Journal: Cell reports

Article Title: Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia

doi: 10.1016/j.celrep.2023.112027

Figure Lengend Snippet:

Article Snippet: Venetoclax , MedChem Express , HY-15531.

Techniques: Purification, Blocking Assay, Recombinant, Lysis, Staining, Saline, DNA Methylation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Binding Assay, Single Cell Gel Electrophoresis, Software, Protein Array, Membrane

Journal: Frontiers in Oncology

Article Title: SNHG9, a Papillary Thyroid Cancer Cell Exosome-Enriched lncRNA, Inhibits Cell Autophagy and Promotes Cell Apoptosis of Normal Thyroid Epithelial Cell Nthy-ori-3 Through YBOX3/P21 Pathway

doi: 10.3389/fonc.2021.647034

Figure Lengend Snippet: Identification and expression of PTC cell exosome-enriched lncRNA SNHG9. (A) High-throughput screening identification of PTC associated exosome lncRNAs. SNHG9 is PTC cell exosome-enriched lncRNA in TPC-1 and K-1 cells compared with Nthy-ori-3 cell. (B, C) Validation of SNHG9 overexpression in both TPC-1 and K-1 cells and their respective exosomes compared with Nthy-ori-3 cell and its exosome by qPCR. (D) Coregulation network of SNHG9 with mRNA/miRNA. SNHG9 had an interaction with autophagy related molecules. (E) Gene ontology enrichment analysis showed the highest regulation scores in autophagy and apoptosis. (F) KEGG-pathway-weighted analysis showed SNHG9 mainly targeted apoptosis and autophagy pathways. (G) SNHG9 in the PTC cell supernatant mainly derived from cell exosomes. QPCR showed significantly lower SNHG9 expression level in supernatant treated with Rnase and Triton compared with supernatant treated with only Rnase and control group. (H) QPCR confirmed no SNHG9 expression in cell supernatants after exosome extraction. (I) SNHG9 expression level between tumor and normal tissues in 70 PTC patients from FUSCC. The results were normalized to β-actin mRNA level. (J) Waterfall plot showed the distribution of SNHG9 expression level in each PTC patients from FUSCC. ***P < 0.001, data were pooled from three independent experiments. FUSCC, Fudan University Shanghai Cancer Center; PTC, papillary thyroid cancer.

Article Snippet: Next, we used the Arraystar Human LncRNA Array v2.0 gene chip to compare expression profile data of lncRNAs in Nthy-ori-3, TPC-1 and K-1 cells and their respective exosomes.

Techniques: Expressing, High Throughput Screening Assay, Biomarker Discovery, Over Expression, Derivative Assay, Control, Extraction

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 1. Physical interactions between TRF2 and BER proteins. A, increasing concentrations of various proteins were spotted in replicate (0.6, 0.8, 0.9, and 1 ng) on a grid of the Discover-Light Protein Array membrane and then hybridized with TRF2 protein (10 ng/mL). After washing, bound TRF2 protein was detected by Western blotting with an anti-TRF2 antibody. B, coimmunoprecipitation of TRF2 and Pol h. HeLa whole-cell extracts (500 ng) were immunoprecipitated with either rabbit anti-TRF2 (lane 3) or control IgG (lane 4) antibodies. The immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis with anti–Pol h or anti-TRF2 antibodies as indicated. TRF2 (lane 1) and Pol h (lane 6) were loaded as markers and positive controls. Input, 10% loaded (lane 2). C, coimmunoprecipitation of TRF2 and FEN-1. HeLa whole-cell extracts (500 ng) were immunoprecipitated with either rabbit anti-TRF2 (lane 3) or control IgG antibodies (lane 1). The immunoprecipitates were probed with anti-FEN-1 or anti-TRF2 antibodies as indicated. Input, 10% loaded (lane 2).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Protein Array, Membrane, Western Blot, Immunoprecipitation, Control, SDS Page

