Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: The expression levels of CSTF2 in human cancers. (A) The expression levels of CSTF2 in different tumor tissues compared to normal tissues in The Cancer Genome Atlas (TCGA) database. (B) The expression of CSTF2 in tumor tissues and matched TCGA normal in TCGA database and GTEx data by GEPIA analysis. * ρ < 0.05, ** ρ < 0.01, *** ρ < 0.001 compared to normal tissues.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: CSTF2 is highly expressed in HCC. (A) Analysis of CSTF2 mRNA expression levels in TCGA, International Cancer Genome Consortium (ICGC), and Chinese patients with hepatitis B virus (CHCC-HBV) databases (B) Representative images of the immunohistochemistry staining of CSTF2 in hepatocellular carcinoma (HCC) and adjacent non-tumor tissues from different HCC patients (tumor indicates HCC tissue; normal indicates adjacent normal tissue). (C) Quantitative analysis of sample numbers in different CSTF2 expression levels in 48 pairs of HCC tissues. (D) Western blot analysis of CSTF2 protein levels in non-tumorigenic LO2 liver cells, hepatocellular carcinoma cell lines Huh7, MHCC-97H, and Hep3B (left panel) and qualification of the relative intensity of CSTF2 protein levels (right panel). The levels of β-actin were used as an internal control. ** ρ < 0.01, *** ρ < 0.001.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Expressing, Virus, Immunohistochemistry, Staining, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: The mRNA levels of CSTF2 were correlated with the advanced clinical stage in HCC in different databases. (A-F) The relationship between CSTF2 expression levels with a histological grade, a clinical stage in TCGA databases, ICGC databases, CHCC-HBV databases, GSE14520 databases, and GSE76427 databases and in GSE36376 databases. ** ρ < 0.01, *** p < 0.001 compared to normal.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: CSTF2 higher expression is associated with poor prognosis in HCC. (A-D) Overall survival and relapse-free survival in TCGA-LIHC, CHCC-HBV, GSE14520, and ICGC databases (E) Univariate and (F) multivariate Cox analyses of CSTF2 expression at different TNM stages, ages, and genders.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: Knockout of CSTF2 inhibited the proliferation, migration, and invasion of HCC cells in vitro . (A) The stable CSTF2 knockout Huh7 and MHCC-97H cells were determined by Western blot (upper panel) and the qualification of the relative intensity of CSTF2 protein levels (bottom panel). The levels of β-actin were used as an internal control. (B) The cell proliferation ability of CSTF2 knockout Huh7 and MHCC-97H cells was measured by the CCK-8 assay. (C) Colony-forming assay of CSTF2 knockout Huh7 cells and MHCC-97H cells (left panel) and the qualification of the number of colonies in these cells (right panel). (D) The migration and invasion capacity were evaluated in CSTF2 knockout Huh7 cells and MHCC-97H cells by the Transwell assay (upper panel) and the qualification of migrated cells and invading cells per field (bottom panel). Scale bar, 50 μm. sgCSTF2 represents CSTF2-sgRNA. An empty vector was used as a control of CSTF2-sgRNA. * ρ < 0.05, ** p < 0.01, *** p < 0.001 compared to vector control.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Knock-Out, Migration, In Vitro, Western Blot, Control, CCK-8 Assay, Transwell Assay, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: Overexpression of CSTF2 promoted the cell proliferation, migration, and invasion of HCC cells in vitro . (A) The stable overexpression of CSTF2 in Hep3B cells was determined by Western blot (left panel) and the qualification of the relative intensity of CSTF2 protein levels (right panel). (B) CCK-8 assay of the cell proliferation rate of the overexpression of CSTF2 Hep3B cells. (C) Colony-forming assay of the overexpression of CSTF2 Hep3B cells (left panel) and the qualification of the number of colonies formed (right panel). (D) The migration and invasion were measured in the overexpression of CSTF2 Hep3B cells by the Transwell assay (left panel) and the qualification of migrated cells and invading cells per field (right panel). CSTF2 represents the overexpression of CSTF2. An empty vector was used as control. Scale bar, 50 μm. * ρ < 0.05, ** ρ < 0.01, *** ρ < 0.001 compared to the vector.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Over Expression, Migration, In Vitro, Western Blot, CCK-8 Assay, Transwell Assay, Plasmid Preparation, Control

Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: Knockout of CSTF2 inhibited the tumorigenesis and progression of HCC. (A) In vivo tumor formation of CSTF2 sgRNA Huh7 and vector control Huh7. Tumorigenesis experiments were performed by subcutaneously injecting Huh7 cells into BALB/c nude mice. (B) Strategy for evaluating the hepatocarcinogenesis function of CSTF2 in immunocompetent C57BL/6 mice. Schematic representation of the hydrodynamic tail vein injection HCC model. Plasmids pT3-EF1A-MYC-IRES-luc, px330-p53 (p53-sgRNA), and CMV-SB13 transposase were delivered together with either CSTF2 sgRNA (sgCSTF2) or the control vector for HCC induction. (C) Representative images of dissected livers in CSTF2 sgRNA and the vector control group. (D) The number of tumor nodules in mice injected with sgCSTF2 was significantly lower than that in the control group. (E) Qualification of the liver weight, and the ratio of liver weight to body weight of these tumor nodules in CSTF2 sgRNA or control groups. At least 6 mice were used in every group. (F) Immunohistochemical staining formed tumors in the liver tissues using CSTF2 and Ki67 antibodies. Scale bar, 100 μm. sgCSTF2 represents CSTF2-sgRNA. The empty vector of sgRNA was used as control. * ρ < 0.05, ** ρ < 0.01 compared to control.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Knock-Out, In Vivo, Plasmid Preparation, Control, Injection, Immunohistochemical staining, Staining

Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: Functional enrichment analysis of CSTF2 in the TCGA cohort. (A) GO classification analysis of gene changes in the biological process. (B) KEGG pathway enrichment analysis of upregulated pathways (C, D) GSEA analysis of activated pathways and hallmarks of CSTF2 related in HCC based on the TCGA database.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Functional Assay

Journal: Frontiers in Oncology

Article Title: CSTF2 Promotes Hepatocarcinogenesis and Hepatocellular Carcinoma Progression via Aerobic Glycolysis

doi: 10.3389/fonc.2022.897804

Figure Lengend Snippet: High expression of CSTF2 enhances aerobic glycolysis in HCC. (A) Heat map of the cluster analysis of the relationship between the CSTF2 expression and glycolysis-related enzymes and glucose transporters. (B) The correlation analysis of CSTF2 expression and glycolysis-related enzymes and glucose transporters. (C-D) The relative expression levels of CSTF2, SLC2A1, HK2, LDHA, PKM, PFKFB3, and PFKM detected by quantitative RT-PCR in Huh7 cells and MHCC-97H cells. (E–F) The protein levels of HK2 in Huh7 cells and MHCC-97H cells determined by Western blot; the levels of β-actin were used as an internal control. (G) 3’ RACE HK2 fragments were amplified in Huh7 cells and detected by agarose gel electrophoresis. (H–J) The glycolytic function of HCC cells was measured by the extracellular acidification rate (EACR), relative glucose uptake, and lactate production in CSTF2 knockout Huh7 and MHCC-97H cells. (K-L) The glycolytic function of HCC cells was measured by EACR, the relative glucose uptake, and lactate production in CSTF2-overexpression Hep3B cells. sgCSTF2 represents CSTF2 sgRNA, CSTF2 represents CSTF2 overexpression, and the vector was used as control. Data were presented as the mean ± SEM. ** ρ < 0.01, *** ρ < 0.001 compared to the vector.

