Fig 2: 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 [122]. 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

Fig 3: 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

Fig 4: 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