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 2. Mapping the sites of Pol h and FEN-1 interactions with TRF2. A, schematic of the known domains and structural motifs of TRF2 and the borders of the various GST-tagged fragments. B, basic NH2 terminus; TRFH, dimerization domain; Myb, Myb-like telomere DNA binding domain. Numbers indicate the amino acid sequence. B, Coomasie staining of the recombinant GST-tagged TRF2 fragments (2 Ag each) used in the binding assay after single-step purification and SDS-PAGE. C, TRF2 domains that interacted with Pol h and FEN-1. HeLa nuclear extracts (400 AL) were incubated with either GST alone (lanes 2 and 9) or GST-tagged TRF2 fragments (lanes 3-6 and 8) that were prebound to glutathione beads. Eluted proteins were separated by SDS-PAGE, transferred to a membrane, and stained with amido black to ensure equal loading of the various TRF2 fragments. The membrane was probed with mouse anti–Pol h or rabbit anti-FEN-1 antibodies.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Binding Assay, Sequencing, Staining, Recombinant, Purification, SDS Page, Incubation, Membrane

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 4. FEN-1 incision activity in the presence of TRF2. A, FEN-1 incision of a 10-nt flap substrate. Reactions contained 120 pmol/L FEN-1 incubated with a 10-nt flap substrate (100 nmol/L) either alone (lane 2) or together with increasing TRF2 concentrations (9, 18, 90, 180, or 900 pmol/L; lanes 3-7, respectively) at 37jC for 10 minutes. The relative percent incision activity was calculated as described in Materials and Methods and normalized to the FEN-1 alone control (lane 2). Values represent the average and SD of at least three independent experiments. B, FEN-1 incision of a telomeric flap substrate. Reactions contained 10 pmol/L FEN-1 incubated with a 15-nt flap substrate harboring telomeric sequence 5V to the flap (10 nmol/L). FEN-1 was incubated alone (lane 2) or together with increasing TRF2 concentrations (100, 300, and 1,000 pmol/L; lanes 3-5) at 37jC for 10 minutes. The relative percent incision activity was calculated as in (A).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Activity Assay, Incubation, Control, Sequencing

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 5. TRF2 specifically stimulates primer extension by Pol h. A, TRF2 effects on Pol h activity. Pol h (0.5 nmol/L) was preincubated with increasing TRF2 amounts (0, 0.5, 1.5, or 3.0 nmol/L; lanes 2-5, respectively) for 5 minutes on ice. Reactions were initiated by adding the nontelomeric mix15/mix34 substrate (25 nmol/L) and were incubated for 15 minutes at 37jC. Reaction products were run on a 20% denaturing polyacrylamide gel and visualized by a Phosphorimager. Lane 1, substrate alone. E, 0.5 nmol/L (lane 6) and 3.0 nmol/L (lane 7) of heat-denatured TRF2 protein. Lanes 8 and 9, TRF2 (0.5 or 3.0 nmol/L, respectively) in the absence of Pol h. B, quantitation of Pol h primer-extension. Percent of total products with the indicated number of nucleotides incorporated was calculated as described in Materials and Methods. Columns, mean from three independent experiments; bars, SD. C, Klenow activity. Increasing Klenow concentrations (lanes 2-7) were incubated with the mix15/mix34 substrate (25 nmol/L) for 15 minutes at 37jC. Lane 1, substrate alone. Products were analyzed as in (A). D, TRF2 affects on Klenow primer extension. Klenow (0.32 nmol/L) was preincubated with increasing TRF2 amounts (0, 0.32, 0.96, 1.92, or 3.84 nmol/L; lanes 2-6, respectively) and Pol h (0.3 nmol/L) was preincubated with 1.8 nmol/L TRF2 (lane 8) for 5 minutes on ice. Reactions were initiated by adding mix15/mix34 substrate (25 nmol/L) and were incubated for 15 minutes at 37jC. Products were analyzed as in (A).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Activity Assay, Incubation, Quantitation Assay

