Journal: Nature Communications

Article Title: Inherited ARPC5 mutations cause an actinopathy impairing cell motility and disrupting cytokine signaling

doi: 10.1038/s41467-023-39272-0

Figure Lengend Snippet: a Family pedigrees from patients with ARPC5 variants. Arrows point to the patients studied (P1 in Family 1, P2 in Family 2); Roman numerals indicate generations. b Table depicting the main clinical and laboratory findings in P1 and P2. Negative symbols (−) denote absence; crosses, from (+) to (+++) indicate less to more severe phenotypes, respectively. c Patient 1 images. In the left image, a standing radiograph of P1 shows biconvex thoracolumbar scoliosis with convex right thoracic spinal curvature and convex left thoracolumbar spinal curvature; a right upper lobe pneumatocele is also identified. In the right images (upper, middle and lower, respectively), lung ground glass opacities, multiple abdominal scars product of abnormal wound healing, and right sided myositis of the thigh, are also detected.

Article Snippet: P1’s fibroblasts were transfected with pCMV3-ARPC5-C-HA plasmid (Sino Biological, Cat. HG16494-CY) or mock-transfected (transient; transfection efficiency 20–40%) with empty vector using Amaxa Human Dermal Fibroblast Nucleofector Kit (Lonza, Cat. VPD-1001), program U-023, following manufacturer’s protocol.

Techniques:

Journal: Nature Communications

Article Title: Inherited ARPC5 mutations cause an actinopathy impairing cell motility and disrupting cytokine signaling

doi: 10.1038/s41467-023-39272-0

Figure Lengend Snippet: a Western blotting analysis of patient 1 (P1), her parents, and healthy controls (HC) T-cell blasts lysates with antibodies specific to Arp2/3 complex subunits, as indicated. Vinculin and β-tubulin were used as loading controls. b Western blotting analysis of cell extracts from P1’s and HC’s fibroblasts after native gel electrophoresis. ARPC5- or ARPC2-specific antibodies were used to probe the Arp2/3 complex; GAPDH was used as a loading control. Blots in this figure are representative of at least two independent experiments. The numbers below the western blotting images ( a and b ) represent protein expression levels, quantitatively measured in relation to healthy controls, after normalization to the loading control. Healthy controls’ average value was set at 10. PAGE polyacrylamide gel electrophoresis. Source data are provided as a Source Data file.

Article Snippet: P1’s fibroblasts were transfected with pCMV3-ARPC5-C-HA plasmid (Sino Biological, Cat. HG16494-CY) or mock-transfected (transient; transfection efficiency 20–40%) with empty vector using Amaxa Human Dermal Fibroblast Nucleofector Kit (Lonza, Cat. VPD-1001), program U-023, following manufacturer’s protocol.

Techniques: Western Blot, Nucleic Acid Electrophoresis, Expressing, Polyacrylamide Gel Electrophoresis

Journal: Nature Communications

Article Title: Inherited ARPC5 mutations cause an actinopathy impairing cell motility and disrupting cytokine signaling

doi: 10.1038/s41467-023-39272-0

Figure Lengend Snippet: a Protein expression by immunoblotting of individual Arp2/3 complex subunits in lysates from P1’s fibroblasts transiently transfected (efficiency 20–40%) with empty vector or a plasmid encoding wild-type (WT) ARPC5 . Vinculin was used as a loading control. b Western blotting analysis after native gel electrophoresis of cell extracts from P1’s and healthy control’s (HC) fibroblasts expressing WT ARPC5 by lentiviral transduction. ARPC5- or ARPC2-specific antibodies were used to probe the Arp2/3 complex. GAPDH was used as a loading control. c Immunofluorescence images of representative fibroblasts from P1 transfected with WT ARPC5 (left column) versus cells transfected with empty vector (right column). Cells were seeded on a retronectin-coated surface and fixed after 120 min. d Real-time, impedance-based monitoring of P1’s ARPC5 -rescued fibroblasts versus ARPC5 mock-transduced cells. Impedance values are reported as cell index (CI). Curves represent the mean (±standard deviation) cell index value from four technical replicates. e Wound healing assay with WT ARPC5 -rescued fibroblasts from P1 (middle column) and mock-transduced P1 cells (right column), HC cells were used as controls (left column). The gap length shown at 0 h corresponds to 0.94 mm. All results in this figure are representative of at least two independent experiments. The numbers below the western blotting images represent protein expression levels relative to WT ARPC5 -rescued P1 fibroblasts ( a ) or HC fibroblasts ( b ), after normalization to the loading control. PAGE polyacrylamide gel electrophoresis. Source data are provided as a Source Data file.

Article Snippet: P1’s fibroblasts were transfected with pCMV3-ARPC5-C-HA plasmid (Sino Biological, Cat. HG16494-CY) or mock-transfected (transient; transfection efficiency 20–40%) with empty vector using Amaxa Human Dermal Fibroblast Nucleofector Kit (Lonza, Cat. VPD-1001), program U-023, following manufacturer’s protocol.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Nucleic Acid Electrophoresis, Transduction, Immunofluorescence, Standard Deviation, Wound Healing Assay, Polyacrylamide Gel Electrophoresis