Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 1 CstF1 interacts with both BARD1 and p53 to form a protein complex. (a) Diagram of BARD1, CstF1 and p53 derivatives. (b) Requirement of p53 C-terminal domain for both CstF1 and BARD1 interaction. Recombinant His-p53 or the indicated His-p53 derivatives were incubated with purified GST, GST-CstF1 or GST-BARD1. Protein samples were treated with RNase A. Bound proteins were eluted and analyzed by western blot with anti-p53 antibodies. Five percent of His-p53 or His-p53 derivatives used in the reaction are shown as input. (c) CstF1, BARD1 and p53 can coexist in complexes. Either GST-BARD1 or GST-CstF1 was immobilized in glutathione-Sepharose beads to test its ability to bind CstF1/BARD1 and p53 from NE of HeLa cells, while increasing concentrations of recombinant His-p53 (5, 10 and 20 ng) were added. Bound proteins were detected by immunoblotting with CstF1 and p53 antibodies. The pellets (PD) and the supernatant (SN) were analyzed. (d) p53, CstF and BARD1 co-immunoprecipitate from NEs of MCF7 cells treated with UV irradiation. CstF and p53 co-immunoprecipitation is irrespective of UV irradiation. Co-immunoprecipitation assays were performed using NEs of cells exposed or not to UV irradiation and allowed to recover for 2 h as described before. NEs were immunoprecipitated with either anti-CstF2, to ensure that any detected interactions were between p53 and intact CstF, or anti-p53 antibodies. Protein samples were treated with RNase A. Equivalent amounts of the pellets (IP) and the supernatants (SN) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and proteins were detected by immunoblotting with antibodies against BARD1, CstF2 and p53. Antibodies against Topo II were used as a control of specificity. Positions of Topo II, CstF2 and p53 are indicated. Twenty percent of the NE used in the immunoprecipitation reaction is shown as input.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Recombinant, Incubation, Western Blot, Irradiation, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Control

Journal: Oncogene

Article Title: p53 inhibits mRNA 3' processing through its interaction with the CstF/BARD1 complex.

doi: 10.1038/onc.2011.29

Figure Lengend Snippet: Figure 4 The S241F mutation in p53 disrupts the BARD1, CstF and p53 interaction and decreases the inhibitory effect of p53 on 30 cleavage, and all this is restored by WT-p53 expression. (a) CstF, p53 and BARD1 do not co-immunoprecipitate from NE of DLD-1 cells that express S241F mutant p53. NEs were immunoprecipitated with either anti-CstF2 or anti-p53 antibodies. Protein samples were treated with RNase A. Equivalent amounts of the pellets (IP) and the supernatants (SN) were resolved by sodium dodecyl sulfate– polyacrylamide gel electrophoresis and proteins were detected by immunoblotting with antibodies against BARD1, CstF2 and p53. Antibodies against Topo II were used as a control of specificity. Positions of Topo II, BARD1, CstF2 and p53 are indicated. Twenty percent of the NE used in the immunoprecipitation reaction is shown as input. (b) CstF, BARD1 and p53 co-immunoprecipitate from NEs of D-A2 cells following induction of WT-p53 expression. D-A2 cells were grown in the absence of Dox to induce the expression of WT-p53. Samples were analyzed as in (a). (c) The induced expression of WT-p53 in D-A2 cells inhibits pre-mRNA 30 cleavage. NEs from DLD-1 (left panel) and D-A2 cells (right panel) non-treated or treated with Dox and/or UV irradiation, and allowed to recover for the times indicated in the figure, were analyzed for L3 pre-mRNA 30 cleavage. Positions of pre-mRNA and the 50 cleavage product are indicated. (d) The S241F mutation in p53 abolishes the inhibition of 30 cleavage. NEs from HeLa cells were preincubated with no addition or increasing amounts of recombinant His-p53 or His-p53 (S241F) derivative (40, 80 and 120 ng). After 15 min, L3 pre-mRNA was added and incubation continued for 90 min. RNAs were purified and analyzed by denaturing PAGE. Positions of pre-mRNA and the 50 cleavage product are indicated.

Article Snippet: Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz). siRNA knockdown of p53 expression in HeLa and MCF7 cells The siRNA specific for human p53 was synthesized by Qiagen (Valencia, CA, USA) and the control RNA duplex used as non-silencing siRNA was obtained from Dharmacon RNA Technologies (Lafayette, CO, USA).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Control, Irradiation, Inhibition, Recombinant, Incubation

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