Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 1 S100A9 promotes prostate cancer cell invasion and β1 integrin expression through interaction with TLR4. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (A) PC-3 and DU-145 cells invasion was measured by transwell invasion assay. (B) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. PC-3 and DU-145 cells were treated with S100A9 (20 µg/ml) for 48 h. (C, D) Cell extracts were immunoprecipitated (IP) with control mouse IgG, mouse anti-S100A9 antibody. Immunoblot (IB) was used to detect S100A9, TLR4 and RAGE. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA. (E) TLR4 expression was examined by Western blot after 48 h siRNA transfection. PC-3 and DU-145 cells were transfected with TLR4 or control siRNA followed by stimulation with S100A9. (F) Tumor cell invasion was measured by transwell invasion assay. (G) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Transwell Invasion Assay, Western Blot, Immunoprecipitation, Control, Transfection

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 2 NF-κB mediates S100A9-induced prostate cancer cell β1 integrin up-regulation. PC-3 and DU-145 cells transfected with or without TLR4 siRNA or control siRNA, were transfected with NF-κB-luciferase reporter plasmid, and treated with S100A9 (20 µg/ml) for 48 h. (A, B) Activity of NF-κB was detected by measuring the relative activity of luciferase. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ ml) for 48 h. (C) The mRNA and protein levels of β1 integrin were determined by qPCR or Western blot. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Transfection, Control, Luciferase, Plasmid Preparation, Activity Assay, Western Blot

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 3 S100A9 promotes prostate cancer cell invasion via integrin β1/FAK signaling. PC-3 and DU-145 cells were treated with or without BAY11-7082 (5µM) for 30 min. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (A) Fibronectin expression was determined by Western blot. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 48 h. (B) Supernatant fibronectin (FN) concentration was determined by ELISA. PC-3 and DU-145 cells were treated with or without S100A9 (20 µg/ml) for 30 min. (C) The phosphorylation of FAK was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 30 min, the expression of integrin β1 (D) or phosphorylation of FAK (E) was measured by Western blot. PC-3 and DU-145 cells were transfected with control siRNA or integrin β1-specific siRNA for 48 h. Then cells were treated with or without S100A9 (20 µg/ml) for 48 h. (F) The invasion activity were measured by transwell invasion assay. (G) PC-3 and DU- 145 cells were treated with β1 integrin functional blocking antibody MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 30 min and the phosphorylation of FAK was measured by Western blot. (H) PC-3 and DU-145 cells were treated with MAB13 (50 μg/mL) or control IgG (50 μg/mL) for 30 min and then treated with or without S100A9 (20 µg/ml) for 48 h for invasion. (I) PC-3 and DU-145 cells were pretreated for 30 min with FAK inhibitor, PF562271 (100 nM) followed by stimulation with S100A9 (20 µg/ml) for 48 h for invasion. Data are represented as the mean ± S.E.M. *p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Transfection, Control, Activity Assay, Transwell Invasion Assay, Functional Assay, Blocking Assay

Journal: OncoTargets and Therapy

Article Title:

S100A9 promotes prostate cancer cell invasion by activating TLR4/NF-κB/integrin β1/FAK signaling

doi: 10.2147/ott.s192250

Figure Lengend Snippet: Figure 4 S100A9 induces prostate cancer cell metastasis in vivo. DU-145 cells were transfected with pcDNA3.1 or pc DNA-S100A9 plasmid. (A, B) The expression or secretion of S100A9 was determined by Western blot and ELISA. The cells were injected to nude mice via tail vein. 30 days after inoculation, nude mice were sacrificed. (C) Expressions of S100A9 and integrin β1 in xenograft tumors were detected by immunohistochemistry. (D) Expressions of FAK, p-FAK, NF-kB p65 and p-NF-kB p65 in xenograft tumors were determined by Western blot. (E) Micrometastatic tumors in the lungs of mouse xenografts were counted and subjected to H and E staining. Scale bar 50 μ. Magnifcation×200. Data are represented as the mean ± S.E.M.*p<0.05.

Article Snippet: Antibodies and reagents The recombinant human S100A9 (9254-S9), human S100A9 ELISA Kit (DY5578) were purchased from R&D Systems (R&D Systems, Shanghai, China).

