Journal: Cell systems

Article Title: Molecular and Functional Networks Linked to Sarcopenia Prevention by Caloric Restriction in Rhesus Monkeys

doi: 10.1016/j.cels.2019.12.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sections were rinsed thoroughly then incubated in biotinylated anti-mouse IgG or biotinylated anti-rabbit IgG (Vector Labs) for 1 h. Sections were rinsed, then incubated in peroxidase labeled avidin biotin complex (ABC) solution (Vector Labs) for 1 h and color developed with ImPACT NovaRED reagent (Vector Labs) for ~5–8 min and rinsed.

Techniques: Plasmid Preparation, Recombinant, Avidin-Biotin Assay, Electron Microscopy, Sample Prep, Microarray, Labeling, Software

Journal: Stem cell research & therapy

Article Title: Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

doi: 10.1186/s13287-017-0507-4

Figure Lengend Snippet: Fig. 2 Expression and promoter methylation of DNA methyltransferases in intestinal epithelium. a, b Decreased Dnmt1 expression is detected in db/db crypts and intestinal epithelium stem cells (IESCs) by qRT-PCR using the FACS cell population (a) and Western blot using the MACS cell population (b left) and quantification of immunoblot bands from Western blot (b right). mRNA and protein levels are expressed relative to β-actin. Mean ± SE; n ≥6; *P < 0.05, **P < 0.01 by t test. C represents the control db/+ group; D represents the diabetic db/db group. c Promoter methylation microarray of Dnmt3b indicates promoter hypermethylation not only in db/db mice (orange bars) but also in db/+ mice (light blue bars). Values in brackets indicate the peak score that reflects the probability of positive enrichment and average P value scores. d, e Validation of Dnmt3b microarray results by bisulfite sequence. The table indicates the results of bisulfite sequencing analyses of 10 bacterial colonies per group (d). The CpG sites validated are the sites that exhibit a peak signal in the microarray. Black circles represent methylated CpG sites; white hollow circles represent unmethylated CpG sites; crosses represents methylation status not determined . Bar graph showing the ratio of methylated cyto- sine in CpG within the regions analyzed (e). f, g Immunohistochemical staining of Dnmt1 (f) and Dnmt3a (g). Scale bars = 50 μm. Diff differenti- ated cells, N.S. not significant

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Methylation, Quantitative RT-PCR, Western Blot, Control, Microarray, Biomarker Discovery, Sequencing, Methylation Sequencing, Immunohistochemical staining, Staining

Journal: Stem cell research & therapy

Article Title: Sox9 transcriptionally regulates Wnt signaling in intestinal epithelial stem cells in hypomethylated crypts in the diabetic state.

doi: 10.1186/s13287-017-0507-4

Figure Lengend Snippet: Fig. 3 Promoter methylation levels and gene expression of Wnt signaling-related genes. a (left) Hierarchical clustering analysis on the basis of the DMEP-related genes related with Wnt signaling. Each row indicates a DMEP-related gene, and the corresponding gene name is indicated on the right. Each column indicates a sample analyzed. Methylation levels range from unmethylated (green) to fully methylated (red), as indicated by the color legend at the top of the graph. a (right) The degree of methylation of Wnt signaling pathway-related genes among DMEP-related genes. The bar height reflects the degree of methylation. b, c The mRNA levels of Wnt signaling-related genes in crypts and intestinal epithelium stem cells (IESCs) using the FACS cell population. d (upper) Western blot analysis of Sox9 using the MACS cell population, and d (lower) quantification of immunoblot bands from three repetitions of western blot experiments. e Immunohistochemical staining of Sox9 in the small intestinal epithelium. The arrows indicate cells expressing low levels of Sox9, which were considered IESCs. Scale bars = 50 μm. mRNA and protein levels are expressed relative to β-actin. Mean ± SE; n ≥6 (mRNA); n ≥3 (protein); *P < 0.05, **P < 0.01, ***P < 0.001 by t test. N.S. not significant

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Dnmt1 antibody (1:2000; Abcam, Cambridge, MA, UK), rabbit anti-mouse Dnmt3a antibody (1:1000; Abcam), rabbit anti-mouse Sox9 antibody (1:1500; Abcam), and rabbit anti-mouse β-actin antibody (1:2000; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Methylation, Gene Expression, Western Blot, Immunohistochemical staining, Staining, Expressing

