Journal:

Article Title: CCN1/Cyr61 Is Regulated by the Canonical Wnt Signal and Plays an Important Role in Wnt3A-Induced Osteoblast Differentiation of Mesenchymal Stem Cells †

doi: 10.1128/MCB.26.8.2955-2964.2006

Figure Lengend Snippet: Regulation of CCN1/Cyr61 by Wnt3A/β-catenin. (A) Relative microarray signal intensities of five members of the CCN family upon Wnt3A stimulation. The acquired microarray hybridization data were filtered and normalized prior to further analysis, as described in Materials and Methods. (B) Time course expression of CCN1/Cyr61 upon Wnt3A stimulation using qPCR. Subconfluent C3H10T1/2 cells were cultured in 0.5% FCS complete medium and infected with AdWnt3A or AdGFP. Total RNA was collected at the indicated time points and subjected to qPCR analysis. All samples were normalized for GAPDH expression. PCRs were done in triplicate. (C) Western blot analysis of Wnt3A-induced CCN1/Cyr61 expression. At the indicated time points, AdWnt3A- or AdGFP-infected C3H10T1/2 cells were lysed and subjected to Western blot analysis using an anti-CCN1/Cyr61 antibody. The expression level of β-actin was used as a loading control. (D) ChIP analysis of the mouse CCN1/Cyr61 promoter. C3H10T1/2 cells were infected with AdWnt3A or AdGFP for 36 h followed by formaldehyde cross-linking. The cross-linked cells were lysed and subjected to sonication and immunoprecipitation using anti-β-catenin (anti-β cat) or control IgG. The recovered chromatin DNA fragments were used for PCR amplifications with primers specific for the mouse CCN1 promoter.

Article Snippet: The remaining two-thirds of the lysate was subjected to immunoprecipitation using anti-β-catenin antibody (BD Pharmingen) or mouse immunoglobulin G (IgG) (Pierce).

Techniques: Microarray, Hybridization, Expressing, Cell Culture, Infection, Western Blot, Sonication, Immunoprecipitation

Journal: Environmental toxicology

Article Title: Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

doi: 10.1002/tox.24087

Figure Lengend Snippet: FIGURE 5 Involvement of miR-1247-3p in the Gallic acid (GA)-inhibited integrin/FAK cascade in DLD-1 cells. Cells were treated with 90 μM GA for 24 h, lysed for total RNA extraction, and subjected to (A) miRNA expression profiling assay via microarray analysis or (B) quantitative analysis of the miRNAs via qPCR. miRNAs with up- and downregulation in response to GA were labeled with red and green, respectively. (C) Cells were transfected with control vector (Ctl-miR) or anti-miR-1247-3p (anti-miR), treated with GA for 24 h, and subjected to the assessment of integrins, FAK, and paxillin by Western blot. Densitometric analysis was performed for semi-quantitation of signals. The tubulin signal was used as internal control. Average signal ratios of phosphorylated protein/total protein compared with the control were indicated.

Article Snippet: Antibodies specifically recognizing human tubulin (SC-134237), integrin αV (SC-376156), integrin β3(SC-365679), focal adhesion kinase (FAK, SC-271126), phosphorylated FAK (pY397-FAK, SC-81493), paxillin (SC-136297), phosphorylated paxillin (pY118-Paxillin, SC-365020), Src (SC-32789), phosphorylated Src (pY416-Src, SC-24621, and CS#2101), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

Techniques: RNA Extraction, Expressing, Microarray, Labeling, Transfection, Control, Plasmid Preparation, Western Blot, Quantitation Assay

Journal: Environmental toxicology

Article Title: Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

doi: 10.1002/tox.24087

Figure Lengend Snippet: FIGURE 4 Gallic acid (GA) reduced integrin expression and inhibited FAK/Paxillin/Src and PI3K/AKT signaling in DLD-1 cells. Cells were treated with GA at the indicated concentrations for 24 h; collected; and lysed for the immunodetection of (A) integrins and the associated signaling proteins, (B) phosphor-paxillin and Src, and (C) PI3K, phosphor-AKT, and AKT via Western blot. Semi-quantitation of signals was conducted by densitometric analysis. DMSO treatment was used as control (C). Densitometric analysis was performed for the semi-quantitation of signals. The tubulin signal was used as internal control. Average signal ratios of phosphorylated protein/total protein compared with the control were indicated.

