Journal: Cancer research

Article Title: PTEN Lipid Phosphatase Activity Suppresses Melanoma Formation by Opposing an AKT/mTOR/FRA1 Signaling Axis

doi: 10.1158/0008-5472.CAN-23-1730

Figure Lengend Snippet: A, Proliferation as measured by relative confluence of M10M6 and M10M1 cells expressing Pten WT or Pten C124S or Pten WT +Fra1. B, Quantification of percentage occupied area of low-density colony formation assays of M10M6 and M10M1 cells expressing Pten WT or Pten C124S or Pten WT +Fra1. C, Quantification of colony number of anchorage-independent growth of M10M6 and M10M1 cells expressing Pten WT or Pten C124S or Pten WT +Fra1. D, Quantification of cell numbers of transwell invasion assays of M10M6 and M10M1 cells expressing Pten WT or Pten C124S or Pten WT +Fra1. E-F, Quantification of cell numbers of transwell invasion assays of FRA1 silenced mouse melanoma cells M10M6 (E) and human melanoma cells WM115, 1205Lu, and WM2664 (F). G-H, M10M6 cells expressing Pten WT or Pten C124S or Pten WT +Fra1 were subcutaneously injected into NSG mice (n=10). Mice were fed chow containing 200 mg/kg Doxycycline to induce expression of PTEN mutants. Tumor volumes were measured every 3 days. The curves of tumor volume (G) and the tumor weight at the end point (H) are shown. Mean ± SEM are shown in (A-H). Data are analyzed with Student’s unpaired t test, * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Human melanoma cell lines WM115 (RRID:CVCL_0040), WM266–4 (RRID:CVCL_2765), and 1205Lu (RRID:CVCL_5239) were obtained from Meenhard Herlyn (Wistar Institute) and SBCL2 (RRID:CVCL_D732) were obtained from David Tuveson (Cold Spring Harbor Laboratory).

Techniques: Over Expression, Expressing, Injection

Journal: Cell Death & Disease

Article Title: Protein dynamics at invadopodia control invasion–migration transitions in melanoma cells

doi: 10.1038/s41419-023-05704-4

Figure Lengend Snippet: A In situ melanoma WM1832 cells were plated on FITC-Gelatin (Grey), fixed, stained with Hoechst and labelled for actin (Red) and cortactin (Cyan). Boxed regions and insets depict invadopodia. Graphs indicate fluorescent intensity in arbitrary units (A.U.) of F-actin (red), Cortactin (Cyan) with respect to Gelatin (green) over the indicated line scan in the inset. Note the colocalisation of actin and cortactin in dot-like structure and the absence of degradation area. Scale bar: 10 µm. B X – Y (top, left) confocal images of in situ melanoma WM1552 showing invadopodia (insets, white arrows) as identified by dot-like F-actin (Red) and cortactin (Cyan) colocalisation. X – Z (bottom, left) images of invadopodia projecting into a collagen/gelatin matrix plated in the top chamber of a 1-μm transwell filter. Histograms represents mean invadopodia length and whiskers with 10–90 percentile with 54–117 invadopodia from 27 to 56 cells analysed per condition. C Metastatic melanoma A375 cells were plated on FITC-Gelatin (Grey), fixed, stained with Hoechst and labelled for actin (Red) and cortactin (Cyan). Boxed regions and insets depict invadopodia (White) and degradation areas were identified as black holes on fluorescent gelatin. Graphs indicate fluorescent intensity in arbitrary units (A.U.) of Actin (red), Cortactin (Cyan) with respect to Gelatin (green) over the indicated line scan in the inset. Dotted line indicates inactive invadopodia whereas straight lines indicate active invadopodia. Note that co-localisation of actin and cortactin in dot-like structure label both active and inactive invadopodia. Scale bar: 10 µm. The mean number of invadopodia/cell was calculated for 57 WM1862 and 123 A375 cells.

Article Snippet: Human melanoma cell lines, were purchased from Rockland and cultured in MCDB153/L-15 medium (M7403, Sigma; 11415064, Invitrogen) supplemented with 2% fetal bovine serum, 1.68 mM CaCl 2 and 1% penicillin/streptomycin.

Techniques: In Situ, Staining

Journal: Cell Death & Disease

Article Title: Protein dynamics at invadopodia control invasion–migration transitions in melanoma cells