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Tissue microarray (TMA) based immunohistochemistry analysis of furin, TGF-β1 and pERK1/2 in CRC patients. CRC TMA spots showing overexpression of ( A ) furin, ( B ) TGF-β1 and ( C ) pERK1/2. In contrast, another set of TMA spots showing reduced expression of ( D ) furin, ( E ) TGF-β1 and ( F ) pERK1/2. 20 X/0.70 objective on an Olympus BX 51 microscope (Olympus America Inc., Center Valley, PA, USA, scale bar = 200 μm).

Article Snippet: The slides were incubated with primary antibodies against furin (ab183495, Abcam, Cambridge, UK) with a dilution of 1:100 (pH 9.0), TGF-β (3C11, 1:2000, pH 6.0; Santa Cruz Biotechnology, Dallas, TX, USA) and phosphorylated ERK1/2 (pERK 1/2, 137F5, 1:1000, pH 6.0; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Microarray, Immunohistochemistry, Over Expression, Expressing, Microscopy

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Furin promotes CRC cell growth through TGF-β/ERK1/2 signaling. ( A , B ) Furin overexpression (Furin-OE) enhances clonogenicity. CRC cells transfected with furin cDNA were selected, and stable clones were subjected to clonogenic assays ( n = 3). ( C ) Furin overexpression activates TGF-β/ERK1/2 signaling. Protein lysates from furin-overexpressing clones were analyzed by immunoblotting. ( D ) Furin knockdown suppresses clonogenicity. CRC cells transfected with two independent furin shRNA constructs were subjected to clonogenic assays ( n = 3). ( E ) Silencing furin decreases TGF-β expression and ERK1/2 phosphorylation. * p < 0.05 compared with controls.

Article Snippet: The slides were incubated with primary antibodies against furin (ab183495, Abcam, Cambridge, UK) with a dilution of 1:100 (pH 9.0), TGF-β (3C11, 1:2000, pH 6.0; Santa Cruz Biotechnology, Dallas, TX, USA) and phosphorylated ERK1/2 (pERK 1/2, 137F5, 1:1000, pH 6.0; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Over Expression, Transfection, Clone Assay, Western Blot, Knockdown, shRNA, Construct, Expressing, Phospho-proteomics

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: A positive feedback loop between furin and TGF-β promotes CRC cell growth. ( A ) TGF-β1 overexpression upregulates furin and enhances ERK1/2 phosphorylation. CRC cells transfected with TGF-β1 cDNA were analyzed by immunoblotting. ( B ) TGF-β1 knockdown decreases furin expression and ERK1/2 activation. Protein lysates from CRC cells transfected with two independent TGF-β1 shRNA constructs were examined by immunoblotting. ( C ) Overexpression of TGF-β1 increases clonogenic growth. Stable TGF-β1-overexpressing CRC clones were subjected to clonogenic assays ( n = 3). ( D ) TGF-β1 silencing reduces clonogenic growth. CRC cells transfected with two distinct TGF-β1 shRNAs were analyzed by clonogenic assays ( n = 3). * p < 0.05 compared with controls.

Article Snippet: The slides were incubated with primary antibodies against furin (ab183495, Abcam, Cambridge, UK) with a dilution of 1:100 (pH 9.0), TGF-β (3C11, 1:2000, pH 6.0; Santa Cruz Biotechnology, Dallas, TX, USA) and phosphorylated ERK1/2 (pERK 1/2, 137F5, 1:1000, pH 6.0; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Over Expression, Phospho-proteomics, Transfection, Western Blot, Knockdown, Expressing, Activation Assay, shRNA, Construct, Clone Assay

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Furin drives CRC chemoresistance through the TGF-β/ERK signaling pathway. ( A , B ) The 5-FuR CRC cell lines exhibit enhanced proliferation. Parental and 5-FuR cells were subjected to clonogenic assays ( n = 3). ( C ) The 5-FuR CRC cell lines show complete resistance to 5-Fu treatment (50 μM), as assessed by Annexin V/PI apoptosis analysis ( n = 3). ( D ) The 5-FuR cells display increased expression of furin, TGF-β1, and ERK1/2 phosphorylation, as determined by immunoblotting. ( E ) Furin knockdown restores 5-Fu sensitivity (50 μM), as shown by apoptosis analysis ( n = 3). * p < 0.05 compared with controls.