Article Snippet: The sections were blocked with 10% normal donkey serum in PBST (Gibco, USA) for 1 h and incubated with primary antibodies against CSTF2 (1:200, Proteintech, China) or Ki67 (1:1000, Abcam, Cambridge, United Kingdom, Rabbit) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Amplification, Agarose Gel Electrophoresis, Knock-Out, Over Expression, Plasmid Preparation

Journal: Genome Biology

Article Title: Alternative polyadenylation factors link cell cycle to migration

doi: 10.1186/s13059-018-1551-9

Figure Lengend Snippet: Use of distal polyadenylation sites and lower levels of cleavage and polyadenylation factors during quiescence. a UCSC Genome browser views showing the long and short isoforms of INF2 and BOC. The differentially expressed exon is highlighted in cyan. b Real-time PCR validation of APA with quiescence. cDNA samples generated from fibroblasts that were proliferating, quiescent by contact inhibition or serum starvation, or induced into quiescence by serum-starvation and then restimulated, were analyzed with real-time PCR. Primers were designed to recognize the short (terminating at the proximal polyadenylation site) or long (terminating at the distal polyadenylation site) isoforms of INF2 or BOC. Transitioning fibroblasts into quiescence resulted in reduced expression of the short isoform of INF2 and increased expression of the long isoform of BOC. Restimulating quiescent fibroblasts resulted in expression patterns of the short and long isoforms that more closely resemble proliferating cells. Plots show individual datapoints as dots. Bar graphs represent mean and average ± S.D. The number of replicates for all conditions for short and long INF2 is 3. The number of replicates for all conditions for long BOC is 3. The number of replicates for P, 7dCI, and 7dCI-R for short BOC is 3. The number of replicates for 7dSS for short BOC is 2. Statistical significance in knockdown cells compared to control cells was determined for long and short isoforms with two-tailed, unpaired t tests. For all figures, one asterisk indicates p value < 0.05. Two asterisks indicate p value < 0.01. Three asterisks indicate p value < 0.001. c A shift toward expression of longer isoforms in quiescent fibroblasts. Proliferating and 7dCI were analyzed by polyadenylation site-enriched RNA-Seq. Relative use of the distal polyadenylation site (RUD) for individual genes in proliferating fibroblasts is plotted on the x-axis and RUD for the same gene in quiescent conditions is plotted on the y-axis. The dashed black line indicates y = x . The first plot (left) displays all genes with two detected polyadenylation sites. The middle plot displays UTR APA genes and the final plot (right) shows the same data for genes that undergo UR APA. d Immunoblotting was performed on protein lysates collected from proliferating, 7dCI and 7dSS fibroblasts for CstF-64, CFIm25, and CPSF73. Phosphorylation of serine 5 on RNA pol II CTD was monitored by immunoblotting and levels decline with quiescence. α-Tubulin was monitored as a loading control

Article Snippet: Slides were treated with primary antibodies against Ki-67 (Abcam, catalog no. ab16667, dilution 1:150), histone H4 (EMD Millipore, 05-858, 1:2000), CstF-64 (Bethyl Laboratories, IHC-00221, 1:1000), CPSF73 (Bethyl, A301-090A, 1:200) or CFIm25 (Sigma, AV40695, 1:200), followed by EnVision+ HRP-conjugated secondary antibody (Dako) and DAB chromogen (Roche) visualization.

Techniques: Real-time Polymerase Chain Reaction, Generated, Inhibition, Expressing, Two Tailed Test, RNA Sequencing Assay, Western Blot

Journal: Genome Biology

Article Title: Alternative polyadenylation factors link cell cycle to migration

doi: 10.1186/s13059-018-1551-9

Figure Lengend Snippet: Knockdown of cleavage and polyadenylation factors results in changes in isoform use and gene expression that overlap with quiescence. a Knockdown of cleavage and polyadenylation factors induces a shift in isoform expression. Real-time PCR was performed for the short and long isoforms of INF2 and BOC in proliferating fibroblasts expressing a control siRNA or an siRNA that targets CFIm25, CstF-64, or CPSF73. The short isoform of INF2 or BOC was significantly reduced in cells transfected with an siRNA against CstF64 or CPSF73. Plots show individual datapoints as dots. Bar graphs represent mean and average ± S.D. The number of replicates for control, CFIm25 and CPSF73 knockdown for short and long INF2 is 6. The number of replicates for CstF64 knockdown for short and long INF2 is 3. The number of replicates for all conditions for long BOC is 2, except the control, which had 3 replicates. The number of replicates for control and CFIm25 knockdown for short BOC is 3. The number of replicates for CstF64 and CPSF73 knockdown for short BOC is 2. Statistical significance in knockdown cells compared to control cells was determined for long and short isoforms with two-tailed, unpaired t-tests. b Overlap among genes that undergo APA with quiescence and knockdown of cleavage and polyadenylation factors. The overlap between genes that use the proximal polyadenylation site with quiescence and use a proximal polyadenylation site preferentially with CFIm25 knockdown is shown on the left. Overlap between genes that use distal polyadenylation sites with quiescence and genes that use distal polyadenylation sites with CPSF73 or CstF64 knockdown are shown in the middle and the right, respectively. c Overlap between genes upregulated with quiescence and genes upregulated with CstF-64 knockdown (left) and overlap between genes downregulated with quiescence and genes downregulated with CstF-64 knockdown (right). The overlap between groups of genes was tested using the hypergeometric test

