Fig 2: Generation of WAVE2 and IRSp53 images from actin filaments and WAVE2 images in IRSp53-expressing U251 cells. (A) Generation of a WAVE2 image from an actin filament image that was visualized by lifeact-mCherry. The IRSp53-knocked-out U251 cells re-expressing GFP-tagged IRSp53 and lifeact-mCherry were stained with an anti-WAVE2 antibody after fixation and permeabilization. The results are depicted as in Figure 2A. Scale bar, 10 µm. (B) Generation of an IRSp53 image from an actin filament-stained image. Scale bar, 10 µm. (C) Image generation into a WAVE2 image from an IRSp53-stained image. Scale bar, 10 µm. (D) Box plot of the MAE of the entire images in four-fold cross-validation (n = 100). (E) Box plot of the MS-SSIM of the entire images in four-fold cross-validation (n = 100). The data points represent the MS-SSIM values for the generated images in panels (A–C). In panels (D,E), the yellow circles indicate the values for images in panels (A–C). Statistical significance is shown by p < 0.05 (*) by two-sample equal variance two-tailed Student’s t-test. (F) Lamellipodia structures (arrows) observed in the live imaging of lifeact-mCherry. Scale bar, 10 µm. (G) Generation of a WAVE2 image from a lifeact-mCherry image. The cells observed in panel (F) were fixed, permeabilized, and immunostained for WAVE2 (truth). The arrows indicate lamellipodia. Scale bar, 10 µm.

Fig 3: VIPR2 mediates the interaction between WAVE2, ARP and actin. (A) MDA-MB-231 cells transfected with either control siRNA or VIPR2 siRNA1 were stimulated with (+) or without (−) 100 nM VIP for 10 min. Cells were evaluated by immunocytochemistry using an anti-WAVE2 antibody. Representative images are shown. (B) Bar graph shows quantification of the fluorescence intensity of WAVE2 on the plasma membrane. The data are presented as means ± SD [VIP (-) and control si, 76 cells; VIP (-) and VIPR2 si1, 104 cells; VIP (+) and control si, 66 cells; VIP (+) and VIPR2 si1, 130 cells in total]. **p < 0.01, ***p < 0.001, between the indicated groups (Kruskal–Wallis test followed by Dunn’s multiple comparison test). n.s. indicates not statistically significant. (C, D) Pull-down assay using GFP-Trap_A beads was performed to evaluate the amount of WAVE2 and ARP3 bound to EGFP-actin. LLC-PK1 cells stably expressing EGFP-actin were transfected with VIPR2 siRNA3; the pull-down fraction was collected using GFP-Trap_A beads and centrifugation. Western blotting of the obtained fractions (pull down, C) and whole cell lysates (D) was performed using the specific antibodies for the indicated proteins. EGFP-actin was detected by a GFP antibody. Similar data were obtained from three independent experiments, and representative images are shown.

Fig 4: Homozygous NCKAP1L mutations identified in the two families. (A) Pedigrees of affected consanguineous families of Iranian (family 1) and Saudi Arabian (family 2) origins. Generations are designated by Roman and subjects by Arabic numerals. Double lines connecting parents indicate consanguinity. Squares, male subjects; circles, female subjects; filled (black) symbols indicate patients, while unfilled (white) symbols indicate unaffected family members. All family members in family 1 and all in family 2, with the exception of II.3 and II.4, were subjected to whole-exome sequencing. (B) Photograph of axillary lymphadenitis in patient 1. (C–G) In patient 2, a thoracoabdominal computed tomography scan showed right lung focal oligemia and brocheactesis (C) and massive hepatosplenomegaly (D). In the same patient, bone marrow aspirates showed hemophagocytosis (E), whereas liver staining with hematoxylin and eosin (F) or CD68 (G) showed sinusoidal dilatation with hemophagocytic histiocytosis. (H) Sanger traces of the disease-associated NCKAP1L mutations confirming autosomal recessive inheritance. Electropherograms show single base pair substitutions c.421G>T of NCKAP1L (NM_005337) causing V141F missense in family 1 (left panel) and a single base pair substitution c.2821+1 G>A disrupting the donor splice site of NCKAP1L exon 26 in family 2. (I) RT-PCR in PBMCs in family 2 shows homozygous skipping of exon 26 in patient II:1. (J) Western blot analysis of NCKAP1L complex proteins expression in PBMCs from patients and controls. NCKAP1L and WAVE2 proteins are absent in both patients. GAPDH was used as a loading control. Molecular weight (MW) markers are shown on the left in kilodaltons. (K–O) Structure modeling of NCKAP1L and its mutants in the context of the WAVE complex. The cartoon representation of the structure model of the NCKAP1L protein was established using NCKAP’s structure (PDB 4n78 chain B) as a template. The N- to -C is shown as a rainbow color from blue to red. NCKAP1L is shown in cyan, and the product of exon 26 (purple), V141F mutation (red), and WIRS (blue) are in close contact with ABI2 (light salmon; K). Zooming to the V141F mutation shows the WT valine as hot pink (L). The residue has close contacts with the other two helices. When mutated to phenylalanine (patient 1), the large side-chain groups result in severe atomic clash, which indicates a reorganization of the helical structure and may have an effect on ABI2 binding (M). Exon 26’s product (purple) forms close contacts (shown as yellow dashed lines) with ABI2 (N). When exon skipping occurs (patient 2), the helix in the C terminus of the exon 26 product changes to a loop region (shown in magenta; O). The contacts with ABI2 disappear in this situation.

Fig 5: WASF2 is a candidate target of EMX1 in SCG. (a) candidate downstream targets of EMX1 predicted in the hTFtarget system; expression of ANKRD17 (b) and WASF2 (c) in glioma patients in the GEPIA database; correlations of the WASF2 (d) and ANKRD17 (e) with the overall survival of glioma patients in the GEPIA database; (f) expression of WASF2 mRNA in the SCG tissues and the adjacent normal tissues examined by RT-qPCR; (g) expression of EMX1 in different grades of SCG; (h) an inverse correlation between WASF2 and EMX1 in SCG tissues. Three repetitions were performed. Differences were analyzed by the paired t test (f) and one-way ANOVA (g). *p < .05