Fig 2: Breast cancer-specific survival (BCSS) according to CAP1 score, stratified for a body fat percentage, b BMI, c waist circumference, and d waist-hip ratio. Patients at risk, number of events (NoE), LogRank trend test, and adjusted hazard ratios (HRs) with 95% CI comparing low CAP1 expression to high CAP1 expression are shown. HR adjusted for age at diagnosis (continuous), tumor size (> 20 mm, yes/no), and any axillary lymph node involvement (yes/no)

Fig 3: Cell cycle analysis of breast cancer cells. (A) Example of flow cytometry gating strategy with cell population identification, doublet discrimination and model fit of the cell cycle phases. (B) Cell cycle profiles of T47D and MDA-MB-231 breast cancer cells in the absence or presence of adipocyte secretome under normal and obese conditions. (C) Effects by CAP1 knockdown on cell cycle phase distribution in T47D and MDA-MB-231 cells exposed to adipocyte secretome from normal or obese conditions. Graphs show the mean ± SEM from three independent experiments. Multiple comparisons between groups were performed with two-way ANOVA with Sidak’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: Subcellular distribution of CAP1 in platelets. (A) Specificity of anti-CAP1 antibody. Platelet and cell line lysates (30 µg total protein) were resolved by 12% SDS-PAGE, blotted onto PVDF membrane and probed with an antibody for CAP1. The blots were probed for β-actin as a loading control. HT29, human colorectal cancer cell line; HEK, human embryonic kidney 293 cell line; HUVEC, human umbilical vein endothelial cell; COS7, fibroblast-like tissue from monkey kidney tissue. (B) Subcellular fractionation. Platelets were lysed by freeze-thaw in liquid nitrogen and spun at 100,000 × g for 1 hour to separate membrane (M) and cytosolic (C) fractions. The fractions were normalised by volume and resolved by 12% SDS-PAGE, blotted onto PVDF membrane and probed with antibodies for the indicated proteins. CD36 was used as membrane marker and Syk as a cytosolic marker in resting platelets. Latrunculin B (LatB; 20 µM, 20 min) was used to depolymerise F-actin prior to lysis. CAP1 distribution was quantified by densitometry and expressed as percentage relative to the total (M + C) CAP1 in the lysate. Data represent mean ± SD of three independent experiments. (C) Association of CAP1 to actin in detergent insoluble pellet. Platelets (8 × 108/mL platelets) were lysed in the presence of 1% TX-100 and lysates spun at low speed (15,600 × g) for 20 min and high speed (100,000 × g) for 1 hour. Supernatant (S) and pellet (P) were normalised by volume and resolved by 12% SDS-PAGE, blotted onto PVDF membrane and probed with antibodies for the indicated proteins. LatB (20 µM, 20 min) was used to depolymerise F-actin prior to lysis. CAP1 concentration in pellet and supernatant were quantified by densitometry as percent of total (P + S). Data represent mean ± SD of three independent experiments. Full-length blots are presented in Supplementary Fig. 1.

Fig 5: Subcellular localisation of CAP1. Platelets were fixed in suspension with paraformaldehyde and spun on poly-L-lysine coated coverslips (A) or were allowed to spread on fibrinogen coated coverslips and fixed with paraformaldehyde (B–E). For (A–D) cells were immunostained with an anti-CAP1 antibody followed by an Alexa568-coupled secondary antibody (red) and counterstained with FITC-phalloidin for filamentous actin (green). For E platelets were treated with 100 nM PGI2 5 minutes prior to fixation in order to increase the proportion of cells displaying actin nodules30. Platelets were then immunostained with anti-CAP1 and anti-vinculin antibodies followed by Alexa568 or Alexa488-coupled secondary antibodies, respectively (red and green), and counterstained with blue CytoPainter for filamentous actin (blue). Actin colour has been changed to red in the double staining merge panel for better visualisation. Optical sections were acquired with a fluorescence microscope equipped with a structured illumination attachment. Sections were 230 nm apart. Shown is a maximum intensity projection image after deconvolution and single planes of the region are indicated with a square. Arrows point at regions of interest: cell cortex (B), filopodia (C) and actin nodules (D,E). Scale bar 5 µm.