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 6. Comparison of TRF2 stimulation of Pol h on telomeric and nontelomeric template substrates. A, reactions contained Pol h (0.25 nmol/L) alone or together with increasing TRF2 concentrations (0.125, 0.25, 0.75, and 1.5 nmol/L). Reactions were initiated by adding 25 nmol/L substrate with either nontelomeric template sequence (mix15/mix34; lanes 1-6) or telomeric template sequence (mix15/tel34; lanes 7-12) and were incubated for 15 minutes at 37jC, followed by analysis on a 20% denaturing gel. Quantitation and calculation of primer extension products for the nontelomeric (B) or telomeric (C) template substrates was as described in Materials and Methods. Columns, mean from three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Comparison, Sequencing, Incubation, Quantitation Assay

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 7. TRF2 promotion of Pol h primer extension on substrates with TRF2 binding sites. A, reactions contained Pol h (0.25 nmol/L) alone or together with increasing TRF2 concentrations (0.125, 0.25, 0.75, and 1.5 nmol/L). The reactions were initiated by adding 25 nmol/L telomeric substrate (tel21/tel40) and were incubated for 15 minutes at 37jC, followed by analysis on a 20% denaturing gel. E, 1.5 nmol/L (lane 7) heat-denatured TRF2 protein. B, the percent of products with the indicated number of nucleotides incorporated was calculated as described in Materials and Methods. Columns, mean from at least three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Binding Assay, Incubation

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 8. TRF2 stimulates Pol h strand displacement DNA synthesis on a nontelomeric BER substrate. A, schematic of the 34-bp DNA substrate containing an 8-oxo-guanine at position 17 is shown both before and after treatment with OGG1 and APE1. OGG1 removes the 8-oxo-guanine base and APE1 incises the DNA strand 5V to the resulting apurinic/apyrimidinic site. B, the substrate was pretreated with OGG1 (128 nmol/L) for 20 minutes at 37jC. The pretreated DNA (100 nmol/L) was incubated with 3.4 ng/AL APE1 for 25 minutes at 37jC together with increasing Pol h concentrations (0.6, 1.2, 2.5, and 5 nmol/L; lanes 2-5 and 6-9, respectively). The reactions in lanes 6 to 9 also contained increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L, respectively). C, the OGG1-pretreated DNA substrate was incubated with 3.4 ng/AL APE1 in the absence () or presence (+) of Pol h (5 nmol/L; lane 1) and with increasing concentrations of TRF2 (0, 5, 15, and 30 nmol/L; lanes 2-5, respectively). D, quantitation of unreacted substrate (0), short-patch (1), and long-patch (2-6) BER intermediates. E, quantitation of individual long-patch BER intermediates. Reaction products were calculated as a function of total radioactivity as described in Materials and Methods for reactions containing 5 nmol/L Pol h alone (solid columns) or together with 30 nmol/L TRF2 (hatched columns). Columns, mean of two independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: DNA Synthesis, Incubation, Quantitation Assay, Radioactivity

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 9. TRF2 stimulation of Pol h on telomeric BER substrates. A, a schematic of the 39-bp DNA substrate containing an 8-oxo-guanine at position 17 within two tandem telomeric repeats is shown both before and after treatment with OGG1 and APE1. B, the substrate was pretreated with OGG1 (128 nmol/L) for 20 minutes at 37jC. The pretreated DNA (100 nmol/L) was incubated with 3.4 ng/AL APE1 for 25 minutes at 37jC together with increasing Pol h concentrations (0.62, 1.2, 2.5, and 5 nmol/L; lanes 2-5 and 6-9, respectively). The reactions in lanes 6 to 9 also contained increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L, respectively). C, the OGG1-pretreated DNA substrate was incubated with 3.4 ng/AL APE1 in the presence (+) or absence () of Pol h (1.2 nmol/L), TRF2 (7.5 nmol/L), or FEN-1 (30 nmol/L) as indicated. E, heat-inactivated control. D, quantitation of unreacted substrate (0), short-patch (1), and long-patch (2-7) BER intermediates. E, quantitation of individual long-patch BER intermediates. Reaction products were calculated as a function of total radioactivity as described in Materials and Methods for reactions containing 1.2 nmol/L Pol h alone (solid columns) or together with 7.5 nmol/L TRF2 (hatched columns). Columns, mean of three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Incubation, Control, Quantitation Assay, Radioactivity

