Fig 2: Protein expression of collagens, fibrillins and collagen modifying enzymes in murine aRMS and eRMS. Representative images showing tissue microarray negative control immunohistochemical staining and positive tumoral immunohistochemical staining of COL18A1, COL4A1, COL4A2, FBN1, FBN2, PLOD1, and PLOD2. Note that the representative aRMS IHC image for FBN1 shown is nearly negative (scoring is 1). Antibody specificity is with tumor specimen ID in parentheses listed on the figure. Models used for IHC include murine aRMS models (U21459, U24988, U48484, U20745, and U23674), a human aRMS model (pcb00380), murine eRMS models (U37125, U34278, U24085, and U24055), and a human eRMS model (pcb00232).

Fig 3: FBXO2 regulates EC proliferation through FBN1. (A) FBXO2 and FBN1 protein levels in RL95-2-shFBN1, and RL95-2-shFBXO2/shFBN1 stably transfected cell lines (left) and in Ishikawa-ovFBN1 and Ishikawa-ovFBXO2/ovFBN1 transiently transfected cell lines (right). *P < 0.05, **P < 0.01, one-way ANOVA. (B,C) Cell proliferation was measured by RTCA (B), and colony formation assay (C) in RL95-2-shNC, RL95-2-shFBN1, and RL95-2-shFBXO2/shFBN1 stable cell lines. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, one-way ANOVA. (D) EdU staining intensity and proportion in RL95-2-NC, RL95-2-shFBXO2, RL95-2-shFBN1, RL95-2-shFBXO2/shFBN1 stable cell lines. (E,F) Apoptotic index (E) and cell cycle analysis (F) as measured by flow cytometric assay in RL95-2-NC, RL95-2-shFBXO2, RL95-2-shFBN1, and RL95-2-shFBXO2/shFBN1 cell lines. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, one-way ANOVA.

Fig 4: FBXO2-FBN1 promote EC cells proliferation by regulating the cell cycle and autophagy signaling pathways. (A) Heatmap and volcano plot of differential genes enrichment based on the RNA sequencing of the RL95-2-NC and RL95-2-shFBXO2 groups. (B) Go functional enrichment analysis. (C) KEGG pathway enrichment analysis. (D,E) GSEA (autophagy signaling pathway) enrichment analysis and the corresponding heatmap. (F,G) GSEA (the cell cycle) enrichment analysis and the corresponding heatmap.

Fig 5: (A–L) Verification of differential genes associated with the cell cycle and autophagy signaling pathways. (A) mRNA levels of MCM7, CDK4, CCND1, SMAC1A, CCND2, CHEK1, CDC14B, and CCNA1 in RL95-2-NC and RL95-2-shFBXO2 groups. CDK4, CCND1, CCND2, and CCNA1 were significantly down-regulated in the RL95-2-shFBXO2 group compared with RL95-2-NC group. Data are presented as mean ± SD, **P < 0.01, t-test. (B) mRNA levels of PPP2CA, ATG4A, ATG4B, ATG4C, and ATG4D in the RL95-2-NC and RL95-2-shFBXO2 groups. ATG4A and ATG4D were significantly up-regulated in the RL95-2-shFBXO2 group compared with the RL95-2-NC group. Data are presented as mean ± SD, **P < 0.01, ***P < 0.001, t-test. (C) Protein levels of CDK4, CyclinD1, CyclinD2, and CyclinA1 in RL95-2-NC, RL95-2-shFBXO2, RL95-2-shFBN1, RL95-2-shFBXO2/shFBN1, and RL95-2-ovFBN1 cells. Knockdown of FBXO2 and overexpression of FBN1 revealed the same tendency in decreasing protein levels of CDK4, CyclinD1, CyclinD2, and CyclinA1 compared with the RL95-2-NC group. Knockdown of both FBXO2 and FBN1 showed increased protein levels of CDK4, CyclinD1, CyclinD2, and CyclinA1 compared with the RL95-2-shFBXO2 group. α-Tublin was used as a loading control. (D) The autophagy-specific inhibitor 3-MA (2 mM) reversed the effects of FBXO2 on the RL95-2 cells proliferation. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, one-way ANOVA. (E) The CDK4/6 inhibitor palbociclib (2 μM) reversed the effects of FBXO2 on the Ishikawa cells proliferation. Data are presented as mean ± SD, **P < 0.01, one-way ANOVA. (F–L), FBXO2 silencing inhibited EC carcinogenicity in vivo via FBN1. (F) RL95-2-NC, RL95-2-shFBXO2, RL95-2-shFBN1, and RL95-2-shFBXO2/shFBN1 cells were injected into BALB/c nude mice subcutaneously (0.2 ml, 5 × 106 cells) and harvested at day 27. (G) Mice weight were monitored every 3 days and recorded (P > 0.05). (H,I) Tumor weight was recorded in a time-dependent manner (H) and at harvest day (I) among the RL95-2-NC, RL95-2-shFBXO2, RL95-2-shFBN1, and RL95-2-shFBXO2/shFBN1 groups. Data are presented as mean ± SD, *P < 0.05, **P < 0.01, one-way ANOVA. (J) IHC staining for FBXO2, FBN1, and Ki67 in histologic sections of transplanted tumors. (K) Inverse correlation between FBXO2 and FBN1 staining, Pearson r2 = –0.77, P < 0.001. (L) Positive correlation between FBXO2 and Ki67 staining, Pearson r2 = 0.857, P < 0.0001.