Article Snippet: The slides were incubated with primary antibodies against furin (ab183495, Abcam, Cambridge, UK) with a dilution of 1:100 (pH 9.0), TGF-β (3C11, 1:2000, pH 6.0; Santa Cruz Biotechnology, Dallas, TX, USA) and phosphorylated ERK1/2 (pERK 1/2, 137F5, 1:1000, pH 6.0; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Knockdown

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Furin overexpression enhances tumor growth in vivo. ( A ) Representative images showing the gross morphology and comparative size of excised xenograft tumors from mice. Although five animals were used per group, one tumor was inadvertently damaged during excision and could not be presented as an intact representative image. Importantly, tumor volume and weight measurements were obtained from all five animals, and quantitative analyses include the full cohort. Briefly, DLD1 cells stably expressing either an empty vector or FURIN cDNA were injected subcutaneously into the right flanks of NU/J mice ( n = 5). ( B ) Tumor volume and body weight were recorded weekly to monitor growth and animal condition. ( C ) After four weeks, mice were euthanized, and the tumors were collected and weighed. Data are shown as mean ± SD ( n = 5). ( D ) Tumor lysates were analyzed by immunoblotting with antibodies against Furin, TGF-β1, pERK1/2, total ERK1/2, and GAPDH. ( E ) Immunohistochemical staining of tumor sections (5 µm) was performed using antibodies against Furin (ab183495, Abcam, 1:100), TGF-β (3C11, Santa Cruz Biotechnology, 1:2000), and pERK1/2 (137F5, Cell Signaling Technology, 1:1000). Staining was developed using the Dako EnVision+ System with 3,3′-diaminobenzidine as the chromogenic substrate. Scale bar = 200 μm. Endpoint tumor volumes and weight were compared using one-way ANOVA; * p < 0.05 was considered statistically significant.

Article Snippet: The slides were incubated with primary antibodies against furin (ab183495, Abcam, Cambridge, UK) with a dilution of 1:100 (pH 9.0), TGF-β (3C11, 1:2000, pH 6.0; Santa Cruz Biotechnology, Dallas, TX, USA) and phosphorylated ERK1/2 (pERK 1/2, 137F5, 1:1000, pH 6.0; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Plasmid Preparation, Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Cells

Article Title: Furin Drives Colorectal Cancer Progression and Chemoresistance Through the TGF-β/ERK Signaling Pathway

doi: 10.3390/cells15010043

Figure Lengend Snippet: Depletion of furin inhibits tumor growth in vivo. ( A ) Representative images illustrating the size and appearance of tumors harvested from mice following the experimental endpoint. Although five animals were used per group, one tumor was inadvertently damaged during excision and could not be presented as an intact representative image. Importantly, tumor volume and weight measurements were obtained from all five animals, and quantitative analyses include the full cohort. Briefly, 5-FuR DLD1 cells expressing either an empty vector or FURIN -targeting shRNA were injected subcutaneously into the right flanks of NU/J mice ( n = 5). ( B ) Tumor volume and body weight were assessed weekly to track tumor progression and overall health. ( C ) After four weeks, mice were euthanized, and tumors were collected and weighed. Data are shown as mean ± SD ( n = 5). ( D ) Tumor lysates were analyzed by immunoblotting with antibodies against Furin, TGF-β1, pERK1/2, total ERK1/2, and GAPDH. ( E ) Immunohistochemical staining of tumor sections (5 µm) was performed using antibodies against Furin, TGF-β, and pERK1/2. Staining was developed using the Dako EnVision+ System with 3,3′-diaminobenzidine as the chromogenic substrate. Scale bar = 200 μm. Endpoint tumor volumes and weight were compared using one-way ANOVA; * p < 0.05 was considered statistically significant.