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 10. TRF2 enhances Pol h extension of the 3V tail of a telomeric D-loop. The telomeric D-loop substrate (25 nmol/L) was incubated with increasing Pol h concentrations alone (0.62, 1.2, 2.5, and 5 nmol/L; lanes 2-5) or together with increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L; lanes 6-9). The reactions were initiated by adding substrate and were incubated for 15 minutes at 37jC. The reaction products were run on a 20% denaturing and were visualized by a Phosphorimager.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Incubation

Journal: Cancer research

Article Title: Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

doi: 10.1158/0008-5472.CAN-19-1036

Figure Lengend Snippet: A) iPOND analysis of newly replicated DNA association with mtp53 R273H and PARP in MDA-MB-468.vector, MDA-MB-468.shp53 cells and mtp53R273H overexpression MDA-MB-468.shp53+R273H cells (left panel) and PANC-1 cells (right panel). 1 × 108 cells were labeled with 10 μM EdU for 45 min. The protein-DNA complexes were cross-linked, nascent DNA was conjugated to biotin using click chemistry, and protein-DNA complexes were purified. The eluted proteins were analyzed using western blot. A sample that did not include biotin-azide was used as a negative control. Representative of two independent experiments. B) Single cell in situ proximity ligation assay (PLA) with pulse EdU labeling showed mtp53 R273H associated with newly replicated DNA throughout S-phase. Cells were labeled with 125 μM EdU for 15 min. The protein-DNA complexes were cross-linked, nascent DNA was conjugated to biotin using click chemistry. Representative maximum intensity projection images of 2 central slices for S-phase progression of MDA-MB-468 (upper left panel) and PANC-1 (upper right panel) are shown; thickness: 2μm. PLA of mtp53 R273H/EdU (red) was performed using anti-biotin and anti-p53 antibodies. Anti-biotin IF (green) indicates cells undergoing DNA synthesis. DNA was counterstained with DAPI (blue). Analysis of mtp53/EdU foci per nuclei by CellProfiler and S-phase progression for each nucleus was manually grouped into Early, Mid, or Late S-phase using GraphPad Prism 8. Statistical analysis for MDA-MB-468 and PANC-1: * represents a p-value ≤ 0.05, ** represents a p-value ≤ 0.01, **** represents a p-value ≤ 0.0001, ns= not significant. The p-value was determined by 2-tailed student t-test. MDA-MB-468: Early: n=86, Mid: n=49, Late: n=69; PANC-1: Early: n=51, Mid: n=23, Late: n=44. Representative of three independent experiments with two technical replicates each. C) The z-stack maximum intensity projection images showed mtp53 R273H and PARP associated with newly replicated DNA in MDA-MB-468 cells. Cells were labeled with 125 μM EdU for 15 min with or without 100 μM thymidine chases for 60 min. PLA with EdU (red) was performed as in Fig. 1B. Three independent experiments were performed. D) PLA with or without 20 min incubation of 10 μM poly(ADP-ribose) glycohydrolase inhibitor (PARGi) and 10 μM EdU labeling. Maximum intensity projection images are shown. Representative of two technical replicates.

Article Snippet: Add 35ul anti-p53 DO1-AC antibody beads (Santa Cruz) or mouse IgG-AC beads to precleared lysates and incubated at 4°C for 2 hr with rotation.

