Figure 1. De novo gene expression is... | Rockefeller University Press

Figure 1.

$Figure 1. De novo gene expression is essential for actin cytoskeleton and FA dynamics during migration. (A) Confluent ECFCs were serum starved for 2 h, and actinomycin D or puromycin were added 1 h into the serum starve to inhibit transcription and translation, respectively. Monolayers were scratched to form an open “wound” to longitudinally quantify migratory closure. (B) Wound closure percentage, measured as (initial wound area − wound area at 8 h)/initial wound area × 100, and wound closure rate, measured as the distance the cell front moved over each imaging period (µm/h). Background color shows de novo gene expression–independent (gray) and –dependent (blue) phases. n = 19–24; P < 0.025; two-way ANOVA with Tukey’s post hoc test. (C) F- and G-actin visualized by Alex Fluor 488 phalloidin, Alexa Fluor 594 DNase I, and nuclei visualized by DAPI. (D) Normalized F-actin and G-actin intensity and F-/G-actin ratio per cell, normalized to DMSO-treated controls. n = 60 cells; P < 0.0001; Kruskal Wallace with Dunn’s post hoc test. (E) Western blot of β-actin in F- and G-fractions of ECFC lysate 24 h after actinomycin D. (F) F-/G-actin ratio of β-actin densitometry measurements in F- and G-fractions. n = 3 samples; P < 0.04; two-tailed Student’s unpaired t test. (G) Vinculin and nuclei visualized by Alexa Fluor 594 secondary and DAPI. (H) Vinculin+ FA length. n = 40 cells; P < 0.009; ANOVA with Tukey’s post hoc test. Repeated significance indicator letters (e.g., a–a) signify P > 0.05, while groups with distinct indicators (a vs. b) signify P < 0.05. Summary statistics are represented as mean ± SEM. Box plots show interquartile range with whiskers at minimum/maximum. n.s., not significant; P > 0.05.$

De novo gene expression is essential for actin cytoskeleton and FA dynamics during migration. (A) Confluent ECFCs were serum starved for 2 h, and actinomycin D or puromycin were added 1 h into the serum starve to inhibit transcription and translation, respectively. Monolayers were scratched to form an open “wound” to longitudinally quantify migratory closure. (B) Wound closure percentage, measured as (initial wound area − wound area at 8 h)/initial wound area × 100, and wound closure rate, measured as the distance the cell front moved over each imaging period (µm/h). Background color shows de novo gene expression–independent (gray) and –dependent (blue) phases. n = 19–24; P < 0.025; two-way ANOVA with Tukey’s post hoc test. (C) F- and G-actin visualized by Alex Fluor 488 phalloidin, Alexa Fluor 594 DNase I, and nuclei visualized by DAPI. (D) Normalized F-actin and G-actin intensity and F-/G-actin ratio per cell, normalized to DMSO-treated controls. n = 60 cells; P < 0.0001; Kruskal Wallace with Dunn’s post hoc test. (E) Western blot of β-actin in F- and G-fractions of ECFC lysate 24 h after actinomycin D. (F) F-/G-actin ratio of β-actin densitometry measurements in F- and G-fractions. n = 3 samples; P < 0.04; two-tailed Student’s unpaired t test. (G) Vinculin and nuclei visualized by Alexa Fluor 594 secondary and DAPI. (H) Vinculin⁺ FA length. n = 40 cells; P < 0.009; ANOVA with Tukey’s post hoc test. Repeated significance indicator letters (e.g., a–a) signify P > 0.05, while groups with distinct indicators (a vs. b) signify P < 0.05. Summary statistics are represented as mean ± SEM. Box plots show interquartile range with whiskers at minimum/maximum. n.s., not significant; P > 0.05.