Journal: Cell Reports Medicine

Article Title: Sequence-dependent splicing dysregulation drives therapy resistance in pediatric AML

doi: 10.1016/j.xcrm.2025.102542

Figure Lengend Snippet: Exploration of genetic and transcriptomic correlations with aberrant splicing in pediatric AML (A) Oncoprint displays somatic driver mutations in Suzhou pediatric AML. Cases are divided into High PSI and Low PSI groups, and the genes are grouped based on biological pathways (Fisher’s exact test). (B) Comparison of mutation frequency between Suzhou and SCMC pediatric AML cohorts. (C) Heatmap showing the overall gene expression landscape of 380 RBPs in Suzhou pediatric AML. RBP genes are further clustered into three groups based on the Euclidean distance. (D) The correlations between the 76 AS events and RBP expressions. Edge width means the R values of Mantel’s statistic, and edge color represents the group based on clusters identified in Figures C and Figure C. (E) Correlation between RBPs and splicing events. In the left protein interaction network, edge color shows interaction strength (from STRING), node color indicates RBP correlation with the overall splicing scores, and node size reflects the proportion of splicing events containing each binding motif of RBPs. The right heatmap shows the correlation between RBPs and splicing events across patients (Pearson’s correlation). ∗ p < 0.05, ∗∗ p <0.01, ∗∗∗ p < 0.001. (F) Scatterplot showing U2AF2 gene expression (log 2 -normalized FPKM) across groups from the Suzhou pediatric AML (Student’s t test). FPKM: fragments per kilobase of transcript per million mapped reads. (G) The heatmap shows validated splicing events following each RBP knockdown in the K562 cell line.

Article Snippet: U2AF2 cDNA , CUSABIO , Cat#CSB-CL025408HU.

Techniques: Comparison, Mutagenesis, Gene Expression, Binding Assay, Knockdown

Journal: Cell Reports Medicine

Article Title: Sequence-dependent splicing dysregulation drives therapy resistance in pediatric AML

doi: 10.1016/j.xcrm.2025.102542

Figure Lengend Snippet: U2AF2 downregulation leads to abnormal splicing events in pediatric AML (A and B) RT-PCR was performed to analyze mis-splicing events upon U2AF2 knockdown (A) or overexpression (B) in K562 cells ( n = 3, mean ± SD; Student’s t test). (C) Schematic representation of U2AF2 binding locations. (D) U2AF2-binding sites in WT and KD conditions by CLIP sequencing data from the ENCODE dataset. (E) INTS3 splicing event and the binding region of the Py-track with U2AF2 were visualized using the CLIP data. (F) Schematic representation of the INTS3 minigenes with different TC score constructs (left). Splicing analysis of different INTS3 minigenes in K562 cells (right) ( n = 3, mean ± SD). (G) RT-PCR was performed to analyze INTS3 mis-splicing events with different Py-tracks upon U2AF2 knockdown ( n = 3, mean ± SD; Student’s t test). (H) HNRNPH1 splicing event and the binding region of the Py-track with U2AF2 were visualized using the CLIP data. (I) Schematic representation of the HNRNPH1 minigenes with different TC score constructs (left). Splicing analysis of different HNRNPH1 minigenes in K562 cells (right) ( n = 3, mean ± SD). (J) RT-PCR was performed to analyze HNRNPH1 mis-splicing events with different Py-tracks upon U2AF2 knockdown ( n = 3, mean ± SD; Student’s t test). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: U2AF2 cDNA , CUSABIO , Cat#CSB-CL025408HU.

Techniques: Reverse Transcription Polymerase Chain Reaction, Knockdown, Over Expression, Binding Assay, Sequencing, Construct

Journal: Cell Reports Medicine

Article Title: Sequence-dependent splicing dysregulation drives therapy resistance in pediatric AML

doi: 10.1016/j.xcrm.2025.102542

Figure Lengend Snippet: Downregulation of U2AF2 leads to chemotherapy resistance in AML in vitro and vivo (A) Oncoprint displays mutated genes associated with drug resistance from the Suzhou pediatric AML cohort. (B) Scatterplot showing U2AF2 expression (log 2 -normalized FPKM) in drug-resistant patients from the Suzhou cohort (Student’s t test). (C) Western blot showing the expression of U2AF2 after U2AF2 knockdown in THP1 and K562 cells. (D–G) The IC 50 value for cytarabine and daunorubicin after U2AF2 knockdown in THP1 and K562 cells. Cell viability was assessed using a CCK8 assay ( n = 3; mean ± SD; Student’s t test). (H) Schematic of Mll-Af9+scramble and Mll-Af9+shU2af2 mouse cells transplantation models in which recipient mice from cohort 1 were euthanized at day 16 post-transplant and mice from cohort 2 were observed for survival analysis until end stage. (I) Representative photograph of spleens from cohort 1 with an inch scale (left) ( n = 5), and spleen weight (right) ( n = 5; mean ± SD; Student’s t test). Scale bar, 1 cm. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (J) Representative percentages of GFP+mcherry+ cells of BM and spleen in the recipient from cohort 1 by flow cytometry analysis ( n = 5; mean ± SD; Student’s t test). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (K) Kaplan-Meier survival analysis of recipient mice from cohort 2 ( n = 5; log rank test).

