Data_Sheet_1_Development and validation of a novel necroptosis-related gene signature for predicting prognosis and therapeutic response in Ewing sarcoma.zip

Ewing sarcoma (ES) is the second most common malignant bone tumor in children and has a poor prognosis due to early metastasis and easy recurrence. Necroptosis is a newly discovered cell death method, and its critical role in tumor immunity and therapy has attracted widespread attention. Thus, the emergence of necroptosis may provide bright prospects for the treatment of ES and deserves our further study. Here, based on the random forest algorithm, we identified 6 key necroptosis-related genes (NRGs) and used them to construct an NRG signature with excellent predictive performance. Subsequent analysis showed that NRGs were closely associated with ES tumor immunity, and the signature was also good at predicting immunotherapy and chemotherapy response. Next, a comprehensive analysis of key genes showed that RIPK1, JAK1, and CHMP7 were potential therapeutic targets. The Cancer Dependency Map (DepMap) results showed that CHMP7 is associated with ES cell growth, and the Gene Set Cancer Analysis (GSCALite) results revealed that the JAK1 mutation frequency was the highest. The expression of 3 genes was all negatively correlated with methylation and positively with copy number variation (CNV). Finally, an accurate nomogram was constructed with this signature and clinical traits. In short, this study constructed an accurate prognostic signature and identified 3 novel therapeutic targets against ES.</p

Gene transduction and Sox9 expression in each treatment groups.

<p>(A–B): Bright light and fluorescence microscope examination showed the transduction efficiency of recombinant adenoviruses in monolayer culture (24 hours after transduction, 100X) and micromass culture (3 days after transduction, 40X), respectively. (C): Recombinant adenoviruses mediated overexpression of BMP2 and Sox9 mRNA were evaluated by semi-quantitative RT-PCR analysis using GAPDH as a house keeping gene. (D): Sox9 expression were evaluated by western blot analysis in each treatment group at day 2, 5, and 7 after transduction (a to c) and relative Sox9 expression were analyzed by quantity one software using β-actin as controls (d), the results are expressed as mean±SD of triplicate experiments, *<i>P<0.05</i>, ^#<i>P<0.01</i>.</p

Sox9 potentiates BMP2-induced cartilage formation and inhibits BMP2-induced endochondral ossification in MSCs implantation in vivo.

<p>(A): Macrographic images of ectopic masses. BMP2 or BMP2 and Sox9 co-infected C3H10T1/2 cells were implanted subcutaneously to the flanks of nude mice. Ectopic masses were retrieved at 5 weeks and 8 weeks (a). The volume of the masses was determined using vernier calipers (b). (B): Histological analysis of the retrieved samples. The retrieved samples were fixed, decalcified, paraffin-embedded and subjected to H&E, Masson’s Trichrome, Alcian blue and safranin O-fast green staining. Representative imagines are shown, magnification, 100X, scale bar = 1 mm.</p

Sox9 potentiates BMP2-induced glycosaminoglycans synthesis in MSCs in micromass cultures.

<p>(A): Real-time PCR for the expression of chondrogenic differentiation marker gene ACAN were conducted on day 7 and day 14 after infection of AdGFP, AdBMP2, and/or AdSox9, using GAPDH as a house keeping gene. (B–C): Alcian blue staining for sulfated glycosaminoglycans in micromass cultures of C3H10T1/2 cells on day 7 and day 14 after transduction of indicated recombinant adenoviruses, gross observation (B) and microscope examination (C, 40X) are shown. (D): Alcian blue staining quantifying: cells were extracted with 6 M guanidine hydrochloride, Optical density of the extracted dye was measured at 630 nm. The results were expressed as mean±SD of triplicate experiments, *<i>P<0.05</i>, ^#<i>P<0.01</i>.</p

Sox9 promotes expansion of the proliferating chondrocyte zone and inhibits BMP2-induced chondrocyte hypertrophy and ossification in organ cultures.

<p>(A): Mouse E18.5 forelimbs (n = 4 each group) were harvested and transduced with AdGFP, AdBMP2, and/or AdSox9. The forelimbs were cultured in organ culture medium and the transduction efficiency was visualized under bright light and fluorescence microscope (40X). (B): Histological analysis of the cultured forelimbs. The forelimbs were fixed, decalcified, paraffin-embedded and subjected to H&E staining. Representative imagines are shown (a), magnification, 100X. The average length of the hypertrophic zones, prehypertropic zones and proliferating zones were also determined by using Image J software (b). Hyp = hypertrophic chondrocyte zone, Pre = pre-hypertrophic chondrocyte zone, Pro =  proliferating chondrocyte zone. *<i>P<</i>0.05, ^#<i>P<</i>0.01, scale bar = 1 mm.</p

Sox9 inhibits BMP2-induced osteogenic markers expression in MSCs in vitro.

<p>(A): Real-time PCR for the expression of early osteogenic differentiation gene Runx2 (Aa), late osteogenic gene osteocalcin (OC) and osteopontin (OPN) (b, c) were conducted at indicated time points after infection with AdGFP, AdBMP2, and/or AdSox9, using GAPDH as a house keeping gene. (B): Western blot for the expression of Runx2 (a), OPN (b) and OC (c) were conducted at indicated time points after transduction of indicated recombinant adenoviruses, respectively. (C) Relative protein expression was analyzed by quantity one software using β-actin as controls respectively. The results are expressed as mean±SD of triplicate experiments, *<i>P<0.05</i>, ^#<i>P<0.01</i>.</p