Fig 2: The inhibitory effect on the osteoclastogenesis caused by circ-0110634 overexpression was rescued by TNFRSF1B up-regulation. (A) The effects of circ-0110634 on TNFRSF1B and its protein were evaluated by RT-qPCR and western blot. (B) TNFRSF1B expression was enhanced by pcDNA3.1/TNFRSF1B in PBMCs. (C) The cellular morphology in PBMCs transfected with pcDNA3.1 or pcDNA3.1/circ-0110634 or pcDNA3.1/circ-0110634+pcDNA3.1/TNFRSF1B was measured through TRAP staining. (D) TRAP activity was examined in PBMCs transfected with pcDNA3.1 or pcDNA3.1/circ-0110634 or pcDNA3.1/circ-0110634+pcDNA3.1/TNFRSF1B. (E) Numbers of resorption pits were counted in PBMCs transfected with pcDNA3.1 or pcDNA3.1/circ-0110634 or pcDNA3.1/circ-0110634+pcDNA3.1/TNFRSF1B. (F–I) RT-qPCR along with western blot was taken to analyze the expression and protein levels of TRAP, NFATc1 and CTSK in PBMCs transfected with pcDNA3.1 or pcDNA3.1/circ-0110634 or pcDNA3.1/circ-0110634+pcDNA3.1/TNFRSF1B. ∗P ​< ​0.05, ∗∗P ​< ​0.01.

Fig 3: Circ-0110634 bound to TNFRII and TRAF2 (A) Potential proteins which could interact with TNFRII (B) The interaction between these proteins and TNFRII in HEK-293T cells with pcDNA3.1/circ-0110634 (C) RNA pull down assay was taken to verify whether circ-0110634 could combine with TRAF2 (D–E) RNA-protein pull-down and RIP assays were taken to verify whether circ-0110634 could bind to TRAF2 and TNFRII (F) TNFRII protein was cut down into 6 pieces (G–H) RNA-protein pull-down assays and gel electrophoresis were taken to verify the specific part circ-0110634 could bind to TNFRII (I) TRAF2 protein was cut into 8 pieces (J–K) RNA-protein pull down assays and gel electrophoresis were performed to confirm the binding capacity between specific pieces of TRAF2 and circ-0110634 (L) The binding capacity between TRAF2 and TNFRII was analyzed in circ-0110634-upregulated cells. ∗∗P ​< ​0.01.

Fig 4: Circ-0110634 was up-regulated in ASMSCs exosomes/PBMCs cells (A) Schematic diagram of the genomic location and splicing pattern of circ-0110634 (B) Gel electrophoresis was performed to verify the circular structure of circ-0110634 (C–D) Rnase R and ActD treated with circ-0110634 and linear TNFRSF1B (E–F) FISH and Nuclear cytoplasm fractionation were carried out to verify circ-0110634 location in ASMSCs and HDMSCs (G) RT-qPCR was used to detect circ-0110634 expression in HDMSCs exosomes and ASMSCs exosomes and PBMCs co-cultured with HDMSC exosomes and ASMSCs exosomes (H) Circ-0110634 location in HDMSCs exosomes and ASMSCs exosomes was identified through FISH assay. ∗∗P ​< ​0.01, n. s. meant no significance.

Fig 5: TNFSF2, TNF-R1, and TNF-R2 in experimental ER+ breast cancer. Oophorectomized athymic mice supplemented with physiological levels of estradiol (E2) were injected with MCF-7 into the dorsal mammary fat pads. At similar tumor sizes, mice either continued with E2 or were additionally treated with fulvestrant (E2+Fulv) (5 mg/mouse every 3 days, s.c.). Size-matched tumors from the different treatment groups underwent microdialysis for sampling of extracellular proteins in vivo, which were quantified using proximity extension assay. Data represents extracellular local protein abundance in linear values (2NPX as described in the Methods section). Tumor sections were subjected to immunohistochemistry. Data are presented as the mean ± SD. *P < 0.05. (A) Extracellular TNFSF2, n = 4 animals per group. (B) Tumor sections from each treatment group were stained for collagen (blue) and quantified as the percentage of area with positive staining. Representative sections are depicted. Scale bar = 20 µm. (C) Soluble TNF-R1, n = 4 animals per group. (D) Tumor sections from each treatment group were stained for TNF-R1 and quantified as the percentage of area with positive staining. Representative sections are depicted. Scale bar = 20 µm. (E) Soluble TNF-R2, n = 4 animals per group. (F) Tumor sections from each treatment group were stained for TNF-R2 and quantified as the percentage of area with positive staining. Representative sections are depicted. Scale bar = 20 µm.