Article Snippet: The slides were incubated with primary antibodies against furin (ab183495, Abcam, Cambridge, UK) with a dilution of 1:100 (pH 9.0), TGF-β (3C11, 1:2000, pH 6.0; Santa Cruz Biotechnology, Dallas, TX, USA) and phosphorylated ERK1/2 (pERK 1/2, 137F5, 1:1000, pH 6.0; Cell Signaling Technology, Danvers, MA, USA).

Techniques: In Vivo, Expressing, Plasmid Preparation, shRNA, Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Journal of Nanobiotechnology

Article Title: Cold exposure-induced plasma exosomes impair bone mass by inhibiting autophagy

doi: 10.1186/s12951-024-02640-z

Figure Lengend Snippet: CT-EXO impairs bone mass by inhibiting autophagy. A , Representative western blot image showing the effect of cold exposure on the protein levels of P21, P62, and LC3 in bone tissues. B , Representative western blot image showing the expression protein levels of RUNX2, P62, and LC3, P21 from control-, Vehicle-, CT-EXO-, RAP-, and RAP + CT-EXO-treated BMSCs. C, D , Representative image of microscopic view (D) and entire plate view (E) ARS staining. Scale bar represents 250 μm. E , Quantification of ARS. n = 3 per group. F , Representative Electron Microscopy Images of control-, Vehicle-, CT-EXO-, RAP-, and RAP + CT-EXO-treated BMSCs. Scale bars represent 5 μm G , Representative image of SA-β-gal staining of BMSCs after exosomes treatment. Scale bar represents 100 μm. The control group represents the group without osteogenic induction, while the vehicle group represents the group undergoing osteogenic induction with solvent intervention. H , Schematic flow diagram representing mice treated with vehicle, CT-EXO, RAP, and RAP + CT-EXO. n = 6 per group. I , Representative µCT images of trabecular (top) and cortical (bottom) bone in femora from mice in the vehicle, CT-EXO, RAP, and RAP + CT-EXO groups. Scale bars represent 500 μm (top) and 1 mm (bottom). J–P , Quantitative analysis of BMD, Tb. BV/TV, Tb. Th, Tb. N, Tb. Sp, Ct. Ar/Tt. Ar, Ct. Th. n = 5 per group. Q , Representative calcein double labelling images of the mineralized surface of mouse femora. Scale bar represents 50 μm. R, S , Quantitation of MAR and BFR/BS. n = 5 per group. T , Representative OCN-stained section. Scale bar represents 100 μm. U , Quantification of the number of osteoblasts (N. OBs) on the trabecular bone surface (BS) in distal femora. n = 5 per group. V , TRAP-stained sections. Scale bar represents 100 μm. W , quantification of the number of osteoclasts (N. OCs) on the trabecular bone surface (BS) in distal femora. n = 5 per group. The vehicle referred to is DMSO. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The membrane was blocked with 5% non-fat milk for 1 h before being incubated with primary antibodies, CD9 (ab92726, 1 : 1000, Abcam), CD81 (ab109201, 1 : 1000, Abcam), TSG101 (bs-1365R, 1 : 1000, Bioss), RUNX2 (ab23981, 1 : 2000, Abcam), GAPDH (10494-1-AP, 1 : 4000, Proteintech), COL-I (14695-1-AP,1 : 1000, Proteintech), BMP2 (ab214821, 1 : 2000, Abcam), SATB2 (bs-1949R, 1 : 1000, Bioss), P62 (18420-1-AP, 1 : 2000, Proteintech), and LC3 (14600-1-AP, 1 : 2000, Proteintech), ATG5(66744-1-Ig,1 : 2000, Proteintech), followed by incubation for 1 h at room temperature with the secondary antibody conjugated to horseradish peroxidase.

