Journal: bioRxiv

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

doi: 10.1101/2022.11.29.518198

Figure Lengend Snippet: A. Heatmap, log 2 -transformed RNA-sequencing read counts of ARF GEFs in ID8 spheroids and 2D monolayers (Wild Type, WT (2D)) across n=4 independent RNA preparations. B-C. ID8 Trp53 -/- ;Pten -/- spheroids treated with 20μΜ SecinH3 quantified for (B) Area and (C) phenotype frequency (Spherical, Hyper-protrusive). Heatmaps, (B) (viridis colour scale) is mean of Z-score normalised values to control (DMSO) (C) Grayscale heatmaps, phenotype proportion (z-score) in control (DMSO). Blue-to-read heatmap, log 2 fold change from control. P-values, bubble size (B). Student’s t-test, Bonferroni adjustment; (C). Cochran-Mantel-Haenszel test with Bonferroni adjustment). Black dot indicates homogenous effect across experiments (Breslow-Day test, Bonferroni adjustment, non-significant p-value). N=3 experiments set up using repeated cultures of each sub-line, 4-5 technical replicates/sub-line per experiment, total number of spheroids analysed in Supplementary Table 1. D. Representative phase contrast images of ID8 Trp53 -/- ;Pten -/- treated with 20μΜ SecinH3, described in B-C . Outlines; Hyper-protrusive, blue; Spherical, green. Magnified spheroids shown for each condition at select time points. Arrowheads, protrusions into ECM. Scale bar, 400μm or 17μm (indicated). E. Representative images of ID8 Trp53 -/- ;Pten -/- 1.15 wounded monolayers treated with DMSO or SecinH3 (20μΜ) in wounded monolayers invading ECM. Yellow lines, initial wound. Arrowheads, invasive protrusions. Outlines of invasive front pseudocoloured by time and overlaid as concatenate over phase image of initial wound. N=3 experiments set up using repeated cultures of each sub-line, 3-5 technical replicates per sub-line in each experiment. Scale bar, 200μm or 45μm (zoomed in boxed areas). F. Quantitation of (E). Graph, Relative Wound Density (RWD) at t 1/2 max (time when DMSO 50% closed). Data, mean (black square) ± SD for 3 repeated experiments (large circles), 3-6 technical replicates per experiment (small circles). Exact p-value annotated, ANOVA with Tukey’s HSD test. G. Spider plots of leader cell movement in the first 19 hrs of invasion of Trp53 -/ - ;Pten -/- 1.15 ID8 cells treated with DMSO or SecinH3. 10-25 leader cells were tracked per experiment (n=2 set up using repeated cultures of each sub-line), across multiple technical replicates per experiment. Representative plots from cells tracked in 1 independent experiment shown. H-P. CYTH2 mRNA levels in (H, I) laser-capture micro-dissected normal ovarian surface epithelium versus HGSOC epithelium or normal ovarian stroma versus ovarian cancer-associated stroma, or (J-P) bulk sequencing of normal ovary versus tumour. Specific dataset, sample size (n) and p-values (Mann-Whitney) annotated. Q-T. Overall survival (% patients, months; TCGA OV dataset), of patients grouped by low (M1) versus high (M2) levels based on a median split of (Q) CYTH2 mRNA, (R) CYTH2 exon 9.1 percentage spliced in ratio (PSI), (S) combination of ARF6 and CYTH2 mRNA, or (T) combination of A RF6 mRNA and CYTH2 Ex9.1 PSI. Median survival, sample size (n) and p-value, Log-rank test (Mantel-Cox) annotated.

Article Snippet: Inhibitors were added at the following concentrations: 10 μΜ Nutlin3A (Sigma-Aldrich SML0580), 10 μM pan-PI3Ki (LY294002, Merck 440204), 200 nM PI3Kα-i (A66, Selleckchem S2636), 200 nm PI3Kβ-i (AZD8186, AstraZeneca), 200 nm PI3Kγ-i (AS605240, Stratech S1410), 200 nm PI3Kδ-i (Cal-101, Stratech S2226), 20 μM SecinH3 (Tocris 2849).

