phosphorylated mapk8  (Novus Biologicals)

Journal: Cell communication and signaling : CCS

Article Title: Matrix stiffness-induced IKBKE and MAPK8 signaling drives a phenotypic switch from DCIS to invasive breast cancer.

doi: 10.1186/s12964-025-02276-y

Figure Lengend Snippet: Fig. 3 MAPK8 and IKBKE mediate the stiffness-induced breast cancer phenotype. a, Venn diagram (left) and table (right) showing overlap between ki nases predicted to be upregulated by stiffness and downregulated by AIIB2 treatment. b, Workflow for the siRNA-based screen used to test the function of predicted kinases, and all conditions were run in duplicates. c, Representative phenotype of CA1a cells on high stiffness hydrogels transfected with siRNA as specified. FAK siRNA serves as positive control. Images are single tiles from 6 × 6 montages. Scale bar = 50 μm. d, Area fraction of segmented cell clusters expressed relative to MOCK (Gene of interest/MOCK). The area fraction is calculated as the ratio between the total area and the area of the thresholded objects. Positive control (FAK siRNA) is shown in red. Data represents the average of two technical replicates. Genes whose knockdown results in a similar or lower relative area fraction as the positive control are considered hits, including MAPK8 and IKBKE

Article Snippet: Membranes were blocked with 5% non-fat dry milk (PanReac AppliChem) in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4) for 1 h at RT, followed by overnight incubation at 4 °C with primary antibodies against IKBKE (Cell Signaling Technology, Cat. #D20G4, 1:1000), phosphorylated-MAPK8 (Novus Biologicals; Cat. #NB100-82009; 1:2000), or total-MAPK8 (Santa Cruz; Cat. #sc-1648; 1:5000).

Techniques: Transfection, Positive Control, Knockdown

Journal: Cell communication and signaling : CCS

Article Title: Matrix stiffness-induced IKBKE and MAPK8 signaling drives a phenotypic switch from DCIS to invasive breast cancer.

doi: 10.1186/s12964-025-02276-y

Figure Lengend Snippet: Fig. 5 Matrix stiffness-induced MAPK8 activity is critical for breast cancer cell proliferation in vitro. a, Immunoblot analysis of MAPK8 (left), and phospho- MAPK8 (right) of CA1a cells cultured on 0.4 kPa or 5 kPa hydrogels. Densitometric analysis shows MAPK8 and phospho-MAPK8 levels normalized to load ing controls (ponceau S staining, Supplementary Fig. 2a and 2c) and expressed relative to the low stiffness levels. Data are presented as mean ± S.E.M., with p-values according to an unpaired t-test. b, Representative Edu staining images of CA1a cells grown on 5 kPa transfected with MAPK8 or control siRNA pool (left) and quantification of three biological repeats (> 1000 cells each; right). Scale bar = 50 μm. Data are mean ± S.E.M and p-values according to an unpaired t-test. c, Representative images of actin (top row) and Edu staining (bottom row) in HCC1143 cells treated with MAPK8 or control siRNAs (left). Quantification of area fraction of segmented cell clusters relative to control siRNA of three biological repeats (right). Scale bar = 50 μm. Data are mean ± S.E.M and p-values derived by one-way Anova with Dunnett’s multiple comparison test. d, e, Spinning disc confocal images of breast cancer cells on 5 kPa treated with 5 µM (CA1a, d) or 10 µM ( HCC1143, e) JNK-IN-8 or vehicle for 24 h(left). Quantification of actin staining (top row) and Edu staining (bottom row) of three or more independent experiments(right). Scale bar = 50 μm. Relative area fraction as in (c). Data and statistics as in (b)

Article Snippet: Membranes were blocked with 5% non-fat dry milk (PanReac AppliChem) in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.4) for 1 h at RT, followed by overnight incubation at 4 °C with primary antibodies against IKBKE (Cell Signaling Technology, Cat. #D20G4, 1:1000), phosphorylated-MAPK8 (Novus Biologicals; Cat. #NB100-82009; 1:2000), or total-MAPK8 (Santa Cruz; Cat. #sc-1648; 1:5000).

Techniques: Activity Assay, In Vitro, Western Blot, Cell Culture, Staining, Transfection, Control, Derivative Assay, Comparison