Article Snippet: U2AF2 cDNA , CUSABIO , Cat#CSB-CL025408HU.

Techniques: In Vitro, Expressing, Western Blot, Knockdown, CCK-8 Assay, Transplantation Assay, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Sequence-dependent splicing dysregulation drives therapy resistance in pediatric AML

doi: 10.1016/j.xcrm.2025.102542

Figure Lengend Snippet: Combined inhibition of PRMT5 and type I PRMTs rescues aberrant splicing events and decreases chemotherapy resistance (A) The heatmap shows the percentage of rescued events among the 76 splicing events in NB4 leukemia cells treated with MS023 alone, GSK591 alone, and a combination of both. “Rescued” events were defined as those showing >1% PSI recovery toward the control level with statistical significance ( p < 0.05, paired Student’s t test). (B) RNA-seq coverage plots of the splicing events in NB4 leukemia cells treated with DMSO, MS023 (MS) alone, GSK591 (GSK) alone, and a combination of both (M + G). The red part in each track below represents aberrant splicing regions in each of the genes. (C) RT-PCR results detecting aberrantly spliced transcripts in THP1 leukemia cells, which were treated with PRMT inhibitor MS023 or GSK591 with or without U2AF2 knockdown ( n = 3; mean ± SD; Student’s t test). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (D) RT-PCR results detecting aberrantly spliced transcripts in THP1 leukemia cells, which were treated with PRMT inhibitor MS023 and GSK591 combinations with or without U2AF2 knockdown ( n = 3; mean ± SD; Student’s t test). ∗ p < 0.05, ∗∗ p <0.01 ,∗∗∗ p < 0.001. (E) The drug response curve and IC 50 value for PRMT inhibitor MS023 and GSK591 combinations after U2AF2 knockdown in THP1 leukemia cells. Cell viability was assessed using a CCK8 assay ( n = 3; mean ± SD; Student’s t test).

Article Snippet: U2AF2 cDNA , CUSABIO , Cat#CSB-CL025408HU.

Techniques: Inhibition, Control, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Knockdown, CCK-8 Assay

Journal: Heliyon

Article Title: LncRNA CECR7 boosts hepatocellular carcinoma progression by recruiting RNA binding protein U2AF2 to enhance the stability of EXO1 mRNA

doi: 10.1016/j.heliyon.2023.e19862

Figure Lengend Snippet: CECR7 stabilize EXO1 mRNA under the mediation of U2AF2 protein. (A) The platform starBase ( http://starbase.sysu.edu.cn ) was applied to predict the potential RNA binding protein for both CECR7 and EXO1 mRNA. ( B, C ) RIP assay using antibody against U2AF2 to determine the enrichment of CECR7 and EXO1 mRNA in HCC cells (Mean ± SD; n = 3). ** P < 0.01, *** P < 0.001, Student's t -test. ( D, E ) RT-qPCR analysis and Western blot were used to detect the effect of CECR7 on U2AF2 expression. ( F, G ) U2AF2-overexpressing reversed the repression of EXO1 mRNA by CECR7 shRNA#1, and the EXO1 mRNA induction induced by CECR7-overexpressing was abrogated by U2AF2 shRNA (Mean ± SD; n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA.

Article Snippet: Lysates of HCC cells were cultured in RIP containing magnetic beads conjugated with antibodies against U2AF2 (NBP2-33397, Novus, USA) or control IgG (NBP2-34250, Novus, USA) for 6h at 4 °C.

Techniques: RNA Binding Assay, Quantitative RT-PCR, Western Blot, Expressing, shRNA

Journal: Bioscience Reports

Article Title: Differential regulation of sFlt-1 splicing by U2AF65 and JMJD6 in placental-derived and endothelial cells

doi: 10.1042/BSR20193252

Figure Lengend Snippet: rtPCR was performed to ensure that adequate knockdown ( A ) and overexpression ( B ) of U2AF65 and JMJD6 was achieved in HUVECs ( N = 4). Western blots were also done to see if the message translated to the protein level. Western blots for knockdown of U2AF65 and JMJD6 are shown in ( C ) and samples from overexpression of the proteins are seen in ( D ) ( N = 2). * P < 0.05; # P < 0.01

Article Snippet: Human U2AF65 (NM_007279) was cloned into pCMV6-Entry vector (Origene).

