Skip to main content
Fig. 8 | Genome Biology

Fig. 8

From: Alternative polyadenylation factors link cell cycle to migration

Fig. 8

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

Back to article page