Article Snippet: Slides were treated with primary antibodies against Ki-67 (Abcam, catalog no. ab16667, dilution 1:150), histone H4 (EMD Millipore, 05-858, 1:2000), CstF-64 (Bethyl Laboratories, IHC-00221, 1:1000), CPSF73 (Bethyl, A301-090A, 1:200) or CFIm25 (Sigma, AV40695, 1:200), followed by EnVision+ HRP-conjugated secondary antibody (Dako) and DAB chromogen (Roche) visualization.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Two Tailed Test

Journal: Genome Biology

Article Title: Alternative polyadenylation factors link cell cycle to migration

doi: 10.1186/s13059-018-1551-9

Figure Lengend Snippet: Cleavage and polyadenylation factors are expressed at higher levels in fibroblasts near a wound than in fibroblasts of healthy skin. Mouse skin was collected 5 days after introduction of a punch biopsy. Normal mouse skin was collected 2 cm away from the wound. Samples were stained with immunohistochemistry for proliferation marker Ki-67, histone H4 as a control, or alternative polyadenylation and cleavage factors CstF-64, CPSF73 or CFIm25 (brown). Samples analyzed with immunohistochemistry were counterstained with hematoxylin (blue nuclei). Individual cells at different positions from the wounds were assigned positive or negative staining and the percentages are shown. Ki-67 does not label all dividing cells, and likely underestimates the fraction of cells that are actively cycling . Levels of all three cleavage and polyadenylation factors were higher in the fibroblasts, myofibroblasts and immune cells proximal to a wound than in the fibroblast-rich dermal areas of healthy skin distal to the wound

Article Snippet: Slides were treated with primary antibodies against Ki-67 (Abcam, catalog no. ab16667, dilution 1:150), histone H4 (EMD Millipore, 05-858, 1:2000), CstF-64 (Bethyl Laboratories, IHC-00221, 1:1000), CPSF73 (Bethyl, A301-090A, 1:200) or CFIm25 (Sigma, AV40695, 1:200), followed by EnVision+ HRP-conjugated secondary antibody (Dako) and DAB chromogen (Roche) visualization.

