Journal: Advanced healthcare materials

Article Title: Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells

doi: 10.1002/adhm.201700641

Figure Lengend Snippet: Western blot of intracellular signaling molecules expressed by hMSCs cultured on Col-GAG and MC-GAG in the absence and presence of DMH1. Western blot of A) phosphorylated Smad1/5 (P-Smad1/5) and total Smad (Smad5), phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 (ERK1/2), phosphorylated JNK1/2 (P-JNK1/2) and total JNK1/2 (JNK1/2), Runx2 and B) phosphorylated p38 (p-p38) and total p38, phosphorylated Akt (p-Akt) and total Akt, and actin in hMSCs cultured on Col-GAG and MC-GAG at days 0, 4, 14, and week 4 of culture with or without 50 μm DMH1.

Article Snippet: Western blot analysis was carried out with antibodies against phosphorylated Smad1/5 (p-Smad1/5), total Smad5, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated JNK1/2 (p-JNK1/2), total JNK1/2, phosphorylated p38 (p-p38), total p38, p-Akt, total Akt, and β -actin followed by 1:4000 dilutions of horseradish peroxidase-conjugated IgG antibodies (Bio-Rad, Hercules, CA) and an enhanced chemiluminescent substrate (Thermo Scientific, Rockford, IL).

Techniques: Western Blot, Cell Culture

Journal: Advanced healthcare materials

Article Title: Nonmineralized and Mineralized Collagen Scaffolds Induce Differential Osteogenic Signaling Pathways in Human Mesenchymal Stem Cells

doi: 10.1002/adhm.201700641

Figure Lengend Snippet: Western blot of intracellular signaling molecules expressed by hMSCs cultured on Col-GAG and MC-GAG in the absence and presence of PD98059. Western blot of A) phosphorylated Smad1/5 (P-Smad1/5) and total Smad (Smad5), phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 (ERK1/2), phosphorylated JNK1/2 (P-JNK1/2) and total JNK1/2 (JNK1/2), Runx2 and B) phosphorylated p38 (p-p38) and total p38, phosphorylated Akt (p-Akt) and total Akt, and actin in hMSCs cultured on Col-GAG and MC-GAG at days 0, 4, 14, and week 4 of culture with and without 50 μm PD98059.

Article Snippet: Western blot analysis was carried out with antibodies against phosphorylated Smad1/5 (p-Smad1/5), total Smad5, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated JNK1/2 (p-JNK1/2), total JNK1/2, phosphorylated p38 (p-p38), total p38, p-Akt, total Akt, and β -actin followed by 1:4000 dilutions of horseradish peroxidase-conjugated IgG antibodies (Bio-Rad, Hercules, CA) and an enhanced chemiluminescent substrate (Thermo Scientific, Rockford, IL).

Techniques: Western Blot, Cell Culture

Journal: Journal for Immunotherapy of Cancer

Article Title: TSH-TSHR axis promotes tumor immune evasion

doi: 10.1136/jitc-2021-004049

Figure Lengend Snippet: Mechanism of TSH-induced tumor PD-L1 expression (A) KTC1 (left) and U87 (right) were treated with the culture supernatant of moDCs from patients with DTC and/or TSHR inhibitor (ML224). Whole-cell lysates were subjected for immunoblot analysis of PD-L1, HIF1α, β-actin, phosphorylated, and total AKT, ERK, JNK, P38, P65. Representative immunoblot picture (up) and quantitative histogram (down) are shown. Expression of PD-L1 and HIF1α was calculated as the ratio between band intensity of these genes and β-actin. Phosphorylation of five kinases was calculated as the ratio between band intensity of phosphorylated protein and total protein. (B–C) KTC1 (up) and U87 (down) were treated with TSH (B) or culture supernatant of moDCs (C) and four inhibitors. Representative histograms (left) and PD-L1 MFI (right) were shown. (D) Transcription factors (TF) enrichment in KTC1 treated with culture supernatant of moDCs based on RNA sequencing data sets. The scatter plot ranked TF from first with increasing enrichment and decreasing ChIP-X enrichment analysis 3 scores. TF from the AP-1 family (blue) and other PD-L1-related TF (red) were colored. (E) KTC1 (up) and U87 (down) were treated with the culture supernatant of moDCs from patients with DTC and/or TSHR inhibitor (ML224). Whole-cell lysates were subjected for immunoblot analysis of β-actin, phosphorylated and total c-JUN, and STAT1. (F) Immunofluorescence staining of KTC1 (left) and U87 (right) for phosphorylated (down) and total (up) c-JUN. DTC, differentiated thyroid cancers; MFI, median fluorescence intensity; moDC, monocyte-derived dendritic cells; PD-L1, programmed death-ligand 1; TSH, thyroid-stimulating hormone; TSHR, TSH receptor.

Article Snippet: Antibodies specific for TSHα (25014–1-AP, 1:1000), total ERK (16443–1-AP, 1:1000), total JNK (24164–1-AP, 1:1000), total p38 (14064–1-AP, 1:1000) and HIF1α (66730–1-Ig, 1:1000) were purchased from Proteintech.

Techniques: Expressing, Western Blot, RNA Sequencing Assay, Immunofluorescence, Staining, Fluorescence, Derivative Assay

Journal: bioRxiv

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

doi: 10.1101/2025.04.16.649065

Figure Lengend Snippet: 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: bioRxiv

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

doi: 10.1101/2025.04.16.649065

Figure Lengend Snippet: 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, and ) 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: Western Blot, Cell Culture, Staining, Transfection, Control, Derivative Assay, Comparison

Journal: Molecular Medicine Reports

Article Title: Vaspin attenuates the progression of atherosclerosis by inhibiting ER stress-induced macrophage apoptosis in apoE −/− mice

doi: 10.3892/mmr.2015.4708

Figure Lengend Snippet: Visceral adipose tissue-derived serine protease inhibitor (vaspin) downregulates the expression levels of proteins associated with endoplasmic reticulum stress-induced apoptosis in macrophages. Activating transcription factor (ATF)6, C/EBP-homologous protein (CHOP), total (t)-c-Jun N-terminal kinases (JNK)1/2, phosphorylated (p)-JNK1/2, cleaved-caspase 12, cleaved-caspase 9, and cleaved-caspase 3 protein expression levels were determined by western blot analysis. Each band density was normalized to control. Data are presented as the mean ± standard error of the mean. * P<0.05 compared with the control cells; # P<0.05 compared with oxidized-low-density lipoprotein (ox-LDL) (50 µ g/ml) alone.

Article Snippet: The membranes were blocked with 5% BSA for 1 h and were then incubated with the following antibodies at 4°C overnight: Anti-activating transcription factor (ATF)6 (1:1,000; ProteinTech Group, Inc., Chicago, IL, USA; cat. no. 24169-AP), anti-CHOP (1:1,000), anti-total (t)-c-Jun N-terminal kinases (JNK)1/2 (1:800; Cell Signaling Technology, Inc., cat. no. 9258, anti-phosphorylated (p)-JNK1/2 (1:800; Cell Signaling Technology, Inc.; cat. no. 4671), anti-cleaved-caspase 12 (1:800; BioVision, Inc., Milpitas, CA, USA; cat. no. 3282-100), anti-cleaved-caspase 9 (1:800; Abcam, Cambridge, MA, USA; cat. no. ab2325) and anti-cleaved-caspase 3 (1:800; Abcam; cat. no. ab2302).

Techniques: Derivative Assay, Protease Inhibitor, Expressing, Western Blot