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adamts5 inhibitor (MedChemExpress)

Name: adamts5 inhibitor
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MedChemExpress adamts5 inhibitor

Rab25 induced <t>ADAMTS5</t> 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.

Adamts5 Inhibitor, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 7 article reviews

adamts5 inhibitor - by Bioz Stars, 2026-03

93/100 stars

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1) Product Images from "The protease ADAMTS 5 controls ovarian cancer cell invasion, downstream of Rab25"

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

Journal: The Febs Journal

doi: 10.1111/febs.70080

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.

Figure Legend 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.

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

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 n m 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% O 2 ) or Hypoxia (1% O 2 ) 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.

Figure Legend Snippet: 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 n m 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% O 2 ) or Hypoxia (1% O 2 ) 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.

Techniques Used: Expressing, Control, Incubation, MANN-WHITNEY

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.

Figure Legend 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.

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

ADAMTS5 was required for Rab25‐dependent pseudopod elongation and directional migration of OC cells on CDM. (A–G) A2780‐DNA3 and A2780‐Rab25 cells were seeded on CDM and treated with DMSO, 5 μ m ADAMTS5 inhibitor (TS5 i), 10 or 50 μ m 4b. Cells were imaged live with a 10× Nikon widefield live‐cell system (Nikon Ti eclipse with Oko‐lab environmental control chamber) for 16 h. The pseudopod length (μm) (B, E), directionality (C, F) and velocity (μm·min −1 ) (D, G) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. * P = 0.0155, **** P < 0.0001, Kruskal–Wallis test. (H–J) A2780‐DNA3 and A2780‐Rab25 cells were seeded on plastic, treated, and imaged as in A. The aspect ratio, measured as cell length/cell width (H), directionality (I) and velocity (μm·min −1 ) (J) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 2 independent experiments. (K–M) A2780‐Rab25 cells were transfected with non‐targeting siRNA control (si‐nt) or ADAMTS5 targeting si‐RNA (si‐TS5) and seeded on CDM. Cells were imaged as in A and the pseudopod length (μm) (K), directionality (L) and velocity (μm·min −1 ) (M) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. **** P < 0.0001, Mann–Whitney test. (N) A2780‐Rab25 cells were transfected as in D and the mRNA levels of ADAMTS5 and GAPDH were measured by qPCR. Data were normalised to si‐nt and presented as mean ± SEM from N = 2 independent experiments. ** P = 0.0022, Mann–Whitney test. (O–R) A2780 cells were transfected with an empty vector control (empty) or ADAMTS5‐FLAG and seeded on CDM. Cells were imaged as in A and the pseudopod length (μm) (O), directionality (P) and velocity (μm·min −1 ) (Q) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 2 independent experiments. **** P < 0.0001, Mann–Whitney test. (R) ADAMTS5‐FLAG and GAPDH levels were measured by Western Blotting. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Figure Legend Snippet: ADAMTS5 was required for Rab25‐dependent pseudopod elongation and directional migration of OC cells on CDM. (A–G) A2780‐DNA3 and A2780‐Rab25 cells were seeded on CDM and treated with DMSO, 5 μ m ADAMTS5 inhibitor (TS5 i), 10 or 50 μ m 4b. Cells were imaged live with a 10× Nikon widefield live‐cell system (Nikon Ti eclipse with Oko‐lab environmental control chamber) for 16 h. The pseudopod length (μm) (B, E), directionality (C, F) and velocity (μm·min −1 ) (D, G) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. * P = 0.0155, **** P < 0.0001, Kruskal–Wallis test. (H–J) A2780‐DNA3 and A2780‐Rab25 cells were seeded on plastic, treated, and imaged as in A. The aspect ratio, measured as cell length/cell width (H), directionality (I) and velocity (μm·min −1 ) (J) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 2 independent experiments. (K–M) A2780‐Rab25 cells were transfected with non‐targeting siRNA control (si‐nt) or ADAMTS5 targeting si‐RNA (si‐TS5) and seeded on CDM. Cells were imaged as in A and the pseudopod length (μm) (K), directionality (L) and velocity (μm·min −1 ) (M) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. **** P < 0.0001, Mann–Whitney test. (N) A2780‐Rab25 cells were transfected as in D and the mRNA levels of ADAMTS5 and GAPDH were measured by qPCR. Data were normalised to si‐nt and presented as mean ± SEM from N = 2 independent experiments. ** P = 0.0022, Mann–Whitney test. (O–R) A2780 cells were transfected with an empty vector control (empty) or ADAMTS5‐FLAG and seeded on CDM. Cells were imaged as in A and the pseudopod length (μm) (O), directionality (P) and velocity (μm·min −1 ) (Q) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 2 independent experiments. **** P < 0.0001, Mann–Whitney test. (R) ADAMTS5‐FLAG and GAPDH levels were measured by Western Blotting. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Techniques Used: Migration, Control, Transfection, MANN-WHITNEY, Plasmid Preparation, Western Blot