Techniques: Staining, Immunohistochemistry, Marker, Negative Staining

Journal: Genome Biology

Article Title: Alternative polyadenylation factors link cell cycle to migration

doi: 10.1186/s13059-018-1551-9

Figure Lengend Snippet: Knockdown of APA factors results in reduced migration. a Example of Incucyte migration assay. Bright-field images from an assay monitoring the rate of migration into a denuded area (marked by a double-arrow) performed with Incucyte real-time imaging are shown. b Proliferating fibroblasts migrate more rapidly into a denuded area than quiescent fibroblasts. Fibroblasts were sampled in proliferating conditions, 7dSS conditions (7dSS), or after 7dSS followed by serum restimulation (7dSS-R). Fibroblasts were plated into 96-well plates and a portion of the well was denuded of cells. Plates were analyzed with an Incucyte real-time imaging instrument and the associated software to monitor the rate at which fibroblasts migrated into the denuded area. The ratio of cell density in the denunded area to the non-denuded area (relative wound density) over a time-course is plotted. Six wells were monitored for each condition and data represent mean and standard deviation. Proliferating versus 7dSS samples ( p value < 0.001, repeated measures two-way ANOVA with Dunnett’s multiple comparison test), proliferating versus 7dSS-restimulated samples ( p value < 0.001), and 7dSS versus 7dSS-R ( p value < 0.001) were statistically significantly different. c Immunoblots demonstrating knockdown of the targeted cleavage and polyadenylation factor by siRNAs in fibroblasts. The percent knockdown of protein level is also shown. d Knockdown of CstF-64 reduces fibroblast migration. Fibroblasts were transfected with a control siRNA or an siRNA against CFIm25, CstF-64, or CPSF73. CstF-64-knockdown fibroblasts exhibited reduced migration into a denuded area than control fibroblasts (CstF64.1 p value = 0.0013). Two additional siRNAs against CstF-64 (CstF64.2 and CstF64.3) reduced migration compared with a matched control siRNA as well (CstF64.2 p value = 0.0021, CstF-64.3 p value = 0.0384). Six replicates were performed for each condition. e Knockdown of CstF-64 or CPSF73 reduced migration of triple negative breast cancer cells. Triple negative breast cancer cell line MDA-MB-231 was transfected with a control siRNA or an siRNA against CstF-64, CPSF73 or CFIm25. Migration into a denuded area on the plate was monitored with an Incucyte instrument. Knockdown of CstF-64 or CPSF73 resulted in reduced migration (CstF64 p value = 0.0002, CPSF73 p value = 0.0013). For all conditions, the number of replicates for each condition was 6. f Schematic diagram showing elevated cleavage and polyadenylation factors in fibroblasts in the wound-healing environment. Increased expression of CstF-64, CPSF73, and CFIm25 in fibroblasts in wounds is expected to result in increased use of proximal polyadenylation sites and may promote fibroblast migration to the wound

Article Snippet: Slides were treated with primary antibodies against Ki-67 (Abcam, catalog no. ab16667, dilution 1:150), histone H4 (EMD Millipore, 05-858, 1:2000), CstF-64 (Bethyl Laboratories, IHC-00221, 1:1000), CPSF73 (Bethyl, A301-090A, 1:200) or CFIm25 (Sigma, AV40695, 1:200), followed by EnVision+ HRP-conjugated secondary antibody (Dako) and DAB chromogen (Roche) visualization.

Techniques: Migration, Imaging, Software, Standard Deviation, Western Blot, Transfection, Expressing

Journal: Scientific reports

Article Title: Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

doi: 10.1038/s41598-024-61951-1

Figure Lengend Snippet: Figure 5. RBM3 increases at 8 h of costimulation of primary human T cells and has its binding motif is enriched around dPAS that are repressed at 8 h. (A) Log2 fold changes of proteins that have been implicated in APA regulation. (B) Western blot analysis of RBM3 and hnRNP A3 protein following 8 h of CD3/CD28 costimulation of CD4+ human T cells. (C) Presence of RBM3 binding motifs around pPAS and dPAS elements for events that exhibit shortening at 8 h versus 48 h or show no change.

Article Snippet: Antibodies used to detect protein expression levels are as follows: CSTF2 (abcam: ab72297), CPSF3 (abcam: ab72299), CPSF6 (Santa Cruz: sc-100692), CPSF7 (Bethyl Laboratories: A301-360A), NUDT21 (abcam: ab183660), hnRNP L (abcam: ab6106), hnRNP A3 (abcam: ab78300), RBM3 (Proteintech: 14363-1-AP).

Techniques: Binding Assay, Western Blot

Journal: Scientific reports

Article Title: Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

doi: 10.1038/s41598-024-61951-1

Figure Lengend Snippet: Figure 6. Examples of genes potentially regulated by RBM3 and a model for RBM3-mediated 3′UTR shortening. (A–C) Left: Schematic of RBM3 motifs adjacent to PAS sequences in ATP6V0A2 (A), PSMD11 (B) and LEPROTL1 (C) genes. Right: 3′RACE showing favoring of pPAS versus dPAS at 8 h of costimulation. (D) Top: 3′RACE of LEPROTL1 in 293T cells transfected with cDNA expressing hnRNP C (control) or RBM3 or vector alone. Asterisks mark products observed only in 293T cells. Bottom: Western blot expression of RBM3, hnRNP C and hnRNP L (loading control) in the same cells as top experiment. Expression of hnRNP C causes a small increase in endogenous RBM3. (E) Model for 3′UTR shortening by RBM3, showing the suggested competitive binding between RBM3 and the cleavage and polyadenylation machinery (CPSF complex).

