Journal: The FEBS journal

Article Title: The protease ADAMTS5 controls ovarian cancer cell invasion, downstream of Rab25.

doi: 10.1111/febs.70080

Figure Lengend Snippet: Fig. 1. Rab25 induced ADAMTS5 expression in OC cells. (A) Schematic, cell lysate (CL) and conditioned media (CM) harvesting. A2780- DNA3 and A2780-Rab25 cells were plated on plastic (PL, N = 3 independent experiments) or telomerase-immortalised fibroblasts (TIF) cell- derived matrix (CDM, N = 4 independent experiments), cell lysates and concentrated conditioned media were extracted, protein levels of ADAMTS5 in CL and CM, and GAPDH in CL were measured by Western Blotting. Membranes were imaged with a Licor Odyssey Sa system, and the band intensity was quantified by IMAGE STUDIO LITE software. Data are presented as mean SEM. *P = 0.0286, Mann–Whitney test. (B) Schematic of the experimental plan. (C, D) A2780-DNA3 and A2780-Rab25 cells were seeded on plastic (C) or TIF- CDM (D) and the mRNA was extracted and quantified by qPCR. The data were normalised to A2780-DNA3 cells (ADAMTS5) or plotted in 2ΔΔCt (Rab25). Data are mean SEM from N = 3 independent experiments. The black dots represent the mean of individual experiments. **P = 0.005, ****P < 0.0001, Mann–Whitney test. (E, F) OVCAR3 cells were transfected with a non-targeting siRNA control (si-nt) or a Rab25-targeting siRNA (si-Rab25) and seeded on plastic (E) or CAF-CDM (F). ADAMTS5 and Rab25 mRNA levels were quantified by qPCR and normalised to si-nt. Data are presented as mean SEM from N = 3 independent experiments. The black dots represent the mean of individual experiments. **P = 0.0012, ***P = 0.0003, ****P < 0.0001, Mann–Whitney test.

Article Snippet: Primary antibodies for western blotting: ADAMTS5 (ab41037; Abcam, Cambridge, UK), Rab25 (18139-1-AP; Proteintech, Manchester, UK), AKT (#4691; Cell Signalling Technology, Leiden, the Netherlands), p-AKT (#4060; Cell Signalling Technology), ERK1/2 (#9102; Cell Signalling Technology), p-ERK (#9101; Cell Signalling Technology), NF-jB (10745-1-AP; Proteintech), GAPDH (SC-47724; Santa Cruz Biotechnology, Dallas, Texas, USA) and a-Tubulin (T9026; Sigma-Aldrich, Merck Life Science UK, Gillingham, Dorset, UK).

Techniques: Expressing, Derivative Assay, Western Blot, Software, MANN-WHITNEY, Transfection, Control

Journal: The FEBS journal

Article Title: The protease ADAMTS5 controls ovarian cancer cell invasion, downstream of Rab25.

doi: 10.1111/febs.70080

Figure Lengend Snippet: Fig. 2. HIF-1 suppressed ADAMTS5 expression in OC cells. (A) Schematic, HIF-1 signalling pathway. (B, C) OVCAR3 (B) and SKOV3 (C) cells were treated with DMSO, 5 or 10 nM Echinomycin for 24 h, and the mRNA levels of ADAMTS5 were measured by qPCR. Data were normalised to the DMSO control and presented as mean SEM from N = 3 independent experiments. The black dots represent the mean of individual experiments. **P = 0.0011, ***P = 0.0003, ****P < 0.0001, Kruskal–Wallis test. (D–G) OVCAR3 (D), SKOV3 (E), A2780-DNA3 (F) and A2780-Rab25 (G) cells were incubated under Normoxia (20% O2) or Hypoxia (1% O2) for 24 h, and the mRNA levels of ADAMTS5 were measured by qPCR. Data were normalised to Normoxia control and presented as mean SEM from N = 3 independent experiments. The black dots represent the mean of individual experiments. ***P < 0.001, ****P < 0.0001, Mann–Whitney test.