Conditioned media derived from A2780‐Rab25 cells enhanced pseudopod elongation and directional migration of A2780‐DNA3 cells on CDM, in an ADAMTS5‐dependent manner. (A, C, E) Schematics of the experimental workflow. Conditioned media (CM) was collected from A2780‐DNA3 and A2780‐Rab25 cells seeded on plastic for 3 days. (B, D) A2780‐DNA3 cells were seeded on CDM and treated with A2780‐DNA3 or Rab25 CM (diluted 1 : 1 in complete media) (B), or with Rab25 CM in the presence of DMSO control or 5 μ m ADAMTS5 inhibitor (TS5 i, D). Cells were 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 arrowheads point to the elongated pseudopods. Scale bar, 100 μm. Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. ** P = 0.0057, **** P < 0.0001, Mann–Whitney test. (E, F) Conditioned media (CM) was collected from A2780‐Rab25 cells transfected with a non‐targeting siRNA control (si‐nt) or ADAMTS5 targeting si‐RNA (si‐TS5) and grown on plastic for 3 days. A2780‐DNA3 cells were seeded on CDM and treated with si‐nt or si‐TS5 A2780‐Rab25 CM (diluted 1 : 1 in complete media). Cells were 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 arrowheads point to the elongated pseudopods. Scale bar, 100 μm. Representative spider plots show the migration paths of manually tracked cells (directionality > 0.5 in black, < 0.5 in red). Scale bar, 200 μm. The pseudopod length (μm), directionality and velocity (μm·min −1 ) of cell migration were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. ** P = 0.0018, *** P = 0.0002, **** P < 0.0001, Mann–Whitney test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Figure Legend Snippet: Conditioned media derived from A2780‐Rab25 cells enhanced pseudopod elongation and directional migration of A2780‐DNA3 cells on CDM, in an ADAMTS5‐dependent manner. (A, C, E) Schematics of the experimental workflow. Conditioned media (CM) was collected from A2780‐DNA3 and A2780‐Rab25 cells seeded on plastic for 3 days. (B, D) A2780‐DNA3 cells were seeded on CDM and treated with A2780‐DNA3 or Rab25 CM (diluted 1 : 1 in complete media) (B), or with Rab25 CM in the presence of DMSO control or 5 μ m ADAMTS5 inhibitor (TS5 i, D). Cells were 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 arrowheads point to the elongated pseudopods. Scale bar, 100 μm. Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. ** P = 0.0057, **** P < 0.0001, Mann–Whitney test. (E, F) Conditioned media (CM) was collected from A2780‐Rab25 cells transfected with a non‐targeting siRNA control (si‐nt) or ADAMTS5 targeting si‐RNA (si‐TS5) and grown on plastic for 3 days. A2780‐DNA3 cells were seeded on CDM and treated with si‐nt or si‐TS5 A2780‐Rab25 CM (diluted 1 : 1 in complete media). Cells were 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 arrowheads point to the elongated pseudopods. Scale bar, 100 μm. Representative spider plots show the migration paths of manually tracked cells (directionality > 0.5 in black, < 0.5 in red). Scale bar, 200 μm. The pseudopod length (μm), directionality and velocity (μm·min −1 ) of cell migration were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. ** P = 0.0018, *** P = 0.0002, **** P < 0.0001, Mann–Whitney test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Techniques Used: Derivative Assay, Migration, Control, MANN-WHITNEY, Transfection

