Fig 2: Akt1 interactors in murine epidermis and in Akt1 immunoprecipitate.Neonatal wild-type murine epidermis stained for Akt1 or pSer404 Lamin A and co-stained for Myh9 (A), Ran (B), STAMBP (C). D IgG1 and Akt1 immunoprecipitate immunoblotted for total Akt, actin, Arp3 and Myh9. E IgG and phospho-(Ser/Thr) Akt substrate (pSubs) IPs immunoblotted for Myh9 and pSubs. F Summary of Akt1 interactors identified by mass spectrometry in an Akt1 co-immunoprecipitate that were also detected to be dispersed with Akt1 and pSer404 Lamin A/C in REKs, murine epidermal sections, and confirmed by western blotting of Akt1 co-immunoprecipitate.

Fig 3: Primary OPCs express Arp2, Arp3 and Pcdh15 at the soma and throughout the processes.a–c Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and Hoechst 33342 (HST; blue). d Single confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and Pcdh15 (green). e–g Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), Pcdh15 (rabbit anti-pan Pcdh15; green) and HST (blue). h Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and Pcdh15 (green). i–k Single z-plane confocal scan of primary mouse OPCs processed to detect Arp2 (mouse anti-Arp2; red) and the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). l Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp2 (red) and the CD3 isoforms of Pcdh15 (green). (m-o) Single z-plane confocal scan of primary mouse OPCs processed to detect Arp3 (mouse anti-Arp3; red), the CD3 isoforms of Pcdh15 (rabbit anti-CD3 Pcdh15; green) and HST (blue). p Single z-plane confocal scan (100×) of primary mouse OPC processes expressing Arp3 (red) and the CD3 isoforms of Pcdh15 (green). Scale bars represent 20 µm (a–c, e–g, i–k, m–o) or 5 µm (d, h, l, p).

Fig 4: Survival map and differential expression of the mRNA and protein level of GMFB in pan-cancer and KIRC. (A) Comparing the survival contribution of GMFB in pan-cancer, estimated using Mantel–Cox test, including overall survival (OS) and disease-free survival (DFS). Tumor types with significant differences (p-value adjustment <0.05) were highlighted. HR, hazard ratio. (B) Comparison expression of Gmfb mRNA level between tumor and normal tissues (pair samples). (C) Comparison expression of Gmfb mRNA level between tumor and normal tissues (unpaired samples). (D) Comparison expression of Gmfb mRNA level between HEK293T and Caki-2 cells tested by RT-qPCR. (E) Comparison of GMFB protein level expression between normal and KIRC tumor tissues (unpaired samples). (F) Immunohistochemistry images in normal and KIRC tumor tissues. (G) Comparison expression of GMFB protein level between 293T and Caki-2 cells tested by Western blotting. (H) Colocalization of GMFB with Arp2 in 293T and Caki-2 cells. (I) Colocalization of GMFB co-localized with Arp3 in 293T and Caki-2 cells. Scale bar = 25 µm. White arrows indicate co-location. KIRC, kidney renal clear cell carcinoma. Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Pcdh15 knockdown promotes F-actin accumulation in OPC veils and increases the expression of genes and proteins that enhance Arp2/3 activity.a Confocal images of OPC processes in control- and Pcdh15 shRNA treated cultures immunolabelled to detect microtubules (a-tubulin, green) and F-actin (Phalloidin, red). b Quantification of phalloidin intensity (integrated pixel density) in control and Pcdh15-knockdown OPCs (normalized to the average intensity of control OPC cultures) (n = 3 independent cultures, =51 veils analysed per condition; one-sample t-test for Pcdh15-shRNA: deviation from 100(%) = 30.25, SD of deviation = 9.19, t (2) = 5.697, p = 0.029). c Degree of colocalization between actin (phalloidin) and microtubule (a-actin) labelling in control and Pcdh15 knockdown OPC veils. Colocalization is expressed as a Pearson correlation coefficient (mean ± SEM, n = 3 independent cultures, =37 veils measured per condition; unpaired t-test, p = 0.81). d Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect Akt, phosphorylated Akt (p-Akt) and ß-actin. e Expression of Akt in control and Pcdh15 knockdown OPC cultures, normalised to ß-actin expression (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t-test, p = 0.12). f Quantification of p-Akt (Ser473) and Akt in control and Pcdh15 knockdown OPCs, normalised to ß-actin (integrated pixel density measured for each protein band; mean ± SEM, n = 3 independent cultures; unpaired t-test, p = 0.20). g Average fold change in cytoskeletal gene expression between OPCs transfected with control- and Pcdh15-shRNA (average from n = 3 independent cultures). h Protein lysates from control and Pcdh15 knockdown OPCs analysed by Western blot to detect CrkII and WAVE2, which are upstream of the Arp2/3 complex, Arp2, Arp3 and Arpc3, which are components of the Arp2/3 complex, and GAPDH. i CrkII, j WAVE2, k Arp2, l Arp3, and m Arpc3 protein expression was quantified by measuring the integrated pixel density of the corresponding Western blot band and normalising expression to GAPDH expression for each sample [mean ± SEM, n = 3 independent cultures, expression in control and Pcdh15 knockdown OPCs was compared by unpaired t-tests: i p = 0.039, j p = 0.004, k p = 0.0001, l p = 0.028, m p = 0.016]. Scale bars represent 5 µm.