Techniques: Plasmid Preparation, Over Expression, Labeling, Purification, Western Blot, Negative Control, In Situ, Proximity Ligation Assay, DNA Synthesis, Incubation

Journal: Cancer research

Article Title: Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

doi: 10.1158/0008-5472.CAN-19-1036

Figure Lengend Snippet: A) Analysis of p53/PARP1 complexes (red) by immunofluorescence microscopy in combination with in situ proximity ligation assay (PLA) in MDA-MB-468 and MCF7 cells. DNA was counterstained with DAPI (blue). The z-stack maximum intensity projection images are shown. Two independent experiments were performed. B) Analysis of fluorescent foci per cell by CellProfiler software and demonstrated by scatter plot using Prism7 GraphPad. **** represents p-value ≤ 0.0001. The p-value was determined by 2-tailed Student t-test. Two independent experiments were performed. C) p53 protein level in MCF7 and MDA-MB-468 cells. Two independent experiments were performed. D) Co-Immunoprecipitation (Co-IP) of PARP and MCM7 with mtp53 in MDA-MB-468 cells. Two independent experiments were performed. E) MTT assay showed reduction of mitochondrial activity after combination treatment 1mM temozolomide plus 10 μM talazoparib for 24hr in cells with mtp53 R273H overexpression compared to cells with endogenous mtp53. Cells were seeded at 1.25 × 105 cells per well in 12-well plates and attached overnight. Cells were treated with either 10 μM talazoparib or 1mM temozolomide, or both for 24 hrs. The absorbance was quantified by measuring the absorbance at 550 nm subtracted from the absorbance at 620 nm. All MTT data are represented as mitochondrial dehydrogenase activity as percentage of a dimethyl sulfoxide (DMSO) vehicle-treated control. Three independent experiments were performed with two technical replicates. F) Chromatin protein levels of mtp53, 53BP1 and Fibrillarin were determined by Western blot analysis with or without combination treatment Temo plus Tal in MDA-MB-468 mtp53 R273H overexpression compared to cells with endogenous mtp53. 50 μg of chromatin protein were loaded on 10% SDS-PAGE gel. Two independent experiments were performed.

Article Snippet: Add 35ul anti-p53 DO1-AC antibody beads (Santa Cruz) or mouse IgG-AC beads to precleared lysates and incubated at 4°C for 2 hr with rotation.

Techniques: Immunofluorescence, Microscopy, In Situ, Proximity Ligation Assay, Software, Immunoprecipitation, Co-Immunoprecipitation Assay, MTT Assay, Activity Assay, Over Expression, Control, Western Blot, SDS Page

Journal: Cancer research

Article Title: Gain-of-Function Mutant p53 R273H Interacts with Replicating DNA and PARP1 in Breast Cancer

doi: 10.1158/0008-5472.CAN-19-1036

Figure Lengend Snippet: A) Analysis of protein expression correlation of p53 and PARP1 in breast invasive carcinoma samples from The Cancer Genome Atlas (TCGA) database40. A total of 817 breast tumor samples were profiled and 633 cases were also profiled by reverse-phase protein array (RPPA). ER, PR and HER2 status was clinically determined by immunohistochemistry. Scatter plots showed p53 and PARP1 protein expression correlation analysis by protein level (z-score) determined by RPPA. Red: TNBC; Green: estrogen receptor–positive (ER+) patients. B) Left panel: Examples of manually scored p53 and PARP1 staining intensity categories: no staining (0), weak staining (1), moderate staining (2) and strong staining (3) at magnification 200x. Right top panel: dot plot diagram showed immunohistochemical (IHC) staining of p53 score in tissue microarray (TMA) from Basal-like, Luminal A and Luminal B subtype of breast cancer. Right lower panel: Dot plot diagram showed immunohistochemical (IHC) staining of PARP1 score in tissue microarray (TMA) from Basel-like and Luminal A subtype of breast cancer grouped by p53 level. C) Live cell imaging of MDA-468 and MCF7 cells PARPi-FL (Green). Cells were imaged by confocal microscopy after 20 min incubation of 500 nM of PARPi-FL. Hoechst staining (blue) used to visualize nuclei. Two independent experiments were performed. D) Protein levels of p53, PARP1 and PAR in breast cancer PDX model WHIM6 and WHIM25 were determined by western blot analysis. Whole cell proteins were extracted from NSG mice xenograft tumors and 50ug of protein was loaded on 10% SDS-PAGE gel. Three technical replicates were performed.

