human melanoma cells wm115  (iCell Bioscience Inc)

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: Biomedicines

Article Title: Screening MT1-MMP Activity and Inhibition in Three-Dimensional Tumor Spheroids

doi: 10.3390/biomedicines11020562

Figure Lengend Snippet: Mean of total relative luminescence units (RLUs) measured in wells containing A172 glioblastoma or WM115 melanoma cells embedded in type I collagen compared to wells without collagen. Statistical analysis was carried out using an unpaired student t-test. *** p ≤ 0.0001.

Article Snippet: A-172 glioblastoma cells (catalog # CRL-1620), H4 astrocytoma cells (catalog # HTB-148), SW 1088 astrocytoma cells (catalog # HTB-12), U-87 glioblastoma cells (catalog # HTB-14), WM-266-4 metastatic melanoma cells (catalog # CRL-1676), and WM115 primary melanoma cells (catalog # CRL-1675) were obtained from ATCC (Manassas, VA, USA).

Techniques:

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

Journal: Cancers

Article Title: Inhibiting FAK–Paxillin Interaction Reduces Migration and Invadopodia-Mediated Matrix Degradation in Metastatic Melanoma Cells

doi: 10.3390/cancers13081871

Figure Lengend Snippet: Invasive potential of melanoma cells is matrix metalloproteinase (MMP)-dependent and regulated by invadopodia activity. ( A ) Melanoma cell lines labelled with Hoechst 33,342 were assessed for invasion into collagen-I over 24 h. Representative images of human epidermal melanocytes (HEM), WM1862 and WM983B melanoma cells in control condition between 0 and 60 µm. Scale bar: 100 µm. The invasion index was calculated by reporting the number of cells above 10 µm on the total number of cells by the field ( B ) in control conditions, or ( C ) in the presence of the non-selective MMP inhibitor, GM6001. Histograms represent the mean ± SD from 3 independent experiments. ***, p < 0.001; unpaired t -test compared to HEM or non-treated cells. ( D ) Melanoma cell lines were plated on FITC–gelatin (gray), fixed, and labelled for actin (red) and cortactin (green). Boxed regions and insets depict the colocalization of actin and cortactin (yellow) as markers for invadopodia. Degradation was identified as black holes on fluorescent gelatin. Scale Bar: 10 µm. ( E ) Histograms represent the mean ± SD of invadopodia numbers per cell ( n = 56–109). ( F ) Histograms represent the mean ± SD of the area of degradation ( n = 40−142), and of the degradation frequency ( G ) from at least 3 independent experiments. ***, p < 0.001; unpaired t -test compared to * WM1552C or †WM862.

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

Techniques: Activity Assay

Journal: Cancers

Article Title: Inhibiting FAK–Paxillin Interaction Reduces Migration and Invadopodia-Mediated Matrix Degradation in Metastatic Melanoma Cells

doi: 10.3390/cancers13081871

Figure Lengend Snippet: FAK expression is increased in melanoma cells. ( A ) Representative Western blot of HEM and melanoma cells. Cellular extracts were analyzed by Western blotting and probed for total FAK and actin as a loading control. ( B ) Histograms represent the mean ± SD from 3 independent experiments ( C ) Melanoma cell lines were immunostained for P-Y118-paxillin (cyan), actin (red) and FAK total (green). Scale bar: 10 µm. FAK localizations in melanoma cells ( n = 46–70) were measured by reporting the FAK signal in FA over FAK signals in the cytoplasm. ( D ) Histograms represent the mean ± SD from 3 independent experiments. **, p < 0.01; ***, p < 0.001; unpaired t -test compared to HEM.

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

Techniques: Expressing, Western Blot

Journal: Cancers

Article Title: Inhibiting FAK–Paxillin Interaction Reduces Migration and Invadopodia-Mediated Matrix Degradation in Metastatic Melanoma Cells

doi: 10.3390/cancers13081871

Figure Lengend Snippet: FAK inhibition reduces the migration of invasive melanoma cells. ( A ) Melanoma cells were transfected for 48 h with control siRNA or FAK siRNA or were treated by PF-573228 at 1 µM for 12 h. Cellular extracts were analyzed by Western blotting and probed for P-Y397 FAK, FAK total, and actin as a loading control. ( B ) Quantification of FAK expression and ( C ) FAK activity after siRNA transfection or PF-573328 from 3 independent experiments. ( D ) Confluent cell layers of melanoma cell lines transiently transfected with control siRNA or FAK siRNA or treated by PF-573228 were wounded and cells were allowed to migrate for 12 h. Images represent WM983B melanoma cells at 0 h and 12 h. ( E ) Histogram represent the mean speed migration normalized to control ± SD from at least 3 independent experiments *, p < 0.05; **, p < 0.01; ***, p < 0.001; unpaired t -test compared to control condition.

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

Techniques: Inhibition, Migration, Transfection, Western Blot, Expressing, Activity Assay

Journal: Cell Death & Disease

Article Title: The novel immunoglobulin super family receptor SLAMF9 identified in TAM of murine and human melanoma influences pro-inflammatory cytokine secretion and migration

doi: 10.1038/s41419-018-1011-1

Figure Lengend Snippet: SLAMF-9 is induced in human pBM by TCM or LPS. a CD14 + pBM were isolated from healthy donor-derived buffy coat samples. pBM were stimulated for 7 days either with control medium or TCM from WM115 human melanoma cells. Expression of human Slamf9 was evaluated by qRT-PCR analysis ( n = 4). Data are presented as mean ± SEM. b pBM were stimulated for 7 days with different combinations of pro- and anti-inflammatory mediators as indicated. The expression level of Slamf9 was determined by qRT-PCR analysis. In both cases the values were normalized to an internal ß-actin control ( n = 3). Data are presented as the mean ± SEM

Article Snippet: The human monocytic cell line U937 (CRL-1593.2TM, ATCC®, Wesel, Germany) and the human melanoma cell line WM115 (CRL-1675TM, ATCC®, Wesel, Germany) were cultured in RPMI-1640 (Gibco by Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS, Biochrom, Berlin, Germany), 100 U penicillin, as well as 100 mg/l streptomycin (Pen/Strep, Biochrom, Berlin, Germany).

Techniques: Isolation, Derivative Assay, Control, Expressing, Quantitative RT-PCR