Techniques: Reverse Transcription Polymerase Chain Reaction, Over Expression, Western Blot

Journal: Bioscience Reports

Article Title: Differential regulation of sFlt-1 splicing by U2AF65 and JMJD6 in placental-derived and endothelial cells

doi: 10.1042/BSR20193252

Figure Lengend Snippet: rtPCR was performed to ensure adequate knockdown ( A ) and overexpression ( B ) was achieved in BeWo placental trophoblasts ( N = 6). Additionally, Western blots were performed to see if the message translated to the protein expression. U2AF65 and JMJD6 knockdown Western blots and their analyses are shown in ( C ), while overexpression can be observed in ( D ) ( N = 3). * P < 0.05; # P < 0.01

Article Snippet: Human U2AF65 (NM_007279) was cloned into pCMV6-Entry vector (Origene).

Techniques: Reverse Transcription Polymerase Chain Reaction, Over Expression, Western Blot, Expressing

Journal: Bioscience Reports

Article Title: Differential regulation of sFlt-1 splicing by U2AF65 and JMJD6 in placental-derived and endothelial cells

doi: 10.1042/BSR20193252

Figure Lengend Snippet: HUVEC knockdown ( A ) and overexpression ( B ) samples were analyzed for impact on sFlt-1 mRNA, with JMJD6 overexpression causing significantly increased sFlt-1 version 3 expression ( N = 4). Media from HUVEC samples were analyzed for sFlt-1 protein. Both knockdown ( C ) and overexpression ( D ) media showed trends of sFlt-1 protein in the same direction as U2AF65 and JMJD6 expression ( N = 4). BeWo knockdown ( E ) or overexpression ( F ) had no effect on sFlt-1 mRNA ( N = 6). Similarly, media collected from U2AF65 or JMJD6 knockdown ( G ) or overexpression ( H ) had no difference in media sFlt-1 concentration ( N = 6). Two-way ANOVA was performed on the rtPCR samples, and one-way ANOVA was performed on media sFlt-1 samples * P < 0.05 vs. control. ND = Not detectable.

Article Snippet: Human U2AF65 (NM_007279) was cloned into pCMV6-Entry vector (Origene).

Techniques: Over Expression, Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Bioscience Reports

Article Title: Differential regulation of sFlt-1 splicing by U2AF65 and JMJD6 in placental-derived and endothelial cells

doi: 10.1042/BSR20193252

Figure Lengend Snippet: HUVEC knockdown samples showed that there was no effect on heparanase mRNA expression ( N = 4) ( A ) or media protein ( N = 3) ( B ). HUVEC JMJD6 overexpression had significant increase in heparanase mRNA ( N = 4) ( C ), but only HUVEC overexpression of U2AF65 had significant increase in media heparanase protein ( N = 3) ( D ). BeWo knockdown of JMJD6 showed significantly decreased heparanase message ( N = 6) ( E ), but none of the BeWo knockdown groups showed any change in heparanase media protein ( N = 3) ( F ). BeWo overexpression of U2AF65 and JMJD6 had no effect on heparanase mRNA ( N = 6) ( G ) or media protein ( N = 3) ( H ). One-way ANOVE was performed for all groups. * P < 0.05 vs. control.

Article Snippet: Human U2AF65 (NM_007279) was cloned into pCMV6-Entry vector (Origene).

Techniques: Expressing, Over Expression

Journal: Bioscience Reports

Article Title: Differential regulation of sFlt-1 splicing by U2AF65 and JMJD6 in placental-derived and endothelial cells

doi: 10.1042/BSR20193252

Figure Lengend Snippet: HUVECs cultured in hypoxia for 24 h had significantly up-regulated JMJD6, with no change in U2AF65 expression ( A ). Similarly, BeWo cells exposed to hypoxia also had unchanged U2AF65 but significantly elevated JMJD6 ( B ). Chronic hypoxia of the RUPP placenta, however, had significantly increased U2AF65 ( C ) and JMJD6 ( D ) compared with sham animals. Two-way ANOVA was performed for both cell groups. Student’s t -test was performed on the placental protein data. N = 6 in all groups. * P < 0.05; # P < 0.01 ## P < 0.001 vs. control/ sham.

Article Snippet: Human U2AF65 (NM_007279) was cloned into pCMV6-Entry vector (Origene).

Techniques: Cell Culture, Expressing