ADAMTS5 inhibition reduced the 3D invasion of OC cells. (A) Schematic of 3D invasion assays. (B) A2780‐DNA3 and A2780‐Rab25 spheroids generated by the hanging drop method were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin and imaged live with a Nikon A1 confocal microscope up to day 2. Scale bar, 200 μm. 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 = 0.0001, two‐way ANOVA, Sidak's multiple comparisons test. (C) A2780‐Rab25 spheroids generated by the hanging drop method were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I and 25 μg·mL −1 fibronectin in the presence of DMSO, 5 or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 2. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to DMSO day 1. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. * P = 0.0207, **** P < 0.0001, two‐way ANOVA, Dunnett's multiple comparisons test. (D) A2780‐Rab25 cells were transfected with an siRNA targeting ADAMTS5 (si‐TS5) or a non‐targeting siRNA control (si‐nt), spheroids were generated, embedded, and imaged as in A. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to si‐nt day 1. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. *** P = 0.0004, two‐way ANOVA, Sidak's multiple comparisons test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Figure Legend Snippet: ADAMTS5 inhibition reduced the 3D invasion of OC cells. (A) Schematic of 3D invasion assays. (B) A2780‐DNA3 and A2780‐Rab25 spheroids generated by the hanging drop method were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin and imaged live with a Nikon A1 confocal microscope up to day 2. Scale bar, 200 μm. 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 = 0.0001, two‐way ANOVA, Sidak's multiple comparisons test. (C) A2780‐Rab25 spheroids generated by the hanging drop method were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I and 25 μg·mL −1 fibronectin in the presence of DMSO, 5 or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 2. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to DMSO day 1. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. * P = 0.0207, **** P < 0.0001, two‐way ANOVA, Dunnett's multiple comparisons test. (D) A2780‐Rab25 cells were transfected with an siRNA targeting ADAMTS5 (si‐TS5) or a non‐targeting siRNA control (si‐nt), spheroids were generated, embedded, and imaged as in A. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to si‐nt day 1. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. *** P = 0.0004, two‐way ANOVA, Sidak's multiple comparisons test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Techniques Used: Inhibition, Generated, Microscopy, Whisker Assay, Transfection, Control

ADAMTS5 was required for OC cell invasion in co‐culture with CAFs. (A) OVCAR3 cells stably expressing nuclear GFP (H2B‐GFP) and cancer‐associated fibroblasts (CAFs) labelled with Cell tracker™ Red CMTPX (2 : 1 ratio) were generated, embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin in the presence of DMSO, 5 or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to DMSO day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments. * P < 0.05, ** P = 0.0035, *** P < 0.001, two‐way ANOVA, Dunnett's multiple comparisons test. (B) Co‐culture spheroids were generated with OVCAR3‐GFP cells (green) transfected with a non‐targeting control (si‐nt), Rab25‐targeting (si‐Rab25) or ADAMTS5 targeting (si‐TS5) si‐RNA and Cell tracker™ Red CMTPX (magenta) labelled CAFs (2 : 1 ratio), embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I and 25 μg·mL −1 fibronectin and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. The spheroid invasion area was calculated with imagej and normalised to si‐nt day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. * P = 0.0353, ** P = 0.0012, *** P = 0.0001, two‐way ANOVA, Dunnett's multiple comparisons test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Figure Legend Snippet: ADAMTS5 was required for OC cell invasion in co‐culture with CAFs. (A) OVCAR3 cells stably expressing nuclear GFP (H2B‐GFP) and cancer‐associated fibroblasts (CAFs) labelled with Cell tracker™ Red CMTPX (2 : 1 ratio) were generated, embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin in the presence of DMSO, 5 or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to DMSO day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments. * P < 0.05, ** P = 0.0035, *** P < 0.001, two‐way ANOVA, Dunnett's multiple comparisons test. (B) Co‐culture spheroids were generated with OVCAR3‐GFP cells (green) transfected with a non‐targeting control (si‐nt), Rab25‐targeting (si‐Rab25) or ADAMTS5 targeting (si‐TS5) si‐RNA and Cell tracker™ Red CMTPX (magenta) labelled CAFs (2 : 1 ratio), embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I and 25 μg·mL −1 fibronectin and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. The spheroid invasion area was calculated with imagej and normalised to si‐nt day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. * P = 0.0353, ** P = 0.0012, *** P = 0.0001, two‐way ANOVA, Dunnett's multiple comparisons test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Techniques Used: Co-Culture Assay, Stable Transfection, Expressing, Generated, Microscopy, Whisker Assay, Transfection, Control

