human phosphokinase array  (R&D Systems)

Journal: Aging Cell

Article Title: Senescent Fibroblasts Drive Melanoma Progression Through GCP ‐2 Induced CREB Phosphorylation Enhancing Glycolysis

doi: 10.1111/acel.70239

Figure Lengend Snippet: GCP‐2 Induced CREBS133 Phosphorylation Drives Melanoma Growth (A) CM from replicative senescent (CM REP SEN) but not from young non‐senescent fibroblasts (CM NON SEN) enhanced CREB phosphorylation in the melanoma cell line A375 as depicted by phosphokinase arrays (upper two panels). Note that the representative experiment of the upper panel was done with A375 wild‐type melanoma cells (A375 WT) and in the lower panel, we employed A375 melanoma cells silenced for GCP‐2/ENA‐78 (A375 KDN). CREB phosphorylation intensities are depicted in red rectangles. Actin served as loading control and is depicted in yellow rectangles. Data are mean ± S.E.M. n = 3. ns, nonsignificant, **** p < 0.0001 by ANOVA and Tukey's post hoc test (lower diagram). (B) Western blot analysis of CREB phosphorylation at the serine residue 133 (pCREBS133) in cell lysates from A375 melanoma cells exposed to CM from non‐senescent (CM NON‐SEN) or replicative senescent fibroblasts (CM REP SEN), and to recombinant GCP‐2 or ENA‐78 at the indicated concentrations (upper panel). Densitometry assessment of western blots with normalization of pCREBS133 to actin (lower panel). Data are mean ± S.E.M. n = 3. ns, nonsignificant, ** p < 0.0001 by ANOVA, and Tukey's post hoc test. (C) Immunostaining of sections from tumors grown after co‐injection of A375 melanoma cells (MM cells GCP 2KDN/ENA‐78KDN) with replicative senescent (REP SEN FIBROBLASTS) and non‐senescent fibroblasts (NON SEN FIBROBLASTS) of different genotypes for phosphorylated CREB (pCREBS133, red) and DAPI staining nuclei (blue, overlay purple) of Melan A positive (green) A375GCP‐2KDN/ENA‐78KDN melanoma cells. (D) Quantification of CREBS133 Melan A‐positive cells of tumors derived from co‐injection of young, non‐senescent (NON SEN) and replicative senescent (REP SEN) fibroblasts of the indicated genotypes ( n = 5) by multiple comparison ANOVA with Tukey's post hoc test, *** p < 0.0001. (E) Representative photomicrographs of skin sections of a healthy young (left) and an old individual (right) stained for Melan A (red) and phosphorylated (active) pCREBS133 (green). Nuclei were stained with DAPI (blue). A total of five young and five old individuals were studied. (F) Representative photomicrographs of melanoma sections of a 24‐year‐old patient (left) and a 84‐year‐old patient (right) stained for Melan A (red) and phosphorylated (active) pCREBS133 (green). Nuclei were stained with DAPI (blue). A total of nine melanomas from young patients and nine melanomas from old patients were studied. (G) The diagram depicts data of (F). Melan A pCREBS133‐positive melanoma cells were counted. Of note, only melanomas from old patients revealed increased CREB activation. Scale bar, 100 μM. At least sections of five different biopsies for each entity were analysed. All data are mean ± S.E.M. *** p < 0.001 by unpaired one‐way ANOVA multiple comparison with Tukey's post hoc test. (H) CREB with a functional phosphorylation site at serine 133 is essential for GCP‐2 and ENA‐78‐dependent melanoma cell migration. The migratory response of either A375 melanoma cells with wild‐type CREB (A375MM CREBWT) or with a replacement mutation at the CREB phosphorylation site at serine 133 (A375MM CREBS133A) was assessed. Mean ± S.E.M. All experiments were repeated three times. *** p < 0.0001 by ANOVA multiple comparison with Tukey's post hoc test. (I) CREB mutated at the phosphorylation site at serine 133 (CREBS133A) abrogates anchorage‐independent growth of A375 melanoma cells. Anchorage‐independent growth of either wild‐type CREB (CREBWT) or melanoma cells with a CREB mutation (A375 MM CREBS133A) were subjected to different concentrations of recombinant GCP‐2 and ENA‐78. The total number of A375 melanoma cells was counted following agar dissolution as indicated in the Materials and Methods section. SYBR green intercalation in the DNA double strand, indicative of viable cells, was quantified by the emission of fluorescence at the ratio of 485/520 nm. All data are the mean ± S.E.M. All experiments were repeated three times. ** p < 0.001 by ANOVA multiple comparison with Tukey's post hoc test.

Article Snippet: 200 μg of cell lysate was dissolved in 2 mL of array buffer 1 and incubated on the phosphokinase array (ARY003B, R&D Systems Inc.) overnight at 4°C with gentle rocking as per the manufacturer's protocol.

Techniques: Phospho-proteomics, Control, Western Blot, Residue, Recombinant, Immunostaining, Injection, Staining, Derivative Assay, Comparison, Activation Assay, Functional Assay, Migration, Mutagenesis, Dissolution, SYBR Green Assay, Fluorescence