Techniques: Western Blot, Expressing, Control, Staining, Electron Microscopy, Solvent, Quantitation Assay

Journal: Journal of Nanobiotechnology

Article Title: Cold exposure-induced plasma exosomes impair bone mass by inhibiting autophagy

doi: 10.1186/s12951-024-02640-z

Figure Lengend Snippet: Exosomal miR-25-3p enriched in CT-EXO regulated osteogenic differentiation and autophagy by tageting SATB2 in BMSCs. A , Volcano diagram of differential expression between between RT-EXO and CT-EXO miRNA according to microarray analysis. B–F , qRT-PCR quantitative results of miRNA (miR-205-5p, miR-30d-5p, miR-320-3p, miR-222-3p, miR-25-3p) level of RT-EXO or CT-EXO. n = 3 per group. G , Representative western blot image showing the expression protein levels of RUNX2, P62, and LC3 from miR-25-3p knocked-in or miR-25-3p knocked-down CT-EXO-treated BMSCs. H, I , Representative image of microscopic view (H) and entire plate view (I) ARS staining from miR-25-3p knocked-in or miR-25-3p knocked-down CT-EXO-treated BMSCs. Scale bar represents 250 μm J , Quantification of ARS. n = 3 per group. K , Venn diagram of the predicted target gene of miR-25-3p from Target scan (blue), miRDB (red), autophagy(yellow), and osteogenesis (orange). L , Luciferase reporter assays were conducted using luciferase constructs carrying a WT or mutant SATB2 3′-UTR co-transfected into BMSCs with miR-25-3p mimics. Firefly luciferase activity was normalized to Renilla luciferase activity. n = 5 per group. M , Transfection efficiency of miR-25-3p detected by qRT-PCR. N , Representative western blot image showing the protein expression levels of SATB2 from miR-25-3p knocked-in miR-25-3p knocked-down BMSCs. O , Knockout efficiency of SATB2 in BMSCs by western blot. P , SATB2, RUNX2, P62, and LC3 expression were measured in the BMSCs treated with siSATB2#3 or siRNA control. Q, R , Representative image of microscopic view (Q) and entire plate view (R)ARS staining. Scale bar represents 250 μm. S , Quantification of ARS. T , Representative western blot image showing the expression protein levels of SATB2, RUNX2, P62, and LC3 from the inhibitor-NC, inhibitor, inhibitor + siRNA-NC, inhibitor + siSATB2 groups. U, V , Representative image of microscopic view (U) and entire plate view (V)ARS staining. Scale bar represents 250 μm. W , Quantification of ARS. n = 3 per group. All groups’ cells have received osteogenic induction, the control group represents the group undergoing osteogenic induction without any intervention. CT-EXO + AgomiR-NC and CT-EXO-AntagomiR-NC served as negative controls for CT-EXO + AgomiR-25-3p and CT-EXO-AntagomiR-25-3p. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The membrane was blocked with 5% non-fat milk for 1 h before being incubated with primary antibodies, CD9 (ab92726, 1 : 1000, Abcam), CD81 (ab109201, 1 : 1000, Abcam), TSG101 (bs-1365R, 1 : 1000, Bioss), RUNX2 (ab23981, 1 : 2000, Abcam), GAPDH (10494-1-AP, 1 : 4000, Proteintech), COL-I (14695-1-AP,1 : 1000, Proteintech), BMP2 (ab214821, 1 : 2000, Abcam), SATB2 (bs-1949R, 1 : 1000, Bioss), P62 (18420-1-AP, 1 : 2000, Proteintech), and LC3 (14600-1-AP, 1 : 2000, Proteintech), ATG5(66744-1-Ig,1 : 2000, Proteintech), followed by incubation for 1 h at room temperature with the secondary antibody conjugated to horseradish peroxidase.

Techniques: Quantitative Proteomics, Microarray, Quantitative RT-PCR, Western Blot, Expressing, Staining, Luciferase, Construct, Mutagenesis, Transfection, Activity Assay, Knock-Out, Control

Journal: Aging (Albany NY)

Article Title: High S100A9 + cell density predicts a poor prognosis in hepatocellular carcinoma patients after curative resection

doi: 10.18632/aging.203162

Figure Lengend Snippet: Association between S100A9 expression and prognosis of HCC patients in the TCGA-LIHC database. ( A ) Violin plot showing the differential expression of the S100A9 gene in tumor ( n = 365) or nontumor ( n = 50) tissues from the TCGA-LIHC database; the Mann- Whitney test was used to analyze the nonparametric test between the two groups. HCC patients in the TCGA dataset were divided into two groups according to the median value of S100A9 expression ( B ) or minimum P -value approach ( C ). The prognostic value of S100A9 was evaluated by the Kaplan-Meier method, and examined by the log-rank test. ( D ) Top 30 GO enrichment results of the 3493 genes highly expressed in the S100A9 high group. ( E – H ) Top enriched signaling pathways based on S100A9 expression identified by GSEA.