Article Snippet: Antibodies used to detect protein expression levels are as follows: CSTF2 (abcam: ab72297), CPSF3 (abcam: ab72299), CPSF6 (Santa Cruz: sc-100692), CPSF7 (Bethyl Laboratories: A301-360A), NUDT21 (abcam: ab183660), hnRNP L (abcam: ab6106), hnRNP A3 (abcam: ab78300), RBM3 (Proteintech: 14363-1-AP).

Techniques: Transfection, Expressing, Control, Plasmid Preparation, Western Blot, Binding Assay

Journal: Functional & Integrative Genomics

Article Title: 3’UTR RNA editing driven by ADAR1 modulates MDM2 expression in breast cancer cells

doi: 10.1007/s10142-025-01611-3

Figure Lengend Snippet: RIP-PCR for MDM2 ( a ), GINS1 ( b ) and F11R ( c ). For RNA-IP, ADAR1 (Abcam, ab168809), and rabbit IgG (Cell Signaling, 2729) antibodies were used. cDNAs were synthesized from RNA samples isolated after RIP with oligo d(T) primers (lanes 1, 2), or gene-specific primers (GSP) for MDM2 (lanes 3,4). Three independent biological replicates are shown for each RIP experiment and PCR. Lane 5 for MDM2 , and lanes 3 for GINS1 and F11R RIP-PCRs had MCF7 cDNA (Input) as positive control for PCR. NT lane was no template/negative control

Article Snippet: Beads were washed, and bound proteins were eluted with SDS loading buffer and analyzed by immunoblotting using CSTF2 (Abcam, ab72297) and ADAR1 (Cell Signaling, 14175) antibodies.

Techniques: Synthesized, Isolation, Positive Control, Negative Control

Journal: Functional & Integrative Genomics

Article Title: 3’UTR RNA editing driven by ADAR1 modulates MDM2 expression in breast cancer cells

doi: 10.1007/s10142-025-01611-3

Figure Lengend Snippet: siRNA-mediated ADAR1 knockdown and RNA editing in MCF7 cells, a . Western blot shows ADAR1 protein levels in ADAR1 siRNA or NT (non-targeting) siRNA transfected cells. UNT: Untransfected cells. The same blots were hybridized with ACTB antibody to test sample loading, b . A-to-I(G) editing ratios of MDM2 , c . A-to-I(G) editing ratios of GINS1 , and A-to-I(G) editing ratios of F11R in ADAR1 knockdown MCF7 cells

Article Snippet: Beads were washed, and bound proteins were eluted with SDS loading buffer and analyzed by immunoblotting using CSTF2 (Abcam, ab72297) and ADAR1 (Cell Signaling, 14175) antibodies.

Techniques: Knockdown, Western Blot, Transfection

Journal: Functional & Integrative Genomics

Article Title: 3’UTR RNA editing driven by ADAR1 modulates MDM2 expression in breast cancer cells

doi: 10.1007/s10142-025-01611-3

Figure Lengend Snippet: Effect of RNA editing on MDM2 protein levels, (a) Dual luciferase reporter assay with MDM2 3’UTR edit rich region. Non-edited (A) and edited (G) oligos were cloned downstream of the luciferase gene in pMIR. MCF7 cells were transiently transfected, and Firefly/Renilla luciferase read-outs from the constructs were normalized to that of empty pMIR (EV) (* p < 0.05, *** p < 0.0005, n = 3 independent transfections, one-way ANOVA, Tukey’s HSD), (b) ADAR1 and MDM2 protein levels in ADAR1 siRNA transfected (24 h, 48 h, 72 h) MCF7 cells. The same blots were hybridized with ACTB antibody to test sample loading. The image is representative of 3 independent experiments. Graphs show densitometric quantification of MDM2 and ADAR1 bands normalized to NT siRNA transfected cells (** p < 0.005, *** p < 0.0005, **** p < 0.0001, one-way ANOVA, Tukey’s HSD). NT: non-targeting siRNA transfected cells, UNT: Untransfected cells, (c) NGS and G% reads for the fifteen RNA editing positions for the 3’UTR of MDM2 in normal breast tissue. Fold changes in RNA editing percentage values between MCF7 cells and normal breast cDNA are shown on the bars

Article Snippet: Beads were washed, and bound proteins were eluted with SDS loading buffer and analyzed by immunoblotting using CSTF2 (Abcam, ab72297) and ADAR1 (Cell Signaling, 14175) antibodies.

