Journal: Oncogene

Article Title: KPNA2 promotes osteosarcoma progression by regulating the alternative splicing of DDX3X mediated by YBX1

doi: 10.1038/s41388-025-03375-3

Figure Lengend Snippet: A Venn diagram showed the overlap of genes between human RBP genes and differential expression genes (DEGs) associated with osteosarcoma. B Hierarchical clustering heatmap showed the FPKM of the differentially expressed RBPs (DERBPs) in three types of tissues. C The top 10 enriched GO terms (biological process) were illustrated for DERBPs co-expression RASEs (pSAR ≥ 50%) genes among different groups. D Co-expression network showed the important pathways of RASEs regulated by DERBPs. E Box plot showed the FPKM value of three representative DERBPs (LEPR, ADD3, and KPNA2). F Boxplot showed the splicing ratios of clualt5p174302 MCM7, clualt5p196175 GSN, cluir62713 PLK1, and cluir60246 POLG. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Then, the antigen was recovered using 2 min microwaved EDTA solution and incubated with goat serum and the primary antibody (KPNA2, ab289858, 1:200; ki67, Proteintech for IHC, 1:2000) overnight at 4 °C, washed with 1X PBS five times, and incubated with the DAKO secondary antibody at room temperature for 40 min.

Techniques: Quantitative Proteomics, Expressing

Journal: Oncogene

Article Title: KPNA2 promotes osteosarcoma progression by regulating the alternative splicing of DDX3X mediated by YBX1

doi: 10.1038/s41388-025-03375-3

Figure Lengend Snippet: A The qRT-PCR results of the co-expression network of DERBPs in osteosarcoma cell lines were shown in the bar plot. B , C Kaplan-Meier survival analysis of overall survival in patients, separately in two independent cohorts (the GSE21257 osteosarcoma cohort and the TCGA-SARC cohort). D Western blot assay showed KPNA2 levels in osteoblast cell line (hFOB1.19) and osteosarcoma cell lines (U2OS, MG63, HOS, MNNG/HOS). E , F Western blotting analysis detected the protein levels of KPNA2 from osteosarcoma specimens and corresponding adjacent non-tumor tissue (NAT) ( n = 8). G The mRNA expression levels of KPNA2 in sarcoma. H The uniform manifold approximation and projection (UMAP) plot of the 15 identified main cell types in 7 primary osteosarcoma lesions. I , J Feature plots of the KPNA2 and Ki-67 genes in the scRNA dataset. The color legend showed the expression levels of the genes. K , L IHC analysis was used to assess the protein levels of KPNA2 in parried osteosarcoma tissue. * P ≤ 0.05, **** P ≤ 0.0001. Log-rank test was used for survival comparison in ( B , C ). Two-tailed paired t tests for ( F , G , L ). Data were presented as mean ± SD (error bars) and representative of at least three independent experiments in ( A , D – G , K – L ).

Article Snippet: Then, the antigen was recovered using 2 min microwaved EDTA solution and incubated with goat serum and the primary antibody (KPNA2, ab289858, 1:200; ki67, Proteintech for IHC, 1:2000) overnight at 4 °C, washed with 1X PBS five times, and incubated with the DAKO secondary antibody at room temperature for 40 min.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Comparison, Two Tailed Test

Journal: Oncogene

Article Title: KPNA2 promotes osteosarcoma progression by regulating the alternative splicing of DDX3X mediated by YBX1

doi: 10.1038/s41388-025-03375-3

Figure Lengend Snippet: A Venn diagram showed the overlap of genes between DEGs in the primary vs. normal group, human RBPs, and KPNA2 binding proteins ranked by expression level in osteosarcoma tissue. B The U2OS and MNNG/HOS osteosarcoma cells underwent infection with the specified shRNAs. Cells were collected for Western blotting analysis after puromycin screening. C Analysis of white blood cells (WCL) or immunoprecipitation (IP) from U2OS, MG63, and MNNG/HOS cells using Western blotting. D – F Colony formation assays detected cell proliferation of U2OS and MNNG/HOS cells ( n = 4). G , H Transwell assays demonstrated the migration and invasion capabilities of U2OS and MNNG/HOS cells ( n = 4). The scale bar was 100 µm. I The tumor growth curve ( n = 5). J MNNG/HOS cells were subcutaneously into the nude mice for xenografts assay ( n = 5). K Ki-67 staining of the tumor. The scale bar was 100 µm. L Excised tumor mass as indicated ( n = 5). M The bar chart showed the quantitative results of Ki-67. Data were shown as the mean ± standard deviation of at least three independent experiments. ns no statistical significance; * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. One-way analysis of variance (ANOVA) was used to compare the difference in ( E , F , H , I , L , M ). Data were presented as mean ± SD (error bars) and representative of at least three independent experiments in ( B – M ).