Article Snippet: A tissue microarray (TMA) was constructed, and the slides were incubated with a rabbit monoclonal primary antibody against S100A9 (34425, 1:2000, Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Expressing, Quantitative Proteomics, MANN-WHITNEY, Protein-Protein interactions

Journal: Aging (Albany NY)

Article Title: High S100A9 + cell density predicts a poor prognosis in hepatocellular carcinoma patients after curative resection

doi: 10.18632/aging.203162

Figure Lengend Snippet: S100A9 expression in HCC tumor. ( A ) Representative images of IHC staining. ( B ) Scatter plot illustrating the correlation between S100A9-expressing cell counts in HCC by computer counting and investigator counting. ( C ) Quantification of S100A9 + cell densities in the N (nontumoral) and T (tumoral) regions ( n = 382), and the Wilcoxon matched-pairs signed-rank test was used to analyze the nonparametric test between the paired two groups.

Article Snippet: A tissue microarray (TMA) was constructed, and the slides were incubated with a rabbit monoclonal primary antibody against S100A9 (34425, 1:2000, Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Expressing, Immunohistochemistry

Journal: Aging (Albany NY)

Article Title: High S100A9 + cell density predicts a poor prognosis in hepatocellular carcinoma patients after curative resection

doi: 10.18632/aging.203162

Figure Lengend Snippet: Prognostic value of S100A9 + cell in HCC patients. A high S100A9 + cell density in nontumor or tumor tissue was associated with poor OS ( A , C ) and a high recurrence rate ( B , D ) in HCC patients. Forest plots showing the association between S100A9 expression in tumors and clinicopathological features in HCC patients using univariate ( E ) or multivariate analysis ( F ). Patients were divided into two groups according to the median value of the S100A9 + cell density in nontumoral and tumoral tissues. The cumulative OS ( G ) and recurrence ( H ) times were calculated using the Kaplan-Meier method and then analyzed with the log-rank test.

Article Snippet: A tissue microarray (TMA) was constructed, and the slides were incubated with a rabbit monoclonal primary antibody against S100A9 (34425, 1:2000, Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: High S100A9 + cell density predicts a poor prognosis in hepatocellular carcinoma patients after curative resection

doi: 10.18632/aging.203162

Figure Lengend Snippet: Correlation between clinicopathological parameters and the density of S100A9 + cells.

Article Snippet: A tissue microarray (TMA) was constructed, and the slides were incubated with a rabbit monoclonal primary antibody against S100A9 (34425, 1:2000, Cell Signaling Technology, USA) at 4°C overnight.

Techniques:

Journal: Aging (Albany NY)

Article Title: High S100A9 + cell density predicts a poor prognosis in hepatocellular carcinoma patients after curative resection

doi: 10.18632/aging.203162

Figure Lengend Snippet: Tumor-infiltrating immune cells associated with S100A9 expression in HCC. ( A ) Differential infiltration of immune cells based on S100A9 expression in groups from the TCGA-LIHC cohort. The proportions of 22 immune infiltrates in tumor ( n = 49) and nontumor tissue ( n = 7) were estimated using the CIBERSORT algorithm. ( B – E ) Prognostic value of M0, M1, M2 and total (M0 + M1 + M2) macrophage densities in HCC ( n = 49). ( F ) The densities of S100A9 + cells and CD15 + neutrophils and CD68 + macrophages were analyzed using Pearson correlation analysis ( n = 382). *** P < 0.001.