ADAMTS5 inhibition did not affect CAF spheroid 3D monoculture invasion nor cell proliferation in 3D. (A, C) Experimental timeline. (B) Spheroids generated with Cell tracker™ Red CMTPX labelled CAFs were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin in the presence of DMSO control or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalized to DMSO day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments. (D) Spheroids generated with OVCAR3‐GFP cells (green) and non‐labelled CAFs (2 : 1 ratio) were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin and cultured in the presence of DMSO or 10 μ m ADAMTS5 inhibitor (TS5 i). EdU was added at day 6, spheroids were fixed on day 8, stained with EdU detection reagent (magenta) and Hoechst 33342 (blue) and imaged with a Nikon A1 confocal microscope. Scale bar, 200 μm. The percentage of EdU positive cells was calculated against Hoechst or GFP. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments.

Figure Legend Snippet: ADAMTS5 inhibition did not affect CAF spheroid 3D monoculture invasion nor cell proliferation in 3D. (A, C) Experimental timeline. (B) Spheroids generated with Cell tracker™ Red CMTPX labelled CAFs were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin in the presence of DMSO control or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalized to DMSO day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments. (D) Spheroids generated with OVCAR3‐GFP cells (green) and non‐labelled CAFs (2 : 1 ratio) were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin and cultured in the presence of DMSO or 10 μ m ADAMTS5 inhibitor (TS5 i). EdU was added at day 6, spheroids were fixed on day 8, stained with EdU detection reagent (magenta) and Hoechst 33342 (blue) and imaged with a Nikon A1 confocal microscope. Scale bar, 200 μm. The percentage of EdU positive cells was calculated against Hoechst or GFP. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments.

Techniques Used: Inhibition, Generated, Control, Microscopy, Whisker Assay, Cell Culture, Staining

ADAMTS5 expression correlated with poor prognosis of OC patients. (A–C) 655 ovarian cancer patients were stratified into low and high ADAMTS5 expression. The Kaplan–Meier analysis compared (A) overall survival (OS), (B) progression‐free survival (PFS) and (C) palliative performance scale (PPS) of OC patients with tumours expressing high ADAMTS5 levels (red) with those expressing low ADAMTS5 levels (black).

Figure Legend Snippet: ADAMTS5 expression correlated with poor prognosis of OC patients. (A–C) 655 ovarian cancer patients were stratified into low and high ADAMTS5 expression. The Kaplan–Meier analysis compared (A) overall survival (OS), (B) progression‐free survival (PFS) and (C) palliative performance scale (PPS) of OC patients with tumours expressing high ADAMTS5 levels (red) with those expressing low ADAMTS5 levels (black).

Techniques Used: Expressing

Working model. Upregulated Rab25 induced ADAMTS5 expression through an NF‐κB‐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.

Figure Legend Snippet: Working model. Upregulated Rab25 induced ADAMTS5 expression through an NF‐κB‐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.

Techniques Used: Expressing, Migration, Activity Assay

Image Search Results

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 ADAMTS5 inhibitor (5‐[[5‐[[(4‐Chlorophenyl)methyl]thio]‐1‐methyl‐3‐(trifluoromethyl)‐1H‐pyrazol‐4‐yl]methylene]‐2‐thioxo‐4‐thiazolidinone;(E)‐5‐((5‐((4‐Chlorobenzyl)thio)‐1‐methyl‐3‐(trifluoromethyl)‐1H‐pyrazol‐4 yl)methylene)‐2‐thioxothiazolidin‐4‐one CAS no.: 929634‐33‐3) was from MedChemExpress, MedChemTronica, Sollentuna, Sweden reported to be a potent ADAMTS‐5 inhibitor, with an IC50 of 1.1 μ m and > 40‐fold functional selectivity over ADAMTS4 [ , ].

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: 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 n m 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% O 2 ) or Hypoxia (1% O 2 ) 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.

