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

Journal: PLoS ONE

Article Title: Prolactin-Induced Protein (PIP) Regulates Proliferation of Luminal A Type Breast Cancer Cells in an Estrogen-Independent Manner

doi: 10.1371/journal.pone.0062361

Figure Lengend Snippet: A, Confocal images showing loss of phalloidin stained actin stress fibers in dox-treated versus vehicle-treated T47D/shPIP dox cells. B, Adhesion assay showing inhibition of T47D cell adhesion to fibronectin after expression of PIP-specific (shPIP/121, shPIP/214) versus a non-specific (shNS) shRNA. Peptide competitors were used to assess specific (RGD) versus non-specific (RGE) binding to fibronectin. The dotted line indicates background binding to bovine serum albumin (BSA). C, Coomassie-stained SDS-polyacrylamide gel showing reduced levels of secreted proteins in the supernatant of dox- versus vehicle-treated T47D/shPIP dox cell cultures. D , Western blot analysis of JNK1/2 phosphorylation in dox- versus vehicle-treated serum-starved T47D/shPIP dox cells at the indicated time points after serum stimulation. Pan anti-JNK1/2 and tubulin antibodies were used as controls, and anti PIP antibodies were used to demonstrate effective dox-mediated PIP knockdown.

Article Snippet: Total JNK1/2 levels were detected by JNK antibody (sc-572) from Santa Cruz (Biotechnology Inc., Santa Cruz, CA.

Techniques: Staining, Cell Adhesion Assay, Inhibition, Expressing, shRNA, Binding Assay, Western Blot

Journal: Microorganisms

Article Title: Monosodium Glutamate Inhibits Pseudomonas aeruginosa -Induced Acute Lung Injury by Targeting the Type III Secretion Systems and Modulating Host Immunity

doi: 10.3390/microorganisms14030725

Figure Lengend Snippet: KEGG pathway enrichment analysis and key protein expression in P. aeruginosa -induced acute lung injury (ALI) in mice. ( A ) KEGG pathway enrichment of differentially expressed genes between the ALI model and control groups; ( B ) protein levels of phospho-JNK, p-p38, p-p65, TLR4, and MyD88 in lung tissues compared between the ALI model and control groups by Western blot analysis (β-actin loading control); ( C ) comparison of protein levels (p-JNK, p-p38, p-p65, TLR4, MyD88) in lung tissues between the model and MSG-treated groups, as detected by Western blot (β-actin control) (n = 12 samples per group; data represent means ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The antibodies for phospho-NF-κB p65 (82335-1-RR), p-65 (80979-1-RR), phospho-P38 (14064-1-AP), phospho-JNK (80024-1-RR), MyD88 (23230-1-AP), TLR-4 (19811-1-AP), P38 (14064-1-AP), JNK (51153-1-AP), and β-actin (66009-1-Ig) were purchased from ProteinTech Group (Wuhan, China).

Techniques: Expressing, Control, Western Blot, Comparison

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: Validation of the targeting relationship between let-7a-5p and MAPK8. (A) Schematic representation of the predicted complementary binding site between let-7a-5p and the 3′-UTR of MAPK8. (B) Relative mRNA expression level of MAPK8 in macrophages overexpressing let-7a-5p detected by RT-qPCR. (C) WB analysis of MAPK8 protein expression in macrophages overexpressing let-7a-5p. (D) Inhibitory effect of let-7a-5p on MAPK8 expression was assessed via a dual luciferase reporter assay. ( **P < 0.01, ***P < 0.001).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: Biomarker Discovery, Binding Assay, Expressing, Quantitative RT-PCR, Luciferase, Reporter Assay

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: Effect of PRP on expression of let-7a-5p and MAPK8. (A) Relative mRNA expression levels of let-7a-5p in knee joint sections of each group detected by RT-qPCR. (B) iNOS and CD206 co-immunolabeld with MAPK8 and counter-stained with DAPI in synovial tissues (Scale bar: 50 μm). (C, D) Quantification of iNOS and CD206 expression co-localized with MAPK8. ( **P < 0.01).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: Expressing, Quantitative RT-PCR, Staining

