myoblasts and myotubes); GSE137168 (FN-RMS cell lines). B Representative western blot (n = 3 biological independent experiments) of protein extracts from FN-RMS control cells and myc-HSPB3 overexpressing FN-RMS cells, 3 and 10 days post-selection. Expression levels of myc and MYOG were analyzed by immunoblotting. Vinculin was used as a loading control. C Total RNA was extracted from FN-RMS control cells and from FN-RMS cells infected with lentiviral particles expressing myc-HSPB3 for 3 and 10 days and the expression levels of MYOG were analyzed by RT-qPCR. n = 3 independent experiments; data are presented as mean value ± SD, Student’s two-tailed t test. Exact P values are reported in the figure. D Representative immunostaining (n = 3 independent experiments) of FN-RMS control cells and myc-HSPB3 overexpressing cells at 10 days post selection in the growth medium, showing the expression of the differentiation marker MyHC. Nuclei were stained with DAPI. Scale bar = 75 µm. E The total RNA was extracted from FN-RMS control cells and myc-HSPB3 overexpressing cells at 3 and 10 days post selection and the expression levels of MyH2 were analyzed by RT-qPCR. n = 3 independent experiments; data are presented as mean value ± SD, Student’s two-tailed t test. Exact P values are reported in the figure. F Cell confluence is decreased in FN-RMS overexpressing myc-HSPB3 for 3 days compared to FN-RMS control cells. Cell growth was assessed by confluence analysis using Celigo Cytometer Nexcelom imaging platform at the reported time points. n = 3 independent experiments; data are presented as mean value ± SD, Student’s two-tailed t test. Exact P values are reported in the figure.