Article Snippet: Primary antibodies for western blotting: ADAMTS5 (ab41037; Abcam, Cambridge, UK), Rab25 (18139-1-AP; Proteintech, Manchester, UK), AKT (#4691; Cell Signalling Technology, Leiden, the Netherlands), p-AKT (#4060; Cell Signalling Technology), ERK1/2 (#9102; Cell Signalling Technology), p-ERK (#9101; Cell Signalling Technology), NF-jB (10745-1-AP; Proteintech), GAPDH (SC-47724; Santa Cruz Biotechnology, Dallas, Texas, USA) and a-Tubulin (T9026; Sigma-Aldrich, Merck Life Science UK, Gillingham, Dorset, UK).

Techniques: Expressing, Control, Incubation, MANN-WHITNEY

Journal: The FEBS journal

Article Title: The protease ADAMTS5 controls ovarian cancer cell invasion, downstream of Rab25.

doi: 10.1111/febs.70080

Figure Lengend Snippet: Fig. 4. Rab25 downregulation did not affect AKT nor ERK phosphorylation. (A) OVCAR3 cells were transfected with a non-targeting siRNA control (si-nt) or Rab25 targeting siRNA (si-Rab25) and the protein levels of AKT, p-AKT, Rab25, and GAPDH were quantified by Western Blotting. Membranes were imaged with a Licor Odyssey Sa system, and the band intensity was quantified by IMAGE STUDIO LITE software. The fold change of normalised p-AKT/AKT and Rab25/GAPDH was plotted. Data are presented as mean SEM from N = 4 independent experiments. *P = 0.0286, Mann–Whitney test. (B) The protein levels of ERK, p-ERK, and a-Tubulin in A2780-DNA3 and A2780-Rab25 cells were quantified by Western blotting as in A. The fold change of normalised p-ERK/ERK was plotted. Data are presented as mean SEM from N = 3 independent experiments. Mann–Whitney test.

Article Snippet: Primary antibodies for western blotting: ADAMTS5 (ab41037; Abcam, Cambridge, UK), Rab25 (18139-1-AP; Proteintech, Manchester, UK), AKT (#4691; Cell Signalling Technology, Leiden, the Netherlands), p-AKT (#4060; Cell Signalling Technology), ERK1/2 (#9102; Cell Signalling Technology), p-ERK (#9101; Cell Signalling Technology), NF-jB (10745-1-AP; Proteintech), GAPDH (SC-47724; Santa Cruz Biotechnology, Dallas, Texas, USA) and a-Tubulin (T9026; Sigma-Aldrich, Merck Life Science UK, Gillingham, Dorset, UK).

Techniques: Phospho-proteomics, Transfection, Control, Western Blot, Software, MANN-WHITNEY

Journal: The FEBS journal

Article Title: The protease ADAMTS5 controls ovarian cancer cell invasion, downstream of Rab25.

doi: 10.1111/febs.70080

Figure Lengend Snippet: Fig. 7. NF-jB inhibition impaired A2780-Rab25 cell migration on CDM. (A) Schematic of the experimental workflow. (B, C) A2780-Rab25 cells were pretreated with DMSO, 2.5, 5, or 10 lM BAY 11-7072 (BAY) for 24 h, seeded on CDM, and imaged live with a Nikon widefield live-cell system (Nikon Ti eclipse with Oko-lab environmental control chamber) for 16 h. Stills extracted from the movies are presented. The arrow-heads point to the elongated pseudopods. Scale bar, 100 lm. Representative spider plots show the migration paths of manually tracked cells (directionality > 0.5 in black, < 0.5 in red). Scale bar, 200 lm. The pseudopod length (lm), directionality and velocity (lmmin1) of cell migration were measured with IMAGEJ. Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. (D) A2780-Rab25 cells were pretreated with DMSO or 2.5 lM BAY 11-7072 (BAY) for 24 h, seeded on CDM, treated with conditioned media (CM) from A2780-DNA3 (DNA3) or Rab25-overexpressing A2780 cells (Rab25) and imaged live with a Nikon widefield live-cell system (Nikon Ti eclipse with Oko-lab environmental control chamber) for 16 h. Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. ****P < 0.0001, Kruskal–Wallis test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Article Snippet: Primary antibodies for western blotting: ADAMTS5 (ab41037; Abcam, Cambridge, UK), Rab25 (18139-1-AP; Proteintech, Manchester, UK), AKT (#4691; Cell Signalling Technology, Leiden, the Netherlands), p-AKT (#4060; Cell Signalling Technology), ERK1/2 (#9102; Cell Signalling Technology), p-ERK (#9101; Cell Signalling Technology), NF-jB (10745-1-AP; Proteintech), GAPDH (SC-47724; Santa Cruz Biotechnology, Dallas, Texas, USA) and a-Tubulin (T9026; Sigma-Aldrich, Merck Life Science UK, Gillingham, Dorset, UK).