Article Snippet: Add 35ul anti-p53 DO1-AC antibody beads (Santa Cruz) or mouse IgG-AC beads to precleared lysates and incubated at 4°C for 2 hr with rotation.

Techniques: Expressing, Protein Array, Immunohistochemistry, Staining, Immunohistochemical staining, Microarray, Live Cell Imaging, Confocal Microscopy, Incubation, Western Blot, SDS Page

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 1. Effects of PAK4 overexpression and knockdown on mesenchymal transition in glioma cells. (a) Western blot analysis of 4910 and U251 glioma cells transfected with EV and PAK4-FL for 48 h along with untreated controls and GAPDH served as a loading control. (b) Representative micrographs of morphological characteristics in EV- or PAK4-FL-treated glioma cells after 48 h transfection. Scale bars: 10 μm. (c) Representative immunoblots from three independent experiments using whole cell lysates of glioma cells transfected with EV or PAK4-FL to assess changes in EMT markers. (d) Immunoblot analysis of effects of PAK4 downregulation using PAK4shRNA when compared with untreated and SV controls in 4910 and U251 cells. (e) Phase contrast micrographs of morphological characteristics of 4910 and U251 cells after SV and PAK4sh treatments for 48 h. Scale bars: 10 μm. (f) Confocal microscopy showing E-cadherin (green) and N-cadherin (red) expression in control, SV- and PAK4sh-treated cells. DAPI was used for nuclear counterstaining. Scale bars: 10 μm.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Over Expression, Knockdown, Western Blot, Transfection, Control, Confocal Microscopy, Expressing

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 2. Radiation-induced nuclear localization of PAK4 and correlation with increased EMT in glioma cells. (a) PAK mRNA levels in control and IR (8 Gy)-treated cells as determined by quantitative PCR. Fold change values of PAK4 are represented as mean ± s.d. of levels obtained from at least five repetitions in three experimental replicates (*P ⩽0.01). (b) Western blotting with cellular lysates showing PAK4 and phospho- PAK4 expression in control and IR (8 Gy)-treated cells. (c) Western blot analysis of PAK4 and phospho-PAK4 levels in cytoplasmic and nuclear fractions with or without IR treatment. GAPDH and HDAC-1 were used as loading controls for cytoplasmic and nuclear fractions respectively. Relative expression levels of cytoplasmic and nuclear PAK4 were estimated by densitometry (ImageJ 1.42) and mean ± s.d. values were presented (*P ⩽0.01). (d) Immunocytochemical analysis to assess sub-cellular localization of PAK4 in control- and IR (8 Gy)-treated cells. Nuclei were counterstained with DAPI. (e) Micrographs showing morphological changes in 4910 and U251 cells after IR treatments. Scale bars: 10 μm. (f) Western blot analysis with whole-cell lysates to assess the expression of epithelial and mesenchymal regulator proteins. (g) Confocal microscopy to examine changes in N-cadherin (red) and E-cadherin (green) levels in IR-treated cells after 48 h. Nuclei were counterstained with DAPI. Scale bars: 10 μm. (h) Assessment of total cellular ROS content in control and IR-treated cells with H2DCFDA staining as described in Materials and methods section 48 h after IR treatment. Relative ROS levels from three independent experiments are shown as mean ± s.d. (*P ⩽0.01).