Article Snippet: A tissue microarray (TMA) was constructed, and the slides were incubated with a rabbit monoclonal primary antibody against S100A9 (34425, 1:2000, Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: High S100A9 + cell density predicts a poor prognosis in hepatocellular carcinoma patients after curative resection

doi: 10.18632/aging.203162

Figure Lengend Snippet: Characterization of patient-derived S100A9 + cells. Multiple immunofluorescence staining shows DAPI (gray), S100A9 (green), CD34 (blue, A ), CD56 (pink, D ), CD20 (purple, G ), and CD3 (orange, J ) expression and coexpression (double-positive cells) in HCC tissue. Quantification of CD34 ( B ), CD56 ( E ), CD20 ( H ), and CD3 ( K ) cell densities in the T and N regions. ( C ) The percentages of S100A9 + CD34 + cells among the total CD34 + cells in the N and T regions. ( F ) The percentages of S100A9 + CD56 + cells among the total CD56 + cells in the N and T regions. ( I ) The percentages of S100A9 + CD20 + cells among the total CD20 + cells in the N and T regions. ( L ) The percentages of S100A9 + CD3 + cells among total CD3 + cells in the N and T regions ( n = 4 - 7). Scale bar = 50 μm. The results are the means ± SEM (bars); NS , no significance; ND , not detected.

Article Snippet: A tissue microarray (TMA) was constructed, and the slides were incubated with a rabbit monoclonal primary antibody against S100A9 (34425, 1:2000, Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Derivative Assay, Immunofluorescence, Staining, Expressing

Journal: Aging (Albany NY)

Article Title: High S100A9 + cell density predicts a poor prognosis in hepatocellular carcinoma patients after curative resection

doi: 10.18632/aging.203162

Figure Lengend Snippet: Myeloid cells are the major source of S100A9. Multiple immunofluorescence staining shows DAPI (gray), S100A9 (green), CD15 (blue, A ), and CD68 (red, D ) expression and coexpression (double-positive cells) in the N and T regions. Quantification of CD15 + ( B ) and CD68 + ( E ) cell densities in the T and N regions ( n = 12). ( C ) The percentages of S100A9 + CD15 + cells among the total S100A9 + cells in the N and T regions. ( F ) The percentages of S100A9 + CD68 + cells among the total S100A9 + cells in the N and T regions. ( n = 12). Scale bar = 25 μm. N, nontumor, T, tumor. * P < 0.05, **** P < 0.0001, NS , no significance.

Article Snippet: A tissue microarray (TMA) was constructed, and the slides were incubated with a rabbit monoclonal primary antibody against S100A9 (34425, 1:2000, Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Immunofluorescence, Staining, Expressing

Journal: eLife

Article Title: Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling

doi: 10.7554/eLife.17047

Figure Lengend Snippet: Transcriptional and biochemical analyses were performed on Cdh16 Cre and Cdh16 Cre ::Tfeb fs mice. ( A , B ) Tables show the relative increase of genes related to the ErbB ( A ) and WNT ( B ) pathways in the microarray analyses performed on kidneys from P0 Cdh16 Cre ::Tfeb fs mice. Graphs show real-time PCR validations performed on kidneys from Cdh16 Cre ::Tfeb fs mice at different stages (P0, P12, P30). Data are shown as the average (± SEM) of at least three Cdh16 Cre ::Tfeb fs mice normalized versus wild-type mice. ( C , D ) Immunoblot analyses performed on ( C ) P30 kidney tissues and ( D ) primary kidney cells isolated from Cdh16 Cre ::Tfeb fs mice to evaluate ErbB and WNT activation status. Each replicate is a distinct biological sample. ErbB signaling was assessed by looking at phosphoAKT (Ser473) to total AKT ratio, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK ratio; WNT signaling was assessed by quantifying β-catenin and CCND1 (Cyclin D1) protein levels. Graphs represent the densitometry quantification of Western blot bands. Values are normalized to actin when not specified and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided, Student’s t test). DOI: http://dx.doi.org/10.7554/eLife.17047.007 10.7554/eLife.17047.008 Figure 3—source data 1. Complete list of 294 genes (represented by 361 probesets) significantly induced (FDR≤0.05) in the KSP_P0 microarray dataset (GSE62977). The genes are ranked by decreasing signed ratio (KSP_P0/CTL). DOI: http://dx.doi.org/10.7554/eLife.17047.008 10.7554/eLife.17047.009 Figure 3—source data 2. Complete list of 628 genes (represented by 729 probesets) significantly induced (FDR≤0.05) in the KSP_P14 microarray dataset (GSE63376). The genes are ranked by decreasing signed ratio (KSP_P14/CTL). DOI: http://dx.doi.org/10.7554/eLife.17047.009