Techniques: Expressing, Control, Incubation, MANN-WHITNEY

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.

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: ADAMTS5 was required for Rab25‐dependent pseudopod elongation and directional migration of OC cells on CDM. (A–G) A2780‐DNA3 and A2780‐Rab25 cells were seeded on CDM and treated with DMSO, 5 μ m ADAMTS5 inhibitor (TS5 i), 10 or 50 μ m 4b. Cells were imaged live with a 10× Nikon widefield live‐cell system (Nikon Ti eclipse with Oko‐lab environmental control chamber) for 16 h. The pseudopod length (μm) (B, E), directionality (C, F) and velocity (μm·min −1 ) (D, G) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. * P = 0.0155, **** P < 0.0001, Kruskal–Wallis test. (H–J) A2780‐DNA3 and A2780‐Rab25 cells were seeded on plastic, treated, and imaged as in A. The aspect ratio, measured as cell length/cell width (H), directionality (I) and velocity (μm·min −1 ) (J) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 2 independent experiments. (K–M) A2780‐Rab25 cells were transfected with non‐targeting siRNA control (si‐nt) or ADAMTS5 targeting si‐RNA (si‐TS5) and seeded on CDM. Cells were imaged as in A and the pseudopod length (μm) (K), directionality (L) and velocity (μm·min −1 ) (M) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. **** P < 0.0001, Mann–Whitney test. (N) A2780‐Rab25 cells were transfected as in D and the mRNA levels of ADAMTS5 and GAPDH were measured by qPCR. Data were normalised to si‐nt and presented as mean ± SEM from N = 2 independent experiments. ** P = 0.0022, Mann–Whitney test. (O–R) A2780 cells were transfected with an empty vector control (empty) or ADAMTS5‐FLAG and seeded on CDM. Cells were imaged as in A and the pseudopod length (μm) (O), directionality (P) and velocity (μm·min −1 ) (Q) were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 2 independent experiments. **** P < 0.0001, Mann–Whitney test. (R) ADAMTS5‐FLAG and GAPDH levels were measured by Western Blotting. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Techniques: Migration, Control, Transfection, MANN-WHITNEY, Plasmid Preparation, Western Blot

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: Conditioned media derived from A2780‐Rab25 cells enhanced pseudopod elongation and directional migration of A2780‐DNA3 cells on CDM, in an ADAMTS5‐dependent manner. (A, C, E) Schematics of the experimental workflow. Conditioned media (CM) was collected from A2780‐DNA3 and A2780‐Rab25 cells seeded on plastic for 3 days. (B, D) A2780‐DNA3 cells were seeded on CDM and treated with A2780‐DNA3 or Rab25 CM (diluted 1 : 1 in complete media) (B), or with Rab25 CM in the presence of DMSO control or 5 μ m ADAMTS5 inhibitor (TS5 i, D). Cells were 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 arrowheads point to the elongated pseudopods. Scale bar, 100 μm. Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. ** P = 0.0057, **** P < 0.0001, Mann–Whitney test. (E, F) Conditioned media (CM) was collected from A2780‐Rab25 cells transfected with a non‐targeting siRNA control (si‐nt) or ADAMTS5 targeting si‐RNA (si‐TS5) and grown on plastic for 3 days. A2780‐DNA3 cells were seeded on CDM and treated with si‐nt or si‐TS5 A2780‐Rab25 CM (diluted 1 : 1 in complete media). Cells were 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 arrowheads point to the elongated pseudopods. Scale bar, 100 μm. Representative spider plots show the migration paths of manually tracked cells (directionality > 0.5 in black, < 0.5 in red). Scale bar, 200 μm. The pseudopod length (μm), directionality and velocity (μm·min −1 ) of cell migration were measured with imagej . Data were plotted as violin plots (median and quartiles) from N = 3 independent experiments. ** P = 0.0018, *** P = 0.0002, **** P < 0.0001, Mann–Whitney test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Techniques: Derivative Assay, Migration, Control, MANN-WHITNEY, Transfection

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: ADAMTS5 inhibition reduced the 3D invasion of OC cells. (A) Schematic of 3D invasion assays. (B) A2780‐DNA3 and A2780‐Rab25 spheroids generated by the hanging drop method were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin and imaged live with a Nikon A1 confocal microscope up to day 2. Scale bar, 200 μm. 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 = 0.0001, two‐way ANOVA, Sidak's multiple comparisons test. (C) A2780‐Rab25 spheroids generated by the hanging drop method were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I and 25 μg·mL −1 fibronectin in the presence of DMSO, 5 or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 2. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to DMSO day 1. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. * P = 0.0207, **** P < 0.0001, two‐way ANOVA, Dunnett's multiple comparisons test. (D) A2780‐Rab25 cells were transfected with an siRNA targeting ADAMTS5 (si‐TS5) or a non‐targeting siRNA control (si‐nt), spheroids were generated, embedded, and imaged as in A. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to si‐nt day 1. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. *** P = 0.0004, two‐way ANOVA, Sidak's multiple comparisons test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Techniques: Inhibition, Generated, Microscopy, Whisker Assay, 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: ADAMTS5 was required for OC cell invasion in co‐culture with CAFs. (A) OVCAR3 cells stably expressing nuclear GFP (H2B‐GFP) and cancer‐associated fibroblasts (CAFs) labelled with Cell tracker™ Red CMTPX (2 : 1 ratio) were generated, embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin in the presence of DMSO, 5 or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalised to DMSO day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments. * P < 0.05, ** P = 0.0035, *** P < 0.001, two‐way ANOVA, Dunnett's multiple comparisons test. (B) Co‐culture spheroids were generated with OVCAR3‐GFP cells (green) transfected with a non‐targeting control (si‐nt), Rab25‐targeting (si‐Rab25) or ADAMTS5 targeting (si‐TS5) si‐RNA and Cell tracker™ Red CMTPX (magenta) labelled CAFs (2 : 1 ratio), embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I and 25 μg·mL −1 fibronectin and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. The spheroid invasion area was calculated with imagej and normalised to si‐nt day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 4 independent experiments. * P = 0.0353, ** P = 0.0012, *** P = 0.0001, two‐way ANOVA, Dunnett's multiple comparisons test. Image created with items adapted from Servier Medical Art, licensed under CC BY 4.0.

Techniques: Co-Culture Assay, Stable Transfection, Expressing, Generated, Microscopy, Whisker Assay, 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: ADAMTS5 inhibition did not affect CAF spheroid 3D monoculture invasion nor cell proliferation in 3D. (A, C) Experimental timeline. (B) Spheroids generated with Cell tracker™ Red CMTPX labelled CAFs were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin in the presence of DMSO control or 10 μ m ADAMTS5 inhibitor (TS5 i) and imaged live with a Nikon A1 confocal microscope up to day 8. Scale bar, 200 μm. Spheroid invasion area was quantified with imagej and normalized to DMSO day 4. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments. (D) Spheroids generated with OVCAR3‐GFP cells (green) and non‐labelled CAFs (2 : 1 ratio) were embedded in 3 mg·mL −1 Geltrex, 3 mg·mL −1 collagen I, and 25 μg·mL −1 fibronectin and cultured in the presence of DMSO or 10 μ m ADAMTS5 inhibitor (TS5 i). EdU was added at day 6, spheroids were fixed on day 8, stained with EdU detection reagent (magenta) and Hoechst 33342 (blue) and imaged with a Nikon A1 confocal microscope. Scale bar, 200 μm. The percentage of EdU positive cells was calculated against Hoechst or GFP. Data are presented as box and whisker plots (Min to Max, + represents the mean) from N = 3 independent experiments.

Techniques: Inhibition, Generated, Control, Microscopy, Whisker Assay, Cell Culture, Staining

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: ADAMTS5 expression correlated with poor prognosis of OC patients. (A–C) 655 ovarian cancer patients were stratified into low and high ADAMTS5 expression. The Kaplan–Meier analysis compared (A) overall survival (OS), (B) progression‐free survival (PFS) and (C) palliative performance scale (PPS) of OC patients with tumours expressing high ADAMTS5 levels (red) with those expressing low ADAMTS5 levels (black).

Techniques: Expressing

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: Working model. Upregulated Rab25 induced ADAMTS5 expression through an NF‐κB‐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.

Techniques: Expressing, Migration, Activity Assay

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