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Immunohistochemistry, Staining

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet: MSCs are fibrosis-driving cells in patients characterized by upregulation of S100A8/A9 (A) Diagnostic BM images of the patients. Representative H&E and reticulin stainings. For additional images (all controls) and detailed patient characteristics, see Figure S5 . (B) UMAP of cells in 1 PMF patient (MF2, n = 243 cells) and two control patients (MF0, n = 255 cells). In the left panel, cells are color coded by their annotated cellular identity, and in the right panel, by their patient source. (C) Top marker genes. Wilcoxon rank-sum test, p < 0.01. (D) Ridgeline plot comparing PMF (blue) versus control (red) condition. Competitive gene set enrichment analysis was used. (E) PROGENy analysis. Sampling-based permutation (10,000 permutations). Pathway activity scores are given as Z scores. (F) Ridgeline plot of S100A8/A9 expression in PMF (blue) or control (red). Significance estimated by modeling the dropout rate as a binomial process with the observed dropout rate per condition as estimator of p for both conditions, respectively. (G) Network plot of ligand-receptor activity in PMF compared to control. (H) Bar plot of top 10 most abundant ligands in all inferred ligand-receptor interactions. (I) Sankey plot of top 20 deregulated TGFB1 -mediated ligand-receptor interactions. The absolute difference in mean LR expression was used as a metric for the extent of deregulation. (J) Sankey plot of top 20 deregulated ligand-receptor interactions mediated by PF4, PF4V1, or PPBP. The absolute difference in mean LR expression was used as a metric for the extent of deregulation. See also Figure S6 and , , , , and .

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Diagnostic Assay, Control, Marker, Sampling, Activity Assay, Expressing

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet: Spatial kinetics of S100A8/S100A9 detects disease progression in MPN and their pharmacological targeting ameliorates the disease (A) ELISA of S100A8 (and S100A9) in MPN (blue) and controls (red) plasma. Two-tailed, two-sample Welch test was used. (B) ELISA of S100A8 (and S100A9) in MPN with different MF grades (blue) and controls (red) plasma. Mean ± SEM. One-way-ANOVA with post hoc Tukey’s was used. (C) Frequency of S100A8 + cells BM biopsies; n = 64 patients. One-way-ANOVA with post hoc Tukey’s HSD was used. (D and E) Grading of S100A8 in the non-hematopoietic compartment in BM biopsies. Scale bar, 100 μm. n = 64 patients. Kruskal-Wallis H test with post hoc Wilcoxon rank-sum test was used. p values were adjusted for multiple hypothesis testing by the Holm-Bonferroni method. (F) White blood cell counts of WT mice transplanted with either JAK2 V617F (blue) or JAK2 WT overexpressing HSPCs (red) each either treated with Tasquinimod 30 mg/kg/day or vehicle control. Two-way repeated ANOVA pairwise comparisons were analyzed by estimated marginal means. (G) Spleens at sacrifice as indicated. (H) Relative spleen weights. Mean ± SEM. One-way-ANOVA with post hoc Tukey’s HSD. (I) Reticulin (MF) grade. Kruskal-Wallis H test with post hoc Wilcoxon rank-sum test. See also Figure S6 .

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Two Tailed Test, Control

Journal: Cell Stem Cell

Article Title: Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis

doi: 10.1016/j.stem.2020.11.004

Figure Lengend Snippet:

Article Snippet: Samples were diluted 1:50 - 1:150 and S100A8 concentration was quantified using the Human S100A8 DuoSet ELISA (R&D Systems, DY4570-05) according to the manufacturer’s instructions.

Techniques: Recombinant, Staining, Plasmid Preparation, Virus, Multiplex Assay, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Avidin-Biotin Assay, Blocking Assay, Retroviral, Software

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: Proteins preferential to either HFpEF or control groups

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Control, Sequencing, Ubiquitin Proteomics

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: Representative MS/MS scan for S100A8 peptide sequence ALNSIIDVYHK. Raw m/z spectral images with peak assignments and b and y ion lists along with a representation of peptide sequencing by tandem mass spectrometry

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Tandem Mass Spectroscopy, Sequencing, Mass Spectrometry

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: Plasma levels of S100A8 in control vs. HFpEF groups. a S100A8 is found in increased levels in the plasma of subjects with HFpEF vs. control subjects as detected by ELISA. The MCW columns include the control (n = 7) and HFpEF (n = 9) from the discovery cohort and the NWU colums include the control (n = 18) and HFpEF (n = 25) samples from the validation cohort. *p < 0.006 vs MCW Control. # p < 0. 002 vs NWU Control

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Clinical Proteomics, Control, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: Overview of primary and secondary screening methods to identify potential mediators of HFpEF. a Platelet proteomes were subject to mass spectral analysis and novel proteins were identified. b Human cardiomyocytes derived from induced pluripotent stem cells were used to determine whether proteins that were identified in a had direct effects on cardiomyocytes function in vitro. Purified recombinant protein S100A8 was tested in this assay

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Derivative Assay, In Vitro, Purification, Recombinant

Journal: Journal of Translational Medicine

Article Title: Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction

doi: 10.1186/s12967-016-0774-3

Figure Lengend Snippet: S100A8-mediated effects on human iPSC-derived cardiomyocytes. a Shows example action potentials recorded from rS100A8 treated iPSC derived human cardiomyocytes. The addition of rS100A8 to the buffer extended the period between action potentials. This period is phase 4; the diastolic membrane potential between action potentials. b rS100A8 exacerbates the arrhythmic tendencies of human cardiomyocytes. c Spontaneous Ca 2+ transients recorded from human cardiomyocytes treated with rS100A8 as indicated by the blue line. rS100A8 significantly delayed the recovery of depolarization. Wash out of rS100A8 reversed these effects

Article Snippet: Recombinant S100A8 was purchased from Creative BioMart (Shirley, NY).

Techniques: Derivative Assay, Membrane

Journal: PLoS ONE

Article Title: Common Interactions between S100A4 and S100A9 Defined by a Novel Chemical Probe

doi: 10.1371/journal.pone.0063012

Figure Lengend Snippet: A: Binding of S100 proteins to immobilized S100A4 and S100A9. S100A1, A4, A7, A9 and A13 (∼1.3 µg/mL) were injected (2 min at 30 µL/min) over S100A4 and S100A9 (density ∼2.5 kRU) in the presence of 1 mM Ca ++ and 20 µM Zn ++ . Responses were calculated at late association phase. B: Formation of homo- and hetero-complexes of S100A4 and S100A9 is Zn ++ dependent. S100A4 and S100A9 were injected over immobilized S100A4 or S100A9 at ∼1.3 µg/mL HBS-P containing 1 mM Ca ++ ±20 µM Zn ++ . Responses at late association phase were calculated. C: HEK293T cells were transfected either with S100A4 or S100A9 cDNA construct alone or the two together, as indicated. After 24 hrs of culture some of the transfected cells were exposed to the membrane permeable cross-linker DSS. Thereafter cell lysates were prepared, equal amounts (30 µg) of protein loaded on an SDS-PAGE gel and western blots were performed using either anti-S100A4 or anti-S100A9 antibodies as indicated. Representative results from one out of two experiments performed are shown.

Article Snippet: The ability of S100A4 and S100A9 to form homo- and hetero-complexes was also tested by injecting S100A4 and S100A9 over these proteins immobilized on the sensor chip and with recombinant human S100A1 (ProSpec, Rehovat, Israel), S100A7 (ProSpec) and S100A13 (R&D Systems) as negative controls.

Techniques: Binding Assay, Injection, Transfection, Construct, Membrane, SDS Page, Western Blot

Journal: Journal of Korean Neurosurgical Society

Article Title: S100ß, Matrix Metalloproteinase-9, D-dimer, and Heat Shock Protein 70 Are Serologic Biomarkers of Acute Cerebral Infarction in a Mouse Model of Transient MCA Occlusion

doi: 10.3340/jkns.2017.0200

Figure Lengend Snippet: Plasma S100β concentration in all groups. The concentration of S100β increases with occlusion time (p=0.001; A) and correlates significantly with infarct volume (r=0.867, p <0.001; b) and neurological deficit (r=0.802, p <0.001; c). * p <0.05.

Article Snippet: The plasma concentrations of S100β, MMP-9, D-dimer, and HSP70 were calculated using mouse protein S100β (CSB-EL-0206643MO; CUSABIO, Wuhan, China), mouse total MMP-9 (MMPT90; R&D Systems, Minneapolis, MN, USA), mouse D-dimer (E03D0224; BlueGene, Shanghai, China), and mouse HSP70 (CSB-E08311m; CUSABIO) ELISA kits.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Journal of Korean Neurosurgical Society

Article Title: S100ß, Matrix Metalloproteinase-9, D-dimer, and Heat Shock Protein 70 Are Serologic Biomarkers of Acute Cerebral Infarction in a Mouse Model of Transient MCA Occlusion

doi: 10.3340/jkns.2017.0200

Figure Lengend Snippet: Sensitivity and specificity of the biomarkers. MMP-9 and HSP70 both have an AUc of 1. S100β has an AUc of 0.98. The AUc for D-dimer is 0.58. MMP-9 : matrix metalloproteinase-9, HSP70 : heat shock protein 70, AUc : the area under the receiver operating characteristic curve.

Article Snippet: The plasma concentrations of S100β, MMP-9, D-dimer, and HSP70 were calculated using mouse protein S100β (CSB-EL-0206643MO; CUSABIO, Wuhan, China), mouse total MMP-9 (MMPT90; R&D Systems, Minneapolis, MN, USA), mouse D-dimer (E03D0224; BlueGene, Shanghai, China), and mouse HSP70 (CSB-E08311m; CUSABIO) ELISA kits.

Techniques:

Journal: Cancer cell

Article Title: Oncogenic KRAS regulates amino acid homeostasis and asparagine biosynthesis via ATF4 and alters sensitivity to L-asparaginase

doi: 10.1016/j.ccell.2017.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: AZD6244, MEK inhibitor , Selleck Chemicals , Cat# S1008; CAS: 606143-52-6.

Techniques: Virus, Plasmid Preparation, Recombinant, Labeling, cDNA Synthesis, SYBR Green Assay, Microarray, Expressing, Gene Expression, Mutagenesis, Software

Journal: Nature Medicine

Article Title: Stratification of radiosensitive brain metastases based on an actionable S100A9/RAGE resistance mechanism

doi: 10.1038/s41591-022-01749-8

Figure Lengend Snippet: a , Heatmap representing color-coded expression levels of commonly deregulated genes in H2030-BrM cell line when cultured under radiosensitive (purple line, adherent; light green line, co-culture with inserts) compared with radioresistant (dark green line, oncospheres; blue line, cell–cell co-culture) conditions in vitro. Only genes with false discovery rate (FDR) < 0.05 and a log 2 ratio >1 were considered. b , Representative images of S100A9 protein expression levels in metastatic lesions growing in brains from mice IC injected with H2030-BrM and E0771-BrM. Three brains were analyzed in each BrM model. Scale bars, 120 µm (H2030-BrM) and 50 µm (E0771-BrM). c , Sixty-six human brain metastases from patients with lung cancer (33 cases) or breast cancer (30 cases) or other primary tumors (3 cases) were stained for S100A9 by immunohistochemistry. Representative images are shown. Scale bar, 50 µm. Quantification of different histological scoring of cancer cells is shown in pie charts. Five cases had to be excluded; 27 of 61 were scored with no staining (score 0), 9 of 61 with weak staining (score 1), 12 of 61 with moderate staining (score 2) and 13 of 61 with strong staining (score 3). d , Quantification of S100A9 expression levels in H2030-BrM after stimulation with 100 ng ml −1 recombinant CXCL1 (rCXCL1) or control. Values are shown in dot plots, and dots represent independent experiments. The line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 7, each experimental condition). P value was calculated Wilcoxon signed rank test, two sided. e , Quantification of S100A9 expression levels in H2030-BrM after stimulation with recombinant transforming growth factor α (rTGF-α) or control. Values are shown in dot plots and dots represent independent experiments. The line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 7, each experimental condition). P value was calculated using two-tailed t -test. f , Quantification by enzyme-linked immunosorbent assay (ELISA) of human S100A9 (hS100A9) in the supernatant of H2030-BrM grown either under adherent conditions in vitro or in organotypic cultures ex vivo. Values are shown in box-and-whisker plots, where every dot represents an independent experiment and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 6, H2030-BrM adherent cultures; n = 8, H2030-BrM growing in organotypic brain cultures). P value was calculated using two-tailed Mann–Whitney test. g , Quantification of in vitro viable cell fraction after irradiation at 10 Gy and 200 ng ml −1 recombinant hS100A9 or control, as determined by manual cell counting of bisbenzimide-positive nuclei. Values are percentages of viable cells respect to unirradiated controls and shown in a dot plot, where each dot represents an independent experiment and the line in the box corresponds to the median ( n = 3, each experimental condition). P value was calculated using a two-tailed t -test. h , Representative pictures of RAGE immunohistochemistry from unirradiated or irradiated established H2030-BrM metastases in vivo. Scale bar, 50 µm. i , Quantification of the percentage of NF-κB + GFP + H2030-BrM cells identified by the expression of an engineered mCherry NF-κB activity reporter. Brain slices with cancer cells were evaluated 72 h after treatment with radiotherapy. Values are shown in box-and-whisker plots where each dot is a brain organotypic culture ( n = 11, nonirradiated; n = 7, irradiated with a single dose of 10 Gy) and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value. P value was calculated using a two-tailed t -test. j , Schema of working model of radioresistance in brain metastasis.

Article Snippet: For stimulation experiments, 200 ng ml −1 recombinant human S100A9 (9254-S9, R&D Systems), 20 ng ml −1 recombinant mouse TNF-α (315-01 A, Peprotech), 50 ng ml −1 recombinant mouse interferon-α (752802, BioLegend), 100 ng ml −1 recombinant TGF-α (239-A-100, R&D Systems) or 100 ng ml −1 recombinant KC/CXCL1 (250-11, Peprotech) was added to the media directly when cells were plated and again when media was changed after irradiation.

Techniques: Expressing, Cell Culture, Co-Culture Assay, In Vitro, Injection, Staining, Immunohistochemistry, Recombinant, Control, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Ex Vivo, Whisker Assay, MANN-WHITNEY, Irradiation, Cell Counting, In Vivo, Activity Assay

Journal: Nature Medicine

Article Title: Stratification of radiosensitive brain metastases based on an actionable S100A9/RAGE resistance mechanism

doi: 10.1038/s41591-022-01749-8

Figure Lengend Snippet: a , Heatmap depicting the gene signature in Fig. scored in the comparison between H2030-BrM growing in adherent conditions in vitro and in brain organotypic cultures ex vivo . Only genes with FDR < 0.05 and a log 2 ratio >1 were considered. b , Quantification of in vitro viable cell fraction after irradiation at 10 Gy and stimulation with 100 ng/ml rCXCL1 or control, as determined by manual cell counting of bisbenzimide+ nuclei. Values are percentages of viable cells respect to unirradiated controls and shown in a dot plot where each dot represents an independent experiment and the line in the box corresponds to the median and whiskers go from the minimum to the maximum value (n = 3, each experimental condition). P value was calculated using two-tailed t test. c , Representative image of the peritumoral microenvironment where CXCL1 mRNA is labelled by RNA in situ hybridization (brown) and GFAP by immunohistochemistry (purple). The dotted line surrounds the metastasis. cc: cancer cells. Scale bar: 50 µm. The experiment was independently repeated three times with similar results. d , Representative image of CXCR2 protein in H2030-BrM brain metastasis. The dotted line surrounds the metastasis. cc: cancer cells. Scale bar: 25 µm. The experiment was independently repeated three times with similar results. e , Quantification of in vitro viable cell fraction after irradiation at 10 Gy and stimulation with rTGFα or control, as determined by manual cell counting of bisbenzimide+ nuclei. Values are percentages of viable cells respect to unirradiated controls and shown in a dot plot where each dot represents an independent experiment and the line in the box corresponds to the median median and whiskers go from the minimum to the maximum value (n = 4, each experimental condition). P value was calculated using two-tailed t test. f , Representative image of the peritumoral microenvironment where TGFα co-localizes with astrocytes (GFAP + ). Scale bar: 10 µm. The experiment was independently repeated three times with similar results. g , Representative image of EGFR immunohistochemistry in experimental H2030-BrM brain metastasis. Scale bar: 50 µm. The experiment was independently repeated three times with similar results. h , Differentially expressed genes in resistant culture preparations (2 biological independent replicas used for each radioresistant preparation (oncospheres, n = 2; co-cultures (cell–cell contact), n = 2), and for radiosensitive preparations, (adherent cultures, n = 2; co-cultures (insert), n = 2)) were used to perform Gene Ontology (GO) analysis using the EnrichR software. The volcano plot represents the significance of each gene set from GO Molecular Function versus its odds ratio. P value is computed from the Fisher exact test. Larger blue points represent significant terms ( P value<0.05); smaller grey points represent non-significant terms. The darker the blue color of a point, the more significant it is. i , Quantification of RAGE expression levels in H2030-BrM 24 hours after a single dose (10 Gy) of radiation. Both adherent (grey) and oncospheres (red) are plotted together. Each circle represents independent cultures (n = 3 nonirradiated adherent cells, n = 3 nonirradiated oncospheres, n = 3 nonirradiated adherent cells, n = 2 nonirradiated oncospheres). Expression values were normalized to their respective nonirradiated control for each culture condition and the line represents the mean. P value was calculated using two-tailed t test. Nonirradiated adherent cells versus irradiated adherent cells, P = 0.1521; nonirradiated oncospheres versus irradiated oncospheres, P = 0.0522. j , 66 human brain metastases from lung cancer patients (33 cases), breast cancer patients (30 cases) or patients with other primary tumors (3 cases) were stained for RAGE by immunohistochemistry. Representative images are shown. Scale bar: 50 µm. Quantification of different histological scoring of cancer cells is shown in pie charts. 6 cases had to be excluded, 15/60 were scored with no staining, 17/60 with weak staining and 28/60 with moderate staining.