Article Snippet: Then, the antigen was recovered using 2 min microwaved EDTA solution and incubated with goat serum and the primary antibody (KPNA2, ab289858, 1:200; ki67, Proteintech for IHC, 1:2000) overnight at 4 °C, washed with 1X PBS five times, and incubated with the DAKO secondary antibody at room temperature for 40 min.

Techniques: Binding Assay, Expressing, Infection, Western Blot, Immunoprecipitation, Migration, Staining, Standard Deviation

Journal: Oncogene

Article Title: KPNA2 promotes osteosarcoma progression by regulating the alternative splicing of DDX3X mediated by YBX1

doi: 10.1038/s41388-025-03375-3

Figure Lengend Snippet: A , B The subcellular distribution of YBX1 in the control transfected cells, KPNA2 knockdown, and KPNA2 overexpression by nuclear/cytoplasmic fractionation via immunofluorescence. The scale bar was 100 µm. C , D Western blotting showed YBX1 levels in different cellular components. The cytoplasmic control employed was MEK1/2, while the nuclear control utilized was Histone H3. E Expressions of YBX1 in the U2OS cells knockdown or overexpressed of KPNA2 were assessed by Western blotting. F , G Confocal microscopy was used to observe the distribution of YBX1 in the cytoplasm and nucleus across the control group, KPNA2 knockdown group, and KPNA2 overexpression group. The scale bar was 10 µm. ns no statistical significance; * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Two-tailed paired t tests for ( B , G ). Data were presented as mean ± SD (error bars) and representative of at least three independent experiments in ( A–G ).

Article Snippet: Then, the antigen was recovered using 2 min microwaved EDTA solution and incubated with goat serum and the primary antibody (KPNA2, ab289858, 1:200; ki67, Proteintech for IHC, 1:2000) overnight at 4 °C, washed with 1X PBS five times, and incubated with the DAKO secondary antibody at room temperature for 40 min.

Techniques: Control, Transfection, Knockdown, Over Expression, Fractionation, Immunofluorescence, Western Blot, Confocal Microscopy, Two Tailed Test

Journal: Oncogene

Article Title: KPNA2 promotes osteosarcoma progression by regulating the alternative splicing of DDX3X mediated by YBX1

doi: 10.1038/s41388-025-03375-3

Figure Lengend Snippet: A Sashimi plots of two osteosarcoma cell lines showed that DDX3X intron retention (IR) decreased after YBX1 knockdown. The DDX3X IR introduced premature termination codons (PTCs) that induce nonsense-mediated mRNA decay. The IR ratios and YBX1 binding motif were indicated. B The IgG and YBX1 groups were subjected to RIP followed by qPCR (RIP-qPCR) analysis to target DDX3X intron 17 ( n = 3). C The interaction of YBX1 protein and DDX3X promoter was identified using the ChIP-qPCR assay and assessed by relative enrichment to input ( n = 3). D qRT-PCR for DDX3X spliced and IR transcripts, following nuclear fractionation of U2OS cell lysates after YBX1 knockdown ( n = 3). E qRT-PCR for DDX3X IR/mRNA transcript level with or without NMD inhibitor (NMDI14) treatment regulated by KPNA2/YBX1 axis (n = 3). F The half-live time (t 1/2 ) of DDX3X mRNA was assessed after Actinomycin D (ActD) treatment regulated by KPNA2/YBX1 axis ( n = 3). G Western blotting showed the DDX3X protein levels in U2OS and MNNG/HOS cell lines regulated by the KPNA2/YBX1 axis (left panel), with quantification of DDX3X expression normalized to GAPDH shown in the right panel ( n = 3). ns no statistical significance; * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Two-tailed paired t tests for ( A ). One-way analysis of variance (ANOVA) was used to compare the difference in ( B – E , G ). Data were presented as mean ± SD (error bars) and representative of three independent experiments in ( A–G ).

Article Snippet: Then, the antigen was recovered using 2 min microwaved EDTA solution and incubated with goat serum and the primary antibody (KPNA2, ab289858, 1:200; ki67, Proteintech for IHC, 1:2000) overnight at 4 °C, washed with 1X PBS five times, and incubated with the DAKO secondary antibody at room temperature for 40 min.

Techniques: Knockdown, Binding Assay, ChIP-qPCR, Quantitative RT-PCR, Fractionation, Western Blot, Expressing, Two Tailed Test

Journal: Oncogene

Article Title: KPNA2 promotes osteosarcoma progression by regulating the alternative splicing of DDX3X mediated by YBX1

doi: 10.1038/s41388-025-03375-3

Figure Lengend Snippet: The graph illustrated the molecular mechanisms by which KPNA2 promotes osteosarcoma through DDX3X AS, mediated by YBX1.

Article Snippet: Then, the antigen was recovered using 2 min microwaved EDTA solution and incubated with goat serum and the primary antibody (KPNA2, ab289858, 1:200; ki67, Proteintech for IHC, 1:2000) overnight at 4 °C, washed with 1X PBS five times, and incubated with the DAKO secondary antibody at room temperature for 40 min.

Techniques: