Journal: Cancers

Article Title: MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells

doi: 10.3390/cancers13020274

Figure Lengend Snippet: Morphology of ovarian cancer cells when co-cultured with mesothelial cells and omental adipocytes. Panels A and B depict the normal cobblestone epithelial morphology of OVCA429 ( A ) and OVCA 433 ( B ) cells when stained with Texas Red Phalloidin for F-actin (and DAPI for nuclei). OVCA429 ( C ) and OVCA433 ( D ) cells were cultured on top of a confluent layer of primary human mesothelial cells and stained with Texas Red phalloidin after 24 h. OVCA429-CD133-GFP ( E ) and OVCA433 CD133-GFP ( F ) cells were cultured on top of confluent differentiated omental adipocytes for 5 days. 40X original magnification. Scale bar = 75 µm. Green = GFP/adipocytes. Arrow = tumor cell projections.

Article Snippet: OVCA429 and OVCA 433 cells were serum starved for 24 h. Cells were then treated with growth factors (as indicated above) for 30 min. Whole-cell lysates were obtained by lysing OVCA429 and OVCA433 cells in RIPA (50 mM Tris pH7.5, 150 mM NaCl, 1% NP-40, 0.5% EDTA, and 1× Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL, USA)).

Techniques: Cell Culture, Staining

Journal: Cancers

Article Title: MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells

doi: 10.3390/cancers13020274

Figure Lengend Snippet: Macrophages alter cellular morphology of ovarian cancer cells during co-culture. THP-1 cells were differentiated and/or differentiated followed by subsequent polarization to M1 and M2 macrophages. After differentiation/polarization CAOV3, OVCA429, and OVCA433 cells were cultured with differentiated THP-1, M1, and M2 polarized macrophages for 48 h. Cultures were stained for CD14 to denote THP-1 derived cells (green), Texas Red Phalloidin as a marker of F-actin (red), and DAPI (blue). 20× original magnification. Scale bar = 150 µm. Arrow = denotes membrane projections produced between tumor cells.

Article Snippet: OVCA429 and OVCA 433 cells were serum starved for 24 h. Cells were then treated with growth factors (as indicated above) for 30 min. Whole-cell lysates were obtained by lysing OVCA429 and OVCA433 cells in RIPA (50 mM Tris pH7.5, 150 mM NaCl, 1% NP-40, 0.5% EDTA, and 1× Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL, USA)).

Techniques: Co-Culture Assay, Cell Culture, Staining, Derivative Assay, Marker, Membrane, Produced

Journal: Cancers

Article Title: MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells

doi: 10.3390/cancers13020274

Figure Lengend Snippet: Macrophage conditioned media induces tunneling nanotubes (TnT)-like projections in ovarian cancer cells. CAOV3, OVCA429, and OVCA433 cells were serum starved for 24 h and left untreated or treated with conditioned media from differentiated THP-1, M1, and M2 macrophages for an additional 24 h. Cells were stained for Texas Red phalloidin (F-actin) and DAPI (blue). 20X original magnification. Scale bar = 150 µm. Arrow = long thin actin-based structures > 30 nM (TnT).

Article Snippet: OVCA429 and OVCA 433 cells were serum starved for 24 h. Cells were then treated with growth factors (as indicated above) for 30 min. Whole-cell lysates were obtained by lysing OVCA429 and OVCA433 cells in RIPA (50 mM Tris pH7.5, 150 mM NaCl, 1% NP-40, 0.5% EDTA, and 1× Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL, USA)).

Techniques: Staining

Journal: Cancers

Article Title: MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells

doi: 10.3390/cancers13020274

Figure Lengend Snippet: EGF and TGFα induce TnTs in ovarian cancer cell lines. CAOV3 ( A – C , J , K ), OVCA429 ( D – F , L , M ) and OVCA433 ( G – I , N , O ) cells were serum starved for 24 h and then treated with 20 nM EGF for 24 h or 50 ng/mL TGFα for 24 h. Cells were stained with Texas Red Phalloidin to visualize F-actin based structures and DAPI (blue). Arrows denote TnTs. ( A – I ) are 20X original magnification and ( J – O ) are 40X original magnification.

Article Snippet: OVCA429 and OVCA 433 cells were serum starved for 24 h. Cells were then treated with growth factors (as indicated above) for 30 min. Whole-cell lysates were obtained by lysing OVCA429 and OVCA433 cells in RIPA (50 mM Tris pH7.5, 150 mM NaCl, 1% NP-40, 0.5% EDTA, and 1× Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL, USA)).

Techniques: Staining

Journal: Cancers

Article Title: MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells

doi: 10.3390/cancers13020274

Figure Lengend Snippet: TnT formation requires MAPK activity. To determine if MAPK or PI3K signaling pathways contributed to TnT formation down stream of EGFR activity OCCs were treated with EGF and pathway inhibitors. ( A ) CAOV3, OVCA429, and OVCA433 cells were serum starved for 24 h and then treated for an additional 24 h with 20 nM EGF + DMSO, EGF + Wortmannin (PI3K inhibitor), EGF + U0126 (MEK inhibitor), EGF + SCH772984 (ERK inhibitor), or EGF + SL0101 (RSK inhibitor). Cells were stained with Texas Red Phalloidin to visualize F-actin based structures and DAPI (blue). Each experiment was performed in triplicate and a minimum of 3 fields of cells were analyzed for each treatment. ( B ) Total cell numbers and number of TnTs were counted for each field. Counts were averaged and error bars denote SEM between at least 3 biological replicates. For statistics, comparisons are between the EGF + DMSO and EGF + treatment. Arrow = TnT. *** p < 0.001, **** p < 0.0001.

Article Snippet: OVCA429 and OVCA 433 cells were serum starved for 24 h. Cells were then treated with growth factors (as indicated above) for 30 min. Whole-cell lysates were obtained by lysing OVCA429 and OVCA433 cells in RIPA (50 mM Tris pH7.5, 150 mM NaCl, 1% NP-40, 0.5% EDTA, and 1× Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL, USA)).

Techniques: Activity Assay, Protein-Protein interactions, Staining

Journal: Cancers

Article Title: MAPK Signaling Is Required for Generation of Tunneling Nanotube-Like Structures in Ovarian Cancer Cells

doi: 10.3390/cancers13020274

Figure Lengend Snippet: Membrane protrusions induction by macrophage conditioned media requires ERK activity. To assess if inhibition of ERK activity can prevent TnT formation in OCCs treated with macrophages, conditioned media (CM), CAOV3, OVCA429, and OVCA433 cells were serum starved for 24 h and then treated with conditioned media from differentiated THP-1, M1, and M2 macrophages in the presence of the ERK inhibitor SCH772984 for an additional 24 h. The top row depicts OCCs treated with M1 CM and DMASO, which results in TnTs and altered morphology (as seen in ). Cells were stained with Texas Red Phalloidin to visualize F-actin based structures and DAPI (blue). Images are representative of 3 independent experiments. 20X original magnification. Scale bar = 150 µm.

Article Snippet: OVCA429 and OVCA 433 cells were serum starved for 24 h. Cells were then treated with growth factors (as indicated above) for 30 min. Whole-cell lysates were obtained by lysing OVCA429 and OVCA433 cells in RIPA (50 mM Tris pH7.5, 150 mM NaCl, 1% NP-40, 0.5% EDTA, and 1× Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL, USA)).

Techniques: Membrane, Activity Assay, Inhibition, Staining