Techniques: Inhibition, Migration, Control

Journal: The FEBS journal

Article Title: The protease ADAMTS5 controls ovarian cancer cell invasion, downstream of Rab25.

doi: 10.1111/febs.70080

Figure Lengend Snippet: Fig. 9. Rab25 expression promoted OC cell invasion in co-culture with CAFs. (A) Schematic of 3D invasion assays. (B) A2780-DNA3 and A2780-Rab25 cells were labelled with Cell Tracker Green and CAFs were labelled with Cell Tracker Red. Co-culture spheroids (1 : 1 ratio) generated by the hanging drop method were embedded in 3 mgmL1 Geltrex, 3 mgmL1 collagen I, and 25 lgmL1 fibronectin and imaged live with a Nikon A1 confocal microscope up to day 2. Scale bar, 200 lm. Spheroid invasion area was quantified with IMAGEJ and normalised to DNA3 day 1. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. *P = 0.0173, **P < 0.008, two-way ANOVA, Sidak’s multiple comparisons test. (C) A2780-DNA3/CAF co-culture spheroids were generated and embedded as in B, treated with conditioned media (CM) generated by A2780-DNA3 (DNA3) or A2780-Rab25 (Rab25) cells and imaged live with a Nikon A1 confocal microscope up to day 2. Scale bar, 200 lm. Spheroid invasion area was quantified with

Article Snippet: Primary antibodies for western blotting: ADAMTS5 (ab41037; Abcam, Cambridge, UK), Rab25 (18139-1-AP; Proteintech, Manchester, UK), AKT (#4691; Cell Signalling Technology, Leiden, the Netherlands), p-AKT (#4060; Cell Signalling Technology), ERK1/2 (#9102; Cell Signalling Technology), p-ERK (#9101; Cell Signalling Technology), NF-jB (10745-1-AP; Proteintech), GAPDH (SC-47724; Santa Cruz Biotechnology, Dallas, Texas, USA) and a-Tubulin (T9026; Sigma-Aldrich, Merck Life Science UK, Gillingham, Dorset, UK).

Techniques: Expressing, Co-Culture Assay, Generated, Microscopy, Whisker Assay

Journal: The FEBS journal

Article Title: The protease ADAMTS5 controls ovarian cancer cell invasion, downstream of Rab25.

doi: 10.1111/febs.70080

Figure Lengend Snippet: Fig. 13. Working model. Upregulated Rab25 induced ADAMTS5 expression through an NF-jB-dependent signalling pathway in OC cells. Secreted ADAMTS5 promoted OC cell migration and invasion in a proteolytic activity-related manner. Rab25 could also be involved in controlling the crosstalk between OC cells and CAFs in the TME. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Article Snippet: Primary antibodies for western blotting: ADAMTS5 (ab41037; Abcam, Cambridge, UK), Rab25 (18139-1-AP; Proteintech, Manchester, UK), AKT (#4691; Cell Signalling Technology, Leiden, the Netherlands), p-AKT (#4060; Cell Signalling Technology), ERK1/2 (#9102; Cell Signalling Technology), p-ERK (#9101; Cell Signalling Technology), NF-jB (10745-1-AP; Proteintech), GAPDH (SC-47724; Santa Cruz Biotechnology, Dallas, Texas, USA) and a-Tubulin (T9026; Sigma-Aldrich, Merck Life Science UK, Gillingham, Dorset, UK).

Techniques: Expressing, Migration, Activity Assay