Journal: The EMBO Journal

Article Title: PTEN deficiency exposes a requirement for an ARF GTPase module for integrin‐dependent invasion in ovarian cancer

doi: 10.15252/embj.2023113987

Figure Lengend Snippet: A, B Immunofluorescence and confocal imaging of Trp53 −/− ; Pten −/− 1.15 spheroids stained for α5‐integrin or β1‐integrin (grey or FIRE LUT), Hoechst (blue) and F‐actin (magenta). Magnified images from boxed regions shown. Arrowheads, labelling at protrusion tips. Scale bars, 5 μm. Representative of n = 3 spheroids imaged. (B) Intensity profiles for integrins (grey) and F‐actin (magenta) from spheroids in (A). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead. C, D Immunofluorescence and confocal imaging of Trp53 −/− ; Pten −/− 1.15 spheroids stained for pFAK (Y379) or pSRC Family Kinases (SFK pY416) (grey or FIRE LUT), Hoechst (blue) and F‐actin (magenta). Magnified images from boxed regions shown. Arrowheads, positive staining. Scale bars, 5 μm. Representative of n = 5 spheroids imaged. (D) Intensity profiles for active FAK and Src (grey) and F‐actin (magenta) from spheroids in (C). Tip measured is annotated, ECM to body, yellow arrow, tip, white arrowhead. E–H Representative capture ELISA graphs (E, G) and associated quantitation (F, H) for recycling of internalised cargoes between Trp53 −/− versus Trp53 −/− ; Pten −/− cells or Trp53 −/− ; Pten −/− cells expressing sh Scramble versus sh Arf6 for active β1‐integrin. Graphs shown are representative of n = 2 (E) or n = 3 (G) independent replicates. Data, mean (black square) ± SD for repeated experiments (large circles), 1–3 technical replicates/experiment/timepoint (small circles), two‐tailed t ‐test, P ‐values are annotated. I–K Overall survival (% patients, months; TCGA OV data set) of patients grouped into combined expression based on median mRNA split. (I) Low (red line, M1) or high (blue line, M2) expression for all mRNA, control, remaining patients (green line), (J), same as (I), but CYTH2 Ex9 PSI, rather than total CYTH2. (K), as for (I), but PTEN protein levels split by quantiles (red and blue, Q1 + Q2, Q3, low PTEN, green Q4, high PTEN). Median survival, sample size ( n ) and P ‐value, Log‐rank test (Mantel‐Cox) annotated. L Differential abundance ( x , Log Ratio between conditions; y , Log 10 q ‐values) of proteins in PIP 3 ‐responsive module (ARF6 HI ‐AGAP1 HI ‐CYTH2 2G ) versus PI(4,5)P 2 ‐responsive ARF module (ARF6 HI ‐AGAP1 HI ‐CYTH2 3G ) protein samples. Reverse Phase Protein Array Data, TCGA OV. Significantly altered components in AKT signalling pathway labelled (−Log 10 q ‐value > 1.3). M Schema, molecular model for ARF GTPase regulation of integrin‐dependent invasion. Source data are available online for this figure.

Article Snippet: The following antibodies were added at 1:200 dilution in PFS and incubated overnight at 4°C with gentle shaking: Collagen IV (Abcam, ab19808), pAKT pS473 (CST, 4060, D9E), pFAK pY397 (CST, 3283), pSRC Family pY416 (CST, 2101), V5‐Tag (ABM, G189), PI3Kβ (Proteintech, 21739‐1‐AP), AGAP1 (TFS, 50542), ITGB1 (Merck, MAB1997), ITGA5 (BD Bioscience, 553319).

Techniques: Immunofluorescence, Imaging, Staining, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Expressing, Two Tailed Test, Protein Array