Fig 2: ACTB Loss-of-Function Mutations Induce Abnormalities of Cellular Morphology and Reduced Migration(A) Immunoblots of cytoplasmic ß-actin. No consistent differences were detected in the cytoplasmic ß-actin amounts in the fibroblasts and LCLs of affected individuals versus controls (sample P1 is from IVa, P2 is from XI, P3 is from II, and P4 is from XXII). Immunoblotting for ß-actin was performed on the cytoplasmic protein fraction, and GAPDH was used as a loading control. Protein samples were isolated using NE-PER nuclear and cytoplasmic extraction reagents (ThermoScientific). 8–10 mg of protein extracts were loaded into the polyacrylamide gel Bolt 10% Bis-Tris Plus Gels (Invitrogen). The membranes were incubated with specific anti-beta actin (ab8227, Abcam) and anti-GAPDH (5174S, Cell Signaling) overnight at 4°C. After washes, the membranes were incubated with a secondary fluorescently labeled goat anti-rabbit antibody (IRDye 800CW Li-Cor), and signal was developed with an Odyssey CLX imaging machine.(B) Fibroblast morphology. ACTB-deficient cells were found to be significantly more circular than non-deficient cells. Phalloidin and DAPI immunostaining was performed in wild-type fibroblasts transfected with 30 nM control siRNA (ON-TARGETplus non-targeting pool, Fisher) and ACTB siRNA (SMARTpool ON TARGET plus ACTB siRNA, Dharmacon) and affected-individual fibroblasts transfected with control siRNA. Cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After blocking solution was washed out, antibody Texas Red-X Phalloidin (T7471, Life Technologies) was applied for 1 hr at room temperature in the dark. Samples were stained with DAPI (4083S, Cell Signaling Technology) for 5 min. Representative pictures show marked difference in the morphology of ß-actin-deficient cells (scale bar: 50 µm). Enlargement of cells is shown in the lower panels. Dashed lines show the outline of the cell boundary. The left bar chart shows that there was no significant difference in the area of each of the cell groups (in µm2) as calculated with ImageJ software. The right bar chart shows the increased circularity of the ACTB-deficient fibroblasts as calculated with ImageJ software (n = 4; **p < 0.01). Values of 1 and 0 stand for a perfect circle and a line, respectively. Error bars indicate mean ± 1 SD.(C) Fibroblast migration. ACTB-deficient cells had impaired migration. A migration assay was performed in wild-type fibroblasts transfected with control siRNA and ACTB siRNA and affected-individual fibroblasts transfected with control siRNA. 96 hr after transfection, a wound was generated in the confluent monolayer of fibroblasts via a p200 pipet tip. Cells were washed with phosphate-buffered saline so that any debris created by the wound would be removed. The first image of the wound was taken with a phase-contrast microscope, and marking the plate under the capture image field created a reference point. Cells were incubated at 37°C in a humidified 5% CO2 incubator for 2 days. After the incubation time, a second image was taken. For quantifying the migration of cells, the cells that crossed into the wound area were counted. Representative pictures show reduced migration in ß-actin-deficient cells (scale bar: 100 µm). The bar chart shows that the numbers of cells in the central wound area are significantly lower in the ß-actin-deficient cells. Data are shown as the mean of absolute cell numbers at 144 hours from two wells of two independent experiments (n = 4; *p < 0.05, **p < 0.01). Error bars indicate mean ± 1 SD.