doi: 10.1038/s41419-023-05704-4

Figure Lengend Snippet: A Metastatic melanoma A375 melanoma cells were fixed stained with Dapi and labelled for P-Pyk2 (Cyan) actin (Red) and cortactin (Magenta). Boxed regions and insets depict focal adhesion (top, white) and typical dot-like invadopodia (bottom white). Graphs indicate fluorescent intensity in arbitrary units (A.U.) of P-Pyk2 (Cyan), actin (red) and cortactin (Magenta) over the indicated line scan in the inset at invadopodia (left) and focal adhesion (right). Note high level of P-Pyk2 in only one of the two invadopodia selected. Note also high level of P-Pyk2 at focal adhesion. Scale bar: 10 µm. B Metastatic melanoma A375 cells were plated on FITC-Gelatin (Grey), fixed, stained with Hoechst and labelled for actin (Red) and P-Pyk2 (Cyan). Boxed regions and insets depict invadopodia (White) and degradation areas were identified as black holes on fluorescent gelatin. Graphs indicate fluorescent intensity in arbitrary units (A.U.) of actin (red), P-Pyk2 (Cyan) with respect to gelatin (green) over the indicated line scan in the inset. Note that all active invadopodia present high level of both actin and P-Pyk2. Scale bar: 10 µm. C Metastatic melanoma A375 cells were plated on FITC-Gelatin (Grey), fixed, stained with Hoechst and labelled for paxillin (Red) and P-Pyk2 (Cyan). Boxed regions and insets depict invadopodia and focal adhesion (White). Degradation areas were identified as black holes on fluorescent gelatin. Graphs indicate fluorescent intensity in arbitrary units (A.U.) of paxillin (red), P-Pyk2 (Cyan) with respect to gelatin (green) over the indicated line scan in the inset. Note that all active invadopodia (straight lines) present high level of P-Pyk2 but background level of paxillin whereas focal adhesions (dotted line) present high level of both paxillin and P-Pyk2 but no degradation. Scale bar: 10 µm.

Article Snippet: Human melanoma cell lines, were purchased from Rockland and cultured in MCDB153/L-15 medium (M7403, Sigma; 11415064, Invitrogen) supplemented with 2% fetal bovine serum, 1.68 mM CaCl 2 and 1% penicillin/streptomycin.

Techniques: Staining

Journal: Cell Death & Disease

Article Title: Protein dynamics at invadopodia control invasion–migration transitions in melanoma cells

doi: 10.1038/s41419-023-05704-4

Figure Lengend Snippet: A Confluent cell layers of A375 cells treated or not with PF-573228 or PF 431396 at 1 µM were wounded and cells were allowed to migrate during 12 h. B A375 melanoma cells lines treated or not with PF-573228 or PF 431396 at 1 µM were plated on Cy3-Gelatin (Grey), fixed, and labelled for actin (Red) and cortactin (Green). Scale bar: 10 µm. C Left, box chart represent the mean migration speed normalised to control of cells and whiskers the 10–90 percentile from at least 3 independent experiments. Right, box chart represent the mean area of degradation ( n = 66–118) from 3 independent experiments and whiskers the 10–90 percentile. *** P < 0.001; unpaired t test compared to control condition.

Article Snippet: Human melanoma cell lines, were purchased from Rockland and cultured in MCDB153/L-15 medium (M7403, Sigma; 11415064, Invitrogen) supplemented with 2% fetal bovine serum, 1.68 mM CaCl 2 and 1% penicillin/streptomycin.

Techniques: Migration, Control

Journal: International Journal of Molecular Sciences

Article Title: Expression of Alternative Splice Variants of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase-4 in Normoxic and Hypoxic Melanoma Cells

doi: 10.3390/ijms22168848

Figure Lengend Snippet: The response of malignant melanoma cells to low oxygen concentration. ( A ) Upper panel: Malignant melanoma lines, WM115 and WM266-4, were cultured with 100 µM pimonidazole (hypoxyprobe) in normoxic (N) and hypoxic conditions (H) for 16 h. The formation of pimonidazole–protein adducts were detected using Western Blot. Lower panel: Ponceau S stained membrane is shown as an internal control for equal protein loading. ( B ) Upper panels: Melanoma cells were cultured for 16 h in normoxia and hypoxia. Then HIF-1 alpha subunit accumulation was verified using the Western Blot. β-actin is shown as an internal control for equal loading. Lower panels: Densitometry analysis of Western Blot bands intensity normalized to β-actin. Relative densitometry value is the average of four independent experiments. The mean ± SEM is shown. Student’s t -test was used to evaluate the influence of hypoxia on HIF-1 alpha subunit stabilization. * p < 0.05 by Student’s t -test, ** p < 0.01 by Student’s t -test, p -value between 0.05 and 0.1 by Student’s t -test was given as an indication of the trend. ( C ) CAIX and PFKFB4 expression was analyzed by RT-qPCR in both melanoma cell lines under hypoxic and normoxic conditions. Expression data for each transcript was normalized to that for the reference gene TBP. Means ± SEM of at least five independent experiments are presented relative to expression in normoxic controls. The Student t -test was used to evaluate the differences between normoxic and hypoxic expression of CAIX and PFKFB4 . ** p < 0.01 by Student’s t -test. ( D ) Upper panels: Melanoma cells were cultured for 16 h in normoxia and hypoxia. Then CAIX and PFKFB4 expression was verified using the Western Blot. β-actin is shown as an internal control for equal loading. Lower panels: Densitometry analysis of Western Blot bands intensity normalized to β-actin. Each relative densitometry value is the average of at least four independent experiments. The mean ± SEM is shown. Studen’st t -test was used to evaluate the influence of hypoxia on CAIX and PFKFB4 expression. * p < 0.05 by Student’s t -test, ** p < 0.01 by Student’s t -test, p -value between 0.05 and 0.1 by Student’s t -test was given as an indication of the trend.

Article Snippet: Two human melanoma cell lines WM115 and WM266-4 were obtained from Rockland Immunochemicals (Limerick, PA, USA).

Techniques: Concentration Assay, Cell Culture, Western Blot, Staining, Membrane, Control, Expressing, Quantitative RT-PCR