Article Snippet: For stimulation experiments, 200 ng ml −1 recombinant human S100A9 (9254-S9, R&D Systems), 20 ng ml −1 recombinant mouse TNF-α (315-01 A, Peprotech), 50 ng ml −1 recombinant mouse interferon-α (752802, BioLegend), 100 ng ml −1 recombinant TGF-α (239-A-100, R&D Systems) or 100 ng ml −1 recombinant KC/CXCL1 (250-11, Peprotech) was added to the media directly when cells were plated and again when media was changed after irradiation.

Techniques: Comparison, In Vitro, Ex Vivo, Irradiation, Control, Cell Counting, Two Tailed Test, RNA In Situ Hybridization, Immunohistochemistry, Software, Expressing, Staining

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 6 PYK2 is a potential target of Entrectinib. (A) The binding model between Entrectinib (pink) and the crucial residues of PYK2 (purple). (B) The bind ing affinity between PYK2 and Entrectinib was analyzed by Microscale thermophoresis (MST). (C) Cellular Thermal Shift Assay and Western blot were performed to evaluate the stability of PYK2 after incubation with or without Entrectinib at different temperatures. (D) Drug Affinity Responsive Target Sta bility and Western blot were performed to evaluate the resistance of PYK2 to enzymatic hydrolysis. (E) The phosphorylation levels of PYK2 in cells treated by TGFβ2(10ng/ml) and different doses of Entrectinib (0.25, 0.5, 1, 2µM) were analyzed by western blot. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. ###P < 0.001 versus control group. ****P < 0.0001 versus TGFβ2 group

Article Snippet: In brief, after dyeing with N-hydroxysuccinimide, the recombinant human PYK2 protein (Solarbio, China) was diluted and mixed with Entrectinib of different concentrations.

Techniques: Binding Assay, Microscale Thermophoresis, Thermal Shift Assay, Western Blot, Incubation, Phospho-proteomics, Control

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: Targeting PYK2, entrectinib allays anterior subcapsular cataracts in mice by regulating TGFβ2 signaling pathway.

doi: 10.1186/s10020-024-00921-9

Figure Lengend Snippet: Fig. 7 The knockdown of PYK2 inhibits the TGFβ2-induced EMT through a non-Smad signaling pathway. (A) The knockdown efficiency of PYK2 was evaluated by western blotting. (B) The knockdown of PYK2 inhibited the EMT induced by TGFβ2. The efficacy of Entrectinib is influenced by PYK2 knock down. (C) PYK2 knockdown inhibited the activation of AKT, JNK, ERK, and P38. GAPDH served as a reference protein. Data was expressed as means ± SD, n = 3. #P < 0.05 and ##P < 0.01 versus the control group

Article Snippet: In brief, after dyeing with N-hydroxysuccinimide, the recombinant human PYK2 protein (Solarbio, China) was diluted and mixed with Entrectinib of different concentrations.

Techniques: Knockdown, Western Blot, Activation Assay, Control