Journal: Frontiers in Immunology

Article Title: Platelet-rich plasma-derived microRNA let-7a-5p alleviates knee osteoarthritis by regulating macrophage polarization and improving inflammatory microenvironment

doi: 10.3389/fimmu.2026.1756467

Figure Lengend Snippet: The let-7a-5p/MAPK8 axis regulates macrophage polarization and inflammatory cytokine release in vitro . (A) IF staining showing expression of iNOS and CD206 in macrophages (Scale bar: 50 μm). (B) Quantification of iNOS-positive cell rate in transfected macrophages. (C) Quantification of CD206-positive cell rate in transfected macrophages. (D-G) Relative mRNA expression levels of pro-inflammatory cytokine (IL-1β and TNF-α) and anti-inflammatory cytokine (IL-4 and IL-10) in transfected macrophages detected by RT-qPCR. ( *P < 0.05, **P < 0.01, ns no significance).

Article Snippet: Subsequently, the tissue sections and cells were incubated overnight at 4°C with primary antibodies against iNOS (Proteintech 22226-1-AP), CD206 (Proteintech 18704-1-AP) and MAPK8 (Proteintech 66210-1-Ig).

Techniques: In Vitro, Staining, Expressing, Transfection, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

doi: 10.1371/journal.pone.0157860

Figure Lengend Snippet: (A) Western blot analysis of endogenous JNK1 present in HEK293T WCE and in NEIL1-FLAG immunoprecipitates. (B) Far-Western analysis to confirm the interaction between NEIL1 and JNK1. Top panel, Coomassie stained SDS-PAGE gel of NEIL1 and its phosphomimetic and ablating mutants (50 pmoles). Bottom panel, membrane after transfer, denaturation, slow refolding, binding to JNK1 from HEK293 WCE, and probing with an anti-JNK1 antibody. BSA was used as a negative control.

Article Snippet: An anti-mouse JNK1 purified monoclonal antibody (for far-Western analysis, clone 228601 from R&D Systems, lot # UQL0209081; cat # MAB17761) and a rabbit polyclonal NEIL1 antibody (for Western Blotting of IP reactions, Abcam, ab21337, lot # GR133490-1) were diluted 1:1000 (final concentration 1μg/ml) in blocking reagent.

Techniques: Western Blot, Staining, SDS Page, Membrane, Binding Assay, Negative Control

Journal: PLoS ONE

Article Title: Phosphorylation Sites Identified in the NEIL1 DNA Glycosylase Are Potential Targets for the JNK1 Kinase

doi: 10.1371/journal.pone.0157860

Figure Lengend Snippet: (A) In vitro kinase assays were performed with NEIL1 constructs expressed from E. coli cells and purified to homogeneity and active JNK1 kinase for 30 minutes at 32°C. γ- 32 P incorporation was quantified using phosphor-autoradiography after SDS-PAGE analysis of the samples. Lane 1, no JNK1 control; lane 2, no NEIL1 control; lane 3–13 are WT, S207E, S207A, S306E, S306A, S61E, S61A, DE, DA, TE, TA, respectively. (B) Graphical representation of two experimental repeats of the in vitro kinase assay. Statistically significant values (at 95% confidence) were determined by a one-way Anova test where * denotes p-values <0.05, *** denotes p-values <0.0005, and **** denotes p values <0.0001. (C) Glycosylase activity assays were performed using Sp:C and AP:C substrates with increasing amounts of in vitro phosphorylated NEIL1 (pNEIL1), unphosphorylated NEIL1 (positive control), and JNK1 (negative control). S and P indicate substrate and product, respectively.

Article Snippet: An anti-mouse JNK1 purified monoclonal antibody (for far-Western analysis, clone 228601 from R&D Systems, lot # UQL0209081; cat # MAB17761) and a rabbit polyclonal NEIL1 antibody (for Western Blotting of IP reactions, Abcam, ab21337, lot # GR133490-1) were diluted 1:1000 (final concentration 1μg/ml) in blocking reagent.

Techniques: In Vitro, Construct, Purification, Autoradiography, SDS Page, Control, Kinase Assay, Activity Assay, Positive Control, Negative Control