Article Snippet: For Western blots, the following antibodies were used: anti-FLAG M2-HRP (Sigma, cat. A8592, RRID: AB_439702 , 1:1000), anti-actin (Sigma, cat. A2066, RRID: AB_476693 , 1:5000), anti-βtubulin (Sigma, cat. T8328, RRID: AB_1844090 1:1000), anti-Human/Mouse/Rat Pan-Akt (R&D, cat. MAB2055, RRID: AB_2224581 , 1:500), Phospho-Akt (Ser473) (D9E) Cell Signaling, cat. #4060, RRID: AB_2315049 , 1:1000), anti-human, mouse, and rat ERK1/ERK2 (R&D, cat.216703, RRID: AB_2140121 , 1:2000), anti-Human/Mouse/Rat Phospho- ERK1(T202/Y204)/ERK2 (T185/Y187) (R&D, cat. AF1018, RRID: AB_354539 1:1000), anti-β-catenin (BD, cat. 610154, RRID: AB_397555 1:500), anti-active β-catenin (Cell Signaling, cat. #8814, RRID: AB_11127203 1:1000), anti-Cyclin D1 (Cell Signaling, cat. #2978, RRID: AB_10692801 1:1000), anti-LRP6 (Cell Signaling, cat. #3395, RRID: AB_1950408 1:1000), anti-phospho-LRP6 (Ser1490) (Cell Signaling, cat. #2568, RRID: AB_2139327 1:1000), anti-GSK3β (Cell Signaling, cat. #9315, RRID: AB_490890 1:1000), anti-phospho-GSK3β (Ser9) (Cell Signaling, cat. #9323, RRID: AB_2115201 1:1000), anti MYC (Cell Signaling, cat. #5605, RRID: AB_1903938 1:1000).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Western Blot, Isolation, Activation Assay

Journal: eLife

Article Title: Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling

doi: 10.7554/eLife.17047

Figure Lengend Snippet: Immunoblot analysis performed on P90 kidneys from Cdh16 Cre ::Tfeb fs mice ( A ) and P90 Cdh16 CreErt2 ::Tfeb fs animals induced with tamoxifen at P14 ( B ) and at P30 ( C ), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test). DOI: http://dx.doi.org/10.7554/eLife.17047.011

Article Snippet: For Western blots, the following antibodies were used: anti-FLAG M2-HRP (Sigma, cat. A8592, RRID: AB_439702 , 1:1000), anti-actin (Sigma, cat. A2066, RRID: AB_476693 , 1:5000), anti-βtubulin (Sigma, cat. T8328, RRID: AB_1844090 1:1000), anti-Human/Mouse/Rat Pan-Akt (R&D, cat. MAB2055, RRID: AB_2224581 , 1:500), Phospho-Akt (Ser473) (D9E) Cell Signaling, cat. #4060, RRID: AB_2315049 , 1:1000), anti-human, mouse, and rat ERK1/ERK2 (R&D, cat.216703, RRID: AB_2140121 , 1:2000), anti-Human/Mouse/Rat Phospho- ERK1(T202/Y204)/ERK2 (T185/Y187) (R&D, cat. AF1018, RRID: AB_354539 1:1000), anti-β-catenin (BD, cat. 610154, RRID: AB_397555 1:500), anti-active β-catenin (Cell Signaling, cat. #8814, RRID: AB_11127203 1:1000), anti-Cyclin D1 (Cell Signaling, cat. #2978, RRID: AB_10692801 1:1000), anti-LRP6 (Cell Signaling, cat. #3395, RRID: AB_1950408 1:1000), anti-phospho-LRP6 (Ser1490) (Cell Signaling, cat. #2568, RRID: AB_2139327 1:1000), anti-GSK3β (Cell Signaling, cat. #9315, RRID: AB_490890 1:1000), anti-phospho-GSK3β (Ser9) (Cell Signaling, cat. #9323, RRID: AB_2115201 1:1000), anti MYC (Cell Signaling, cat. #5605, RRID: AB_1903938 1:1000).

Techniques: Western Blot