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Control, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Confocal Microscopy, Staining

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 3. Association of PAK4 with PPARγ in the nuclear compartment. (a) Identification of potential PAK4 associating TFs using TF–TF Interaction Array. PAK4 was immunoprecipitated from nuclear extracts of 4910 cells with anti-PAK4 antibody and used as a bait. Isotype- specific IgG precipitates were used as negative control. PAK4 association with various TFs is identified as horizontal duplicate spots on the x-ray film. ‘+’ indicates the positive control signals. (b) 4910 cells were treated with EV and PAK4-FL for 48 h and IP experiments was performed with nuclear lysates (500 μg) from 4910 cells with specific antibodies against PAK4 and non-specific IgG followed by immunoblotting with PPARγ. Inputs indicate 10% of each pre-IP samples. (c) PPARγ IP using anti-FLAG and anti-IgG antibody from nuclear lysates of 4910 cells at 48 h post transfection with EV or FLAG-PPARγ constructs followed by immunoblotting for PAK4. (d) IP experiments using 4910 lysates with either a PAK4 specific antibody or non-specific IgG followed by immunoprobing for PPARγ. Reciprocal IPs were performed with anti-PPARγ antibody and subsequent immunoblotting with PAK4 to confirm PAK4/PPARγ association in the nucleus. (e) Identification of minimal PPARγ- interaction domain of PAK4 using bacterially expressed GST, and GST-PPARγ purified using MagneGST Pull-Down System following manufacturer’s protocol. Biotin-labeled PAK4 truncated mutants (1–290aa, 291–591aa and 1–591aa) were incubated with GST-PPARγ, separated on 10% SDS–PAGE and detected as described in Materials and methods section (Top panel). Inputs (10% samples) were analyzed by SDS–PAGE (Bottom panel). (f) Schematic representation of mapping PPARγ interacting domain on PAK4 using different truncation mutants. CRIB: Cdc42-and Rac-interactive binding motif; GID: GEF-H1 Interaction Domain; KD: Kinase Domain. (g) EMSA was performed with 4910 Nuclear extracts (5 μg) to detect PPARγ activity. For the supershift analyses, specific antibodies against PAK4 and PPARγ were incubated with control sample before adding the biotin-labeled probe.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Immunoprecipitation, Negative Control, Positive Control, Western Blot, Transfection, Construct, Labeling, Incubation, SDS Page, Binding Assay, Activity Assay, Control

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 4. Radiation-enhanced PAK4/PPARγ binding in nucleus. (a) IP with antibodies against non-specific IgG and PPARγ using nuclear lysates prepared from control and IR (8 Gy)-treated 4910 and U251 cells followed by immunoprobing with PAK4 antibody (Top panels). Reciprocal IPs were performed with anti-IgG and anti-PAK4, and subsequently immunoprobed with PPARγ to confirm changes in radiation-induced PAK4/ PPARγ interaction in these cells (Bottom panels). Representative blots from three independent experiments are shown. (b) Sub-cellular localization analyses of PAK4 (green) and PPARγ (red) by confocal microscopy in control and IR-treated cells. Scale bars: 10 μm. (c) Protein-DNA array (version II) interaction analysis performed with immunoprecipitated PAK4 from 4910 nuclear extracts as described in Materials and methods section. PAK4 binding with TF consensus sequences was detected by duplicate spots on the membrane. ‘+’ indicates positive control signal.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Binding Assay, Control, Confocal Microscopy, DNA Array, Immunoprecipitation, Membrane, Positive Control

