Fig. 2

Rad18 is a determinant of doxorubicin sensitivity. A Induction of doxorubicin resistance 143B cells(143B-Res) from 143B-parental by doxorubicin treatment. RNA-Seq was performed in 143B-Res in parallel with 143B-parental. B 143B-Res and 143B-parental cells were treated with doxorubicin with the gradient concentrations. IC50was assessed by cell counting kit-8(CCK-8) at 48 h. C KEGG enrichment analysis of RNA-seq. Top20 signaling pathways were listed, among which a variety of drug-resistant related signaling pathways were enriched. D GSEA showed that DNA packaging complex, epoxygenaseP450 pathway, ABC transporters and oxidative phosphorylation pathways were significantly upregulated in 143B-Res cells when compare to the parental cells. E The HEAT map showed the differentially expressed genes of 143B-Res and parental cells. F Venn diagram showed that CRISPR/Cas9 negative screening genes were intersected with up-regulated genes of RNA-seq, and four potential drug resistance genes were obtained, which were Rad18, GPRC5B, FOXN4 and KIF1A. G Apoptosis rate was detected by flow cytometry in 143B cells after 5 μM doxorubicin treatment for 48 h, with which GPRC5B, KIF1A, FOXN4 and Rad18 knocked down by siRNAs. H IC50 and relative mRNA expression of Rad18 to doxorubicin were detected in hFOB1.19 cells and OS cell lines. I Representative images of immunohistochemical staining of Rad18 in normal and OS tissue sections. Scale bar =500 μm, up; Scale bar =100 μm down. J Online datasets analysis of the Rad18 expression in normal and OS tissue sections from GSE39058 and Rad18 expression in groups of good or poor response to chemotherapy from GSE87437. K Linear regression analysis of relative mRNA expression of Rad18 and IC50 in cells as mentioned above(H). L Protein and relative mRNA expression of Rad18 in143B-Res cells and parental. M Representative images of immunofluorescence staining of Rad18 on orthotopic osteosarcoma tissues of mice before and after doxorubicin treatment. Scale bar =100 μm. N. Representative images of IHC staining of Rad18 in OS patients before and post chemotherapy. Scale bar =50 μm