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 Confocal images (single slice) of Trp53 −/− or Trp53 −/− ; Pten −/− (1.15) spheroids expressing mNeonGreen‐tagged (mNG) biosensors for PI(4,5)P 2 (PH‐PLCδ1) or PIP 3 (CYTH3 2G /GRP1). Magnified images from boxed regions, max projection of 8 (PH‐PLCδ1) or 3 (CYTH3 2G /GRP1) z‐slices, pseudocoloured in FIRE LUT. Arrowheads: red, cell–cell contact; yellow, nucleus; green, protrusion tip. Scale bar, 7 μm. Representative of 8 ( Trp53 −/− ) or 10 ( Trp53 −/− ; Pten −/− ) spheroids imaged across n = 2 independent experiments (PH‐PLCδ1) and 22 ( Trp53 −/− ) or 23 ( Trp53 −/− ; Pten −/− ) spheroids imaged across n = 4 independent experiments (CYTH3 2G /GRP1). B Intensity profiles for PH‐PLCδ1 and PH‐CYTH3 from spheroids shown in (A). Protrusions measured are annotated on images in upper panels, yellow lines. Arrowheads: red, protrusion tips. C Schema, select PI‐kinases and phosphatases and their inhibitors participating in PIP 3 production and downstream AKT phosphorylation. D Western blotting and quantitation for S6RP pS235/236, S6RP, GAPDH (sample integrity control) in Trp53 −/− ; Pten −/− 1.15 spheroids treated with DMSO or inhibitors annotated in (B) for 2 days. Representative of n = 3 independent lysate preparations. Data, mean ± SD of pS235/236:total S6RP ratio, normalised to DMSO. P ‐values, unpaired, two‐tailed t ‐tests, as annotated. E, F Quantitation of Trp53 −/− ; Pten −/− 1.15 spheroids treated with DMSO, AKTi (AKT inhibitor II) or pan‐PI3Ki (LY294002), 6‐h time intervals over 72 h. (E) Heatmap (viridis)—area presented as mean of Z ‐score values, normalised to control (DMSO). (F) Frequency of Spherical and Hyper‐protrusive phenotypes. Heatmap (grayscale)—phenotype proportion ( z ‐score) in control. Heatmap (blue‐red)—log 2 fold change from control. P ‐values, bubble size (Cochran–Mantel–Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow–Day test, Bonferroni adjustment, non‐significant). N = 2 independent experiments, 4–5 technical replicates/experiment. Total spheroid number per condition, Table . G Representative phase contrast images of spheroids described in (E). Outlines pseudocoloured for classification (Spherical, green; Hyper‐protrusive, blue). Magnified individual spheroids from boxed regions at indicated timepoints. Arrowheads, protrusions into ECM. Scale bar, 400 or 17 μm (indicated). H, I Quantitation of ID8 Trp53 −/− ; Pten −/− spheroids treated with PI3K isoform specific inhibitors: A66 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ) or CAL‐101 (PI3Kδ), 6‐h time intervals over 72 h. (H) Heatmap (viridis)—area presented as mean of Z ‐score values, normalised to control (DMSO). (I) Frequency of Spherical and Hyper‐protrusive phenotypes. Heatmap (grayscale)—phenotype proportion ( z ‐score) in control. Heatmap (blue‐red)—log 2 fold change from control. P ‐values, bubble size (Cochran–Mantel–Haenszel test with Bonferroni adjustment). Black dot, homogenous effect across independent experiments (Breslow–Day test, Bonferroni adjustment, non‐significant). N = 2 independent experiments, 3–5 technical replicates/experiment. Total spheroid number per condition, Table . J Representative phase contrast images of spheroids described in (H, I). Outlines pseudocoloured for classification (Spherical, green; Hyper‐protrusive, blue). Magnified individual spheroids from boxed regions at indicated timepoints. Arrowheads, protrusions into ECM. Scale bars, 400 or 17 μm, as indicated. K Confocal image of Trp53 −/− ; Pten −/− (1.15) spheroids stained for PI3Kβ (green), F‐actin (magenta) and Hoechst (grey). Magnified images from boxed regions, pseudocoloured in inverted grayscale (F‐actin) or FIRE LUT (PI3Kβ). Yellow or white/black arrowheads, enrichment of F‐actin or PI3Kβ at protrusion tips respectively. Scale bar, 5 μm. Representative of n = 5 spheroids. L Intensity profiles for PI3Kβ (green) and F‐Actin (magenta) from spheroid in (K). Tip measured is annotated, ECM to body, yellow arrow, tip, arrowhead. 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: Expressing, Phospho-proteomics, Western Blot, Quantitation Assay, Control, Two Tailed Test, Staining