Techniques: Transformation Assay, RNA Sequencing, Control, Quantitation Assay, Sequencing, MANN-WHITNEY

Journal: Oncology Letters

Article Title: Function and mode of action of cytohesins in the epidermal growth factor pathway in colorectal cancer cells

doi: 10.3892/ol.2012.1064

Figure Lengend Snippet: Cytohesin-2 (ARNO) enhances the activation of EGFR. (A) The cytohesin inhibitor SecinH3 reduces EGFR signaling. Western blot analysis of HT-29 cells treated with SecinH3 or solvent and stimulated with EGF, respectively, is shown. Phosphorylation of the indicated proteins was determined by immunodetection using phosphospecific antibodies. Glyceraldehyde phosphate dehydrogenase (GAPDH) served as a loading control. (B) ARNO siRNA reduces EGFR signaling. Western blot analysis of HT-29 cells treated with ARNO-siRNA or Lipofectamine 2000 and stimulated with EGF, respectively, is shown.

Article Snippet: SecinH3 (cat. no. 565725/sc-203260) was purchased from Merck and siRNA oligo was purchased from Shanghai Gene Pharma (China).

Techniques: Activation Assay, Western Blot, Immunodetection

Journal: Oncology Letters

Article Title: Function and mode of action of cytohesins in the epidermal growth factor pathway in colorectal cancer cells

doi: 10.3892/ol.2012.1064

Figure Lengend Snippet: SecinH3 and ARNO siRNA inhibit the proliferation of HT-29 cells. (A) SecinH3 inhibits the proliferation of HT-29 cells. The graph shows the relative cell number (from an MTT assay) after 24, 48 and 72 h treatment with either SecinH3 or DMSO. * P<0.05 for the SecinH3 blocking group vs. the control group; n=3. (B) ARNO siRNA inhibits the proliferation of HT-29 cells. The graph shows the relative cell number (from an MTT assay) following 24 and 48 h treatment with ARNO siRNA or negative siRNA. * P<0.05 for ARNO siRNA vs. negative siRNA at 48 h; n=3.

Article Snippet: SecinH3 (cat. no. 565725/sc-203260) was purchased from Merck and siRNA oligo was purchased from Shanghai Gene Pharma (China).

Techniques: MTT Assay, Blocking Assay

Journal: Journal of Cell Science

Article Title: Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6

doi: 10.1242/jcs.213314

Figure Lengend Snippet: Endogenous and synthetic sphingolipids that downregulate nutrient transporters also inactivate the ARF6 GTPase. (A) ARF6-GTP levels normalized to total ARF6 in FL5.12 cells treated with the cytohesin inhibitor SecinH3 (30 µM), C2-ceramide (50 µM), bacterial sphingomyelinase (bSMase; 100 mU/ml), FTY720 (5 µM) or 893 (5 µM) for 3 h. (B,C) ARF6-GTP levels in FL5.12 cells treated with FTY720 (0, 1.25, 2.5, 5 or 10 µM) for 30 min (B) or with 10 µM FTY720 for 5, 15 or 30 min (C). (D) ARF6-GTP levels in FL5.12, MDA-MB-231, HeLa, SW620, PC3 or SupB15 treated for 3 h with FTY720 (10 µM). (E) Steady state surface CD98 levels in FL5.12 cells treated with C2-ceramide or C2-dihydroceramide at the indicated concentrations for 3 h. (F) Viability of C2-ceramide- or C2-dihydroceramide-treated FL5.12 cells at 48 h. (G) ARF6-GTP levels in FL5.12 cells treated with C2-ceramide (50 µM) or C2-dihydroceramide (50 µM). (H) ARF6-GTP levels in FL5.12 cells treated with CalyculinA (10 nM) for 1 h. Means±s.e.m. shown of n≥3 in all panels. Using two-tailed t-test (D-F,H) or ordinary one-way ANOVA (A-C,G): n.s., not significant; *P≤0.05; **P≤0.01; ***P≤0.001. Dunnett's test was used to correct for multiple comparisons (A-C,G). See also Fig. S5.

