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: MAPK8 and IKBKE mediate the stiffness-induced breast cancer phenotype. a , Venn diagram (left) and table (right) showing overlap between kinases 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: 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 loading controls (ponceau S staining, Supplementary Fig. a and c) 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

Journal: bioRxiv

Article Title: Glioma-associated fibroblasts promote glioblastoma resistance to temozolomide through CCL2-CCR2 paracrine signaling

doi: 10.1101/2024.03.05.581575

Figure Lengend Snippet: (A) Gene Set Enrichment analysis of differentially expressed genes on the MAPK pathway in glioma cells (U87 and A172) cultured in control medium or GAF conditioned medium. Adjusted p value, the estimated probability that a set with a given Normalized Enrichment Score represents a false positive finding (n = 6 per group). (B) WB analysis of three key molecules, such as phosphorylated ERK1/2(Thr202/Tyr204) and total ERK1/2, phosphorylated p38MAPK and total p38MAPK, and phosphorylated JNK1/2/3 and total JNK1/2/3, of MAPK signaling pathway of U87 cells cultured with Ctr-CM or GAF-CM for 48 hours following TMZ treatment respectively (C) Quantification of protein levels (4 different GAFs were used for this experiment). (D) Representative flow cytometry plots of apoptotic analyses of U87 cells following TMZ treatment for 48 hours. U87 cells were pretreated with Trametinib (100nmol/L) or DMSO for 6 hours, followed by U87 cells or GAFs coculture for 24 hours. (E) Quantification of apoptotic GBM cells (n = 4 per group). (F) Representative flow cytometry plots of apoptotic analyses of U87 cells following TMZ treatment for 48 hours. U87 cells were pretreated with Trametinib (100nmol/L) or DMSO for six hours, then cultured with Ctr-CM or GAF-CM for 48 hours. (G) Quantification of apoptotic GBM cells (n = 4 per group). (H) Representative flow cytometry plots of apoptotic analyses of U87 cells. U87 cells were pretreated with Ravoxertinib (20µmol/L) or DMSO for six hours, then cultured with Ctr-CM or GAF-CM for 48 hours. (I) Quantification of apoptotic GBM cells (n = 4 per group). (J) WB analysis of cleaved-caspase9, cleaved-caspase3, and phosphorylated ERK1/2 (Thr202/Tyr204) and total ERK1/2 in U87 cells with indicated treatment. (K-M) Quantification of protein levels (4 different GAFs were used for this experiment). (N) WB analysis of cleaved-caspase3 and phosphorylated ERK1/2 (Thr202/Tyr204), and total ERK1/2 in U87 cells with indicated treatment. (O-P) Quantification of protein levels (3 different GAFs were used for this experiment). Data in C, E, G, I, K-M, and N-P were mean±SEM. p values from empirical phenotype-based permutation tests and adjusted using the False Discovery Rate (FDR) procedure (A), two-tailed student-t-test (C, E, G, I, K-M, and N-P). * P < 0.05, ** P < 0.005, *** P < 0.001, **** P < 0.0001, ns = no significant.

Article Snippet: Information of primary antibodies was as follows: Fibronectin (BD Biosciences #610077), α-SMA (Proteintech #55135-1-AP), Cleaved-caspase3 (Cell Signaling Technology #9661), Cleaved-caspase9 (Cell Signaling Technology #9509), Bax (Millipore #ABC11), β-Tublin (Millipore #MAB3408), β-Actin (Cell Signaling Technology #3700), GAPDH (Proteintech #CL594-60004), Phospho-ERK1/2(Thr202/Tyr204) (Cell Signaling Technology #4370S), total-ERK1/2 (Cell Signaling Technology #4696), Phospho-p38 MAPK (T180/Y182) (Abclonal #AP1165), total-p38 MAPK (Abcam #ab308333), Phospho-JNK1-T183/Y185+JNK2-T183/Y185+JNK3-T221/Y223 (Abclonal #AP0276), total-JNK1/2/3 (Abclonal #A4867), CCL2/MCP1 (Abclonal # A23288).

Techniques: Cell Culture, Flow Cytometry, Two Tailed Test