Techniques: Luciferase, Reporter Assay, Clone Assay, Transfection, Construct

Journal: Functional & Integrative Genomics

Article Title: 3’UTR RNA editing driven by ADAR1 modulates MDM2 expression in breast cancer cells

doi: 10.1007/s10142-025-01611-3

Figure Lengend Snippet: ADAR1 interacts with CSTF2, a. Schematic shows the proximity biotinylation approach to detect protein interactions at 3’UTRs using CSTF2_TurboID_HA fusion protein expression and biotinylation in MCF7 cells. Biotinylated target proteins were immunoprecipitated (B-IP) and analyzed by LC-MS/MS. Western blot analysis of CSTF2-TurboID transfected MCF7 cells treated with 100 nM E2 or ethanol for 45 min–3 h, and 50 µM biotin. Nuclear and cytoplasmic lysates were subjected to western blot analysis using anti-CSTF2 antibody to detect CSTF2_TurboID_HA fusion and endogenous CSTF2. HDAC1 and TUBA1A antibodies were used to validate the nuclear and cytoplasmic fractions. Anti-Biotin antibody was used for biotinylation assessment, b. Biotinylated nuclear proteins were identified through LC-MS/MS analysis. ADAR1 was found to be significantly enriched among the biotinylated nuclear proteins (SAINT score 1), c. ADAR1 was confirmed to be biotinylated by the CSTF2-TurboID fusion. MCF7 cells were transfected with CSTF2-TurboID. After 24 h, cells were incubated with 50 µM Biotin in the presence or absence of E2 for 45 min and 3 h. Isolated nuclear and cytoplasmic lysates were used for affinity capture with streptavidin beads. Eluted proteins were used for western blot analysis with anti-ADAR1, anti-CSTF2 and anti-Biotin antibodies, d . Nuclear extracts (500 µg) of MCF7 cells were subjected to Co-IP with CSTF2 or isotype-matched IgG. Input was 25 µg nuclear lysate. Immunoprecipitated proteins were then subjected to immunoblotting using CSTF2 or ADAR1 antibodies. WB: western blotting, e. Upset plot showing intersection of RNA editing events on mRNAs. Majority of RNA editing events were only detected on 3’UTRs. Each row represents a genomic feature, and the filled dots indicate the intersection of editing regions in individual mRNAs. The vertical bars indicate the number of genes in each category

Article Snippet: Beads were washed, and bound proteins were eluted with SDS loading buffer and analyzed by immunoblotting using CSTF2 (Abcam, ab72297) and ADAR1 (Cell Signaling, 14175) antibodies.

Techniques: Expressing, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transfection, Incubation, Isolation, Co-Immunoprecipitation Assay

Journal: Functional & Integrative Genomics

Article Title: 3’UTR RNA editing driven by ADAR1 modulates MDM2 expression in breast cancer cells

doi: 10.1007/s10142-025-01611-3

Figure Lengend Snippet: Effect of CSTF2 knockdown on ADAR1 and MDM2 protein levels. Western blot analysis of CSTF2 siRNA transfected MCF7 cells (24, 48, and 72 h) showed decreased ADAR1 and MDM2 protein levels. The same blots were hybridized with ACTB antibody to test sample loading. The image is representative of 3 independent experiments. Graphs show densitometric quantification of bands normalized to NT bands (ns: not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001; n = 3 biological replicates, one-way ANOVA, Tukey’s HSD). NT: non-targeting siRNA transfected cells

Article Snippet: Beads were washed, and bound proteins were eluted with SDS loading buffer and analyzed by immunoblotting using CSTF2 (Abcam, ab72297) and ADAR1 (Cell Signaling, 14175) antibodies.

Techniques: Knockdown, Western Blot, Transfection