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 5. Radiation-induced PAK4/PPARγ recruitment on the PPARγ-binding site on Nox1 promoter. (a) Real-time PCR showing Nox1 transcriptional levels in control- and IR-treated cells. The fold change values are represented as mean ± s.d. (n = 5) obtained from at least three independent experiments (*P ⩽0.01). (b) Immunoblotting shows Nox1 expression with GAPDH served as an internal control. (c) Whole-cell lysates were subjected immunoblotting and representative blots from three independent experiments were shown. (d) Schematic representation of putative PPARγ binding sites on Nox1 promoter. Seven putative PPRE sites were identified located in the promoter (4 sites), exon-1 (1 site) and intron-1 (2 sites) of human Nox1 based on analysis of a 2.9-kb 5ʹ-flanking region of Nox1 (GenBank: ABC40742.1). ChIP primers specific for R-1, R-2, R-3 and R-4 regions (blue arrows) were used to determine PPARγ recruitment on Nox1 promoter. (e) ChIP analysis of PPARγ occupancy around PPREs on the Nox1 promoter using DNA from 4910 and U251 cells and IP with anti-IgG and anti-PPARγ antibodies with and without IR treatment. 5% of pre-ChIP DNA samples served as input controls for each sample. ChIP DNA from control and IR-treated cells were analyzed by quantitative PCR using ChIP-specific primers covering Nox1 promoter regions (R-1 to R-4). ChIP amplification is shown as percent input from three different experiments (n = 5; *P ⩽0.05, **P ⩽0.01). (f) ChIP assay was performed with R-2 primers and antibodies against non-specific IgG and PPARγ using ChIP DNA as described above in both 4910 and U251 cells. Subsequently, re-ChIP assay was performed using primary ChIP amplicons with anti-IgG and anti-PAK4 antibodies; results from three experimental replicates are shown.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Control, Western Blot, Expressing

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 6. Role of PAK4 in the regulation of PPARγ-mediated Nox1 and EMT in glioma cells. (a) Cells were subjected to SV and PAK4sh for 24 h and subsequently treated with IR for an additional 24 h. At the end of the treatments, whole-cell lysates were subjected to western blotting with GAPDH as internal loading control. (b) Cells were treated with EV and PPARγ-FL for 24 h followed by treatment with PAK4sh for an additional 24 h. Western blotting was performed with whole cell lysates; representative blots from three independent experiments are shown. (c) 4910 cells were treated independently with SV-control or PAK4sh or GW9662 (10 μM) or IR (8 Gy) or with combinations of SV+IR, PAK4sh+IR and GW9662+IR for 48 h. Total ROS levels were estimated as described in Materials and methods section and are presented as mean ± s.d. from three experimental replicates (*P ⩽0.01).

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Western Blot, Control

Journal: Oncogene

Article Title: A novel interaction of PAK4 with PPARγ to regulate Nox1 and radiation-induced epithelial-to-mesenchymal transition in glioma.

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: Figure 7. Effect of PAK4 downregulation on orthotopic tumor growth in nude mice. (a) Paraffin-embedded brain tumor sections were stained and tumor volumes were measured as described in Materials and methods section. Relative tumor size is shown as mean ± s.d. obtained from different groups as indicated (n = 6; *P ⩽0.05, **P ⩽0.01). (b) Immunohistochemical analysis of brain tumors from nude mice that were intracranially implanted with SV or PAK4sh cells and subjected to IR treatments as described in Materials and methods section; representative micrographs are shown. Inset: staining with Non-specific IgG. (c) Confocal microscopy was performed in tumor sections to determine N-cadherin (red) and E-cadherin expression (green) levels. (d) Schematic diagram represents the radiation-induced PAK4 nuclear translocation, binding with PPARγ and co-recruitment of PAK4/PPARγ complex on to Nox1 promoter, which further results in Nox1 transactivation, ROS generation and EMT induction in glioma cells.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD, USA), kinase-dead PAK4 plasmid (PAK4K350M),64 PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa; PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549),65 FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895)66 and respective empty vector controls were used in the study.

Techniques: Staining, Immunohistochemical staining, Confocal Microscopy, Expressing, Translocation Assay, Binding Assay

Journal: JCI Insight

Article Title: CCL3 in the bone marrow microenvironment causes bone loss and bone marrow adiposity in aged mice

doi: 10.1172/jci.insight.159107

Figure Lengend Snippet: ( A ) Representative image of antibody array against CCL family members in peripheral blood serum from young and aged mice. ( B ) Levels of CCL family members in peripheral blood serum from young and aged mice ( n = 12). ( C ) CCL3 level in femur bone marrow of young and aged mice ( n = 12). ( D ) Correlation analysis of serum with bone marrow CCL3 levels. ( E ) CCL3 expression in femur bone marrow of young and aged mice and quantification of CCL3 + cells. Scale bars: 40 