Article Snippet: C2-ceramide (VWR Cat #89164-564); dihydro-C2-ceramide (VWR Cat #89164-568); FTY720 (Fisher Cat #50148635); cantharidin (VWR Cat #89147-162); calyculinA (VWR Cat #89157-750); tautomycin (VWR Cat #89149-880); YM201636 (Cayman Chemical Cat #13576); SecinH3 (Cayman Chemical Cat #10009570); NAV2729 (Tocris Cat #5986).

Techniques: Two Tailed Test

Journal: Journal of Cell Science

Article Title: Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6

doi: 10.1242/jcs.213314

Figure Lengend Snippet: ARF6 inactivation is sufficient to inhibit nutrient transporter recycling. (A) HeLa cells expressing HA-ARF6 or HA-ARF6-T27N stained for CD98. (B) Quantification of intracellular CD98 intensity in A. (C) Surface CD98 quantification and ARF6-GTP levels in FL5.12 cells treated with FTY720 (5 µM) or SecinH3 (30 µM) for 3 h. (D) HeLa cells treated with SecinH3 (30 µM) or NAV2729 (12.5 µM) for 16 h and stained for CD98. (E) HeLa cells treated with SecinH3 (30 µM) or NAV2729 (12.5 µM) for 1 or 6 h and stained for MICAL-L1. (F) Quantification of E. (G) Cytoplasmic intensity quantification of CD98 and GLUT1 in HeLa cells treated with vehicle, 893 (10 µM), or C2-ceramide (50 µM) for 1 h, washed with PBS, then returned to 893 (10 µM) or placed in vehicle or SecinH3 (30 µM) for 2 h. Means±s.e.m. shown for n≥3 in all panels. Using a two-tailed t-test (B) or an ordinary one-way ANOVA (C,F,G) to compare treated samples with controls: n.s., not significant; *P≤0.05; **P≤0.01; ***P≤0.001. Dunnett's test was used in C,F and Tukey's test was used in G to correct for multiple comparisons. Scale bars: 10 µm. See also Figs S6 and S7.

Article Snippet: C2-ceramide (VWR Cat #89164-564); dihydro-C2-ceramide (VWR Cat #89164-568); FTY720 (Fisher Cat #50148635); cantharidin (VWR Cat #89147-162); calyculinA (VWR Cat #89157-750); tautomycin (VWR Cat #89149-880); YM201636 (Cayman Chemical Cat #13576); SecinH3 (Cayman Chemical Cat #10009570); NAV2729 (Tocris Cat #5986).

Techniques: Expressing, Staining, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Sphingolipids inhibit endosomal recycling of nutrient transporters by inactivating ARF6

doi: 10.1242/jcs.213314

Figure Lengend Snippet: ARF6 inhibition enhances the anti-neoplastic effects of synthetic sphingolipids. (A) Viability of SupB15 cells treated with FTY720 (5 µM), SecinH3 (30 µM) or both. (B-E) Viability of different cell lines treated with 893 (4 µM), SecinH3 (30 µM) or a combination. (B) SW620 cells at 120 h. (C) PC3 cells at 144 h. (D) MCF7 cells at 120 h. (E) MDA-MB-231 cells at 96 h. (F) MDA-MB-231 cells at 96 h treated with 893 (4 µM) and NAV2729 (6.25 µM). (G) Peripheral blood mononuclear cell (PBMC) colony formation assay at the indicated doses of 893 and SecinH3. Means±s.e.m. shown for n≥3 in all panels. Using two-tailed t-test (A) or ordinary one-way ANOVA (B-G) to compare with controls: n.s., not significant; *P≤0.05; **P≤0.01; ***P≤0.001. Tukey's test was used to correct for multiple comparisons in B-G; statistics are relative to the respective control.

Article Snippet: C2-ceramide (VWR Cat #89164-564); dihydro-C2-ceramide (VWR Cat #89164-568); FTY720 (Fisher Cat #50148635); cantharidin (VWR Cat #89147-162); calyculinA (VWR Cat #89157-750); tautomycin (VWR Cat #89149-880); YM201636 (Cayman Chemical Cat #13576); SecinH3 (Cayman Chemical Cat #10009570); NAV2729 (Tocris Cat #5986).

Techniques: Inhibition, Colony Assay, Two Tailed Test