Fig. 4

Actin isoform regulation in ACTB-AST patient fibroblasts. a RNA-Seq analysis of actin transcripts in P4 and P5 compared to the healthy control (log₂ fold change ± s.e.m.). Bar graphs show the combined results from both patients relative to the control. Overlayed dot plots show the individual patient data calculated from three technical replicates (p < 0.0001 unless stated as ns); b Representative western blots show reduced β-CYA, increased γ-CYA and α-SMA in P4 and P5 fibroblasts compared to the control (C). Double the sample amount was required to detect α-SMA (#); c Densitometry analysis of P4 and P5 signals expressed as mean (±s.d.) relative to the control (9–10 replicates from 6 lysates; Kruskal–Wallis test); d Reduced β-CYA:γ-CYA ratios are detected in P4 and P5 fibroblasts by immunofluorescence microscopy (mean ± s.d.; image numbers analyzed from 3 experiments are given in brackets; one-way ANOVA); e Representative maximum intensity projections show β-CYA (top) and γ-CYA (bottom) distribution within z-stack slices 1–2 of C, P4, P5, and BWCFF control fibroblasts. Yellow arrows indicate cells where thick basal β-CYA bundles are abundant. Scale bars are 15 µm; f Immunofluorescence microscopy shows increased α-SMA expression in P4 and P5 fibroblasts. Inserts show α-SMA incorporation in ACTB-AST sub-nuclear basal filaments. Scale bars are 15 µm; g Normalized α-SMA fluorescence intensity in individual cells (mean ± s.d.; the number of cells analyzed from 3 experiments is given in brackets; Kruskal–Wallis test); h The percentage of cells per image in which α-SMA is incorporated into basal sub-nuclear filaments (median and IQR; 14–16 images from 3 experiments; Kruskal-Wallis test); i P5 cells co-stained for β-CYA or γ-CYA (left, green in merge) and α-SMA (mid, magenta in merge). Scale bars are 20 µm; j Greater overlap of α-SMA with β-CYA is observed in P5 cells (median and IQR; 33–36 cells from 3 experiments; Mann–Whitney test). In all cases, *p < 0.05, ***p < 0.001, ****p < 0.0001 and ns, not significant