Article Snippet: Antibodies specifically recognizing human tubulin (SC-134237), integrin αV (SC-376156), integrin β3(SC-365679), focal adhesion kinase (FAK, SC-271126), phosphorylated FAK (pY397-FAK, SC-81493), paxillin (SC-136297), phosphorylated paxillin (pY118-Paxillin, SC-365020), Src (SC-32789), phosphorylated Src (pY416-Src, SC-24621, and CS#2101), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

Techniques: Expressing, Immunodetection, Western Blot, Quantitation Assay, Control

Journal: Scientific reports

Article Title: AEG-1 silencing attenuates M2-polarization of glioma-associated microglia/macrophages and sensitizes glioma cells to temozolomide.

doi: 10.1038/s41598-021-96647-3

Figure Lengend Snippet: Figure 6. AEG-1 activates Wnt/β-catenin signaling via targeting GSK-3β in glioma cells. (A) Enriched top 10 KEGG pathways for Affymetrix microarray of AEG-1 NC (N = 3) and KD (N = 3) glioma cells. 22 DEGs were enriched in KEGG_WNT_SIGNALING PATHWAY. P-value = 1.02E−06. [drawn by R 3.6.0 (https://cran.r- project.org/doc/FAQ/R-FAQ.html#Citing-R)]. (B) Enrichment plot of the Wnt signaling pathway from GSEA; ‘h’ and ‘l’ represented AEG-1 high and low expression, respectively. NES = 1.523, NOM P-value = 0.008, FDR q-value = 0.216 [drawn by GSEA tool (version 4.1.0)]. (C) Western blot bands of AEG-1, β-catenin, GSK-3β, cyclin D1, and CD44 in NC and shAEG-1 glioma cell lines. (D) Relative protein abundance was calculated by ImageJ and GraphPad Prism 8.2.1 software. (E, F) In U251 and U87 cells, Co-IP assays showed the direct interaction of AEG-1 and GSK-3β. (G) Immunofluorescence assays were used to detect the localization of AEG-1 (red) and GSK-3β (green) in U251 and U87 cells. All data were presented as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Cells were incubated with rabbit anti-AEG-1 antibody (Proteintech, 1:1000), mouse anti-GSK-3β antibody (Cell Signaling Technology, 1:1000), and rabbit anti-γH2AX (Cell Signaling Technology, 1:400), diluted using 1% FBS, at 4 °C overnight.

Techniques: Microarray, Expressing, Western Blot, Quantitative Proteomics, Software, Co-Immunoprecipitation Assay, Immunofluorescence

Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 3 Validation of cba-miR-222-3p targeting TRAF7 and TRAF7 expression in the testes of striped hamsters. (a) Sequences and peak maps of cba-miR-222-3p, TRAF7-WT, and TRAF7-MT. (b) Relative luciferase activity detected by Dual-Luciferase Reporter Assay. (c) Immunohistochemistry (IHC, tissue microarray [TMA]) of TRAF7 in testes. (d) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (e) Protein expression levels of TRAF7 in the testes detected by western blot (n = 4). (f) Pearson correlation analysis of cba-miR-222-3p and TRAF7. LD, long daylength; MD, moderate daylength; SD, short daylength; ∗, P < 0.05; ∗∗, P < 0.01.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Biomarker Discovery, Expressing, Luciferase, Activity Assay, Reporter Assay, Immunohistochemistry, Microarray, Western Blot

Journal: Integrative zoology

Article Title: cba-miR-222-3p involved in photoperiod-induced apoptosis in testes of striped hamsters by targeting TRAF7.

doi: 10.1111/1749-4877.12918

Figure Lengend Snippet: Figure 4 Expression of cba-miR-222-3p and TRAF7 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis after in vivo injection in the testes of striped hamsters. (a) Schematic diagram of in vivo injection experiment of miRNA mimics. (b) Fluorescence in situ hybridization (FISH, tissue microarray [TMA]) of cba-miR-222-3p in testes after in vivo injection. (c) Fluorescence intensity of cba-miR-222-3p detected by FISH (TMA; n = 4). (d) Immunohistochemistry (IHC, TMA) of TRAF7 in testes after in vivo injection. (e) Integrated density of TRAF7 detected by IHC (TMA; n = 4). (f) TUNEL (TMA) staining of the testes after in vivo injection. (g) The proportion of TUNEL (TMA) staining with apoptotic activity in the testes (n = 4). (h) Relative expression of MEKK3 (n = 6). (i) Relative expression of p38 (n = 6). (j) Relative expression of p53 (n = 6). AG, agomir injection; NC, agomir negative control injection; DAPI, 4′6′-diamidino-2-phenylindole. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: After performing electrophoresis, the proteins were transferred to PVDF membranes, which were then incubated with TRAF7 antibodies (11780-1-AP, Proteintech, China, RRID:AB_2877793) at a 1:1000 dilution and β-actin antibodies (20536-1-AP, Proteintech, China, RRID:AB_10700003) at a 1:1000 dilution, and subsequently incubated with IRDye 800 CW goat anti-rabbit secondary antibodies (31 460, Thermo Fisher, USA).

Techniques: Expressing, TUNEL Assay, In Vivo, Injection, Fluorescence, In Situ Hybridization, Microarray, Immunohistochemistry, Staining, Activity Assay, Negative Control

Journal: BMC cancer

Article Title: Integrated multi-omics reveal lactate metabolism-related gene signatures and PYGL in predicting HNSCC prognosis and immunotherapy efficacy.

doi: 10.1186/s12885-025-13982-8

Figure Lengend Snippet: Fig. 9 Associations between PYGL, the immune infiltration, and the responsiveness to immunotherapy in HNSCC. (A) Relationship between PYGL and infiltration levels of CD8 + T cell in the TCGA cohort. (B) Association of PYGL with infiltration levels of B cell in the TCGA cohort. (C) Inverse link between PYGL and CD8 in OC tissue microarray (n = 48) by IHC. (D) The HNSCC samples were classified into two groups depending on the existence or absence of CD8 + T cells into inflamed and non-inflamed phenotypes. Immunofluorescence showed lower PYGL positivity and higher CD8 and PD-L1 positivity in inflammatory TME. (E) Mean densitometric analysis revealed that PYGL was inversely linked to CD8 + T cell infiltrates. (F) Relationship between PYGL and chemokines. (G) Relationship between PYGL and chemokine receptors. (H) Inverse correlation between PYGL and CD8 in OC tissue microarray (n = 48) by IHC. (I) Correlation analysis of PYGL with CD274 (PD-L1) in the TCGA cohort. (J) Analyses of correlation between PYGL and TIDE score. (K) Mulberry plot showing the relationship between risk signature, PYGL, immune subtype, and predicted immune efficacy

Article Snippet: Primary antibodies were used as follows: anti-PYGL rabbit antibody (15851- 1-AP, Proteintech, Wuhan, China), anti-PD-L1 rabbit antibody (ab205921, Abcam, US), and anti-CD8 rabbit antibody (66868-1-IG, Proteintech, Wuhan, China).

Techniques: Microarray, Immunofluorescence

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: mRNA expression analysis in Nf1 mutant mouse Schwann cells. (A) Microarray analysis was used to compare genome-wide expression levels between normal mouse Schwann cells and Nf1 mutant Schwann cells. The control for each comparison was Cy3-labeled cDNA generated from normal mouse Schwann cell mRNA. For each of four Nf1 mutant Schwann cell samples (Nf1+/−, Nf1−/−, Nf1−/− TXF, and Nf1−/− TXF treated with FTI), mRNA was used as a template to synthesize Cy5-labeled cDNA. Cy3- and Cy5-labeled cDNA probes were hybridized simultaneously to the Incyte Genomics MouseGEM 1.0 cDNA microarray. Relative intensities of Cy3 versus Cy5 fluorescent signals for each cDNA target sequence were analyzed with GeneSpring software. The most changes were observed in the Nf1−/− TXF cells (genes upregulated in Nf1−/− TXF are red; genes downregulated in Nf1−/− TXF are green). Expression of one target cDNA, BLBP (black line), was 26-fold above normal in the Nf1−/− TXF cells and not normalized by FTI treatment. (B) RT-PCR analysis confirmedthe microarray result of elevated BLBP expression in Nf1−/− TXF cells. Reverse transcriptase (RT) was omitted from duplicate samples to control for DNA contamination. Primers for BLBP (∼200-bp amplicon) and actin control primers (∼500-bp amplicon) were included in the mixture for each 40-cycle reaction. The plasmid positive control for BLBP amplification is the UniGEM clone (Incyte Genomics) containing the BLBP cDNA insert spotted on the microarray. (C) Quantitative real-time PCR of BLBP normalized to GAPDH resulted in a 145-fold change over expression in Nf1−/− TXF cells compared to wild-type mouse Schwann cells. Rn, fluorescent signal intensity; horizontal starred line, chosen threshold at geometric phase of amplification.

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Expressing, Mutagenesis, Microarray, Genome Wide, Labeling, Generated, Sequencing, Software, Reverse Transcription Polymerase Chain Reaction, Amplification, Plasmid Preparation, Positive Control, Real-time Polymerase Chain Reaction, Over Expression

Journal:

Article Title: Brain Lipid Binding Protein in Axon-Schwann Cell Interactions and Peripheral Nerve Tumorigenesis

doi: 10.1128/MCB.23.6.2213-2224.2003

Figure Lengend Snippet: Mouse neuron-Schwann cell coculture. Anti-BLBP promotes extension of Nf1−/− TXF cell processes along axons. Wild-type (A and B) or Nf1−/− TXF (C and D) mouse Schwann cells labeled with Cell Tracker green were preincubated with rabbit IgG (A and C) or anti-BLBP antibodies (B and D) and seeded onto DRGN cultures stripped of endogenous Schwann cells. Two days after seeding, cocultures were fixed and stained with antineurofilament antibodies followed by Cy3 (red)-conjugated secondary antibodies. Confocal images obtained with Zeiss LSM Image Browser software are shown. Single cells are representative of the majority observed with each treatment. Arrowheads indicate Schwann cell processes. The asterisk indicates the region which is magnified fivefold in the inset. The scale bar in panel C equals 5 μm and also applies to panels A, B, and D. (E) Lower magnification (scale bar, 5 μm) of Nf1−/− TXF on DRGN cultures in the presence of anti-BLBP antibodies. Arrowheads indicate processes from two cells extending along neurites; other cells lack processes. (F) Extension of Nf1−/− TXF cell processes in the presence of anti-BLBP antibodies is statistically significant. The percentages of Nf1−/− TXF cells extending processes along axons in the presence of control IgG (gray bar) or anti-BLBP antibodies (black bar) are graphed. Error bars reflect standard deviations in a Student t test (P = 0.003).

Article Snippet: In DRGN cocultures, neurofilament was visualized by incubation with anti-NF-15g1 antibodies ( 38 ) diluted 1:5, followed by donkey anti-mouse Cy3-labeled secondary antibodies (Jackson Immunoresearch).

Techniques: Labeling, Staining, Software