Journal: The Febs Journal

Article Title: The protease ADAMTS 5 controls ovarian cancer cell invasion, downstream of Rab25

doi: 10.1111/febs.70080

Figure Lengend Snippet: 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 2 −∆∆Ct (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: The membranes were then imaged with a LICOR Odyssey Sa system, and the intensity of the protein bands was quantified with image studio lite software (LICOR Biotechnology, Lincoln, Nebraska USA).

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

Journal: The Febs Journal

Article Title: The protease ADAMTS 5 controls ovarian cancer cell invasion, downstream of Rab25

doi: 10.1111/febs.70080

Figure Lengend Snippet: Rab25 induced ADAMTS5 expression in an NF‐κB dependent manner. (A) Schematic, NF‐κB signalling pathway. (B, C) OVCAR3 cells were treated with DMSO, 2.5, 5 or 10 μ m BAY 11‐7082 for 24 h and the mRNA levels of ADAMTS5 (B) and Rab25 (C) 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 < 0.0001, Kruskal–Wallis test. (D, E) A2780‐DNA3, A2780‐Rab25 cells (D), OVCAR3 cells transfected with non‐targeting (si‐nt) or Rab25 targeting (si‐Rab25) si‐RNA (E) were seeded on glass‐bottom dishes, fixed, stained for NF‐κB (green), nuclei (blue) and Actin (grey), and imaged with a Nikon A1 confocal microscope. Scale bar, 50 μm. NF‐κB integrated density for the whole cell and the nucleus was measured with imagej and normalised to the cell area. Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. **** P < 0.0001, Mann–Whitney test. (F) A2780‐Rab25 were stimulated with 40 ng·mL −1 TNFα for 40 min, fixed, stained for NF‐κB (green), Actin and nuclei and imaged with a Nikon A1 confocal microscope. Scale bar, 20 μm. NF‐κB integrated density for the whole cell and the nucleus was measured with imagej and normalised to the cell area. Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. **** P < 0.0001, Mann–Whitney test. (G, H) NF‐κB and GAPDH protein levels in A2780‐DNA3 and A2780‐Rab25 cells (G) or OVCAR3 cells transfected with non‐targeting (si‐nt) or Rab25 targeting (si‐Rab25) si‐RNA (H) 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 NF‐κB/GAPDH was plotted. Data are presented as mean ± SEM from N = 5 independent experiments. ** P = 0.0079 for both G and H, Mann–Whitney test. (I) CXCL8 mRNA levels in A2780‐DNA3 and Rab25 cells were quantified by qPCR and normalised to A2780‐DNA3 cells. Data are presented as mean ± SEM from N = 3 independent experiments. **** P < 0.0001, Mann–Whitney test. (J) CXCL8 mRNA levels in OVCAR3 cells transfected with non‐targeting (si‐nt) or Rab25 targeting (si‐Rab25) si‐RNA were quantified by qPCR and normalised to si‐nt. Data are presented as mean ± SEM from N = 3 independent experiments. *** P < 0.001, Mann–Whitney test. (K) CXCL8 mRNA levels in OVCAR3 cells treated with DMSO or 10 μ m BAY 11‐7082 for 24 h were quantified by qPCR and normalised to DMSO control. Data are presented as mean ± SEM from N = 3 independent experiments. **** P < 0.0001, Mann–Whitney test.

Article Snippet: The membranes were then imaged with a LICOR Odyssey Sa system, and the intensity of the protein bands was quantified with image studio lite software (LICOR Biotechnology, Lincoln, Nebraska USA).

Techniques: Expressing, Control, Transfection, Staining, Microscopy, MANN-WHITNEY, Western Blot, Software

Journal: The Febs Journal

Article Title: The protease ADAMTS 5 controls ovarian cancer cell invasion, downstream of Rab25

doi: 10.1111/febs.70080

Figure Lengend Snippet: 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 α‐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: The membranes were then imaged with a LICOR Odyssey Sa system, and the intensity of the protein bands was quantified with image studio lite software (LICOR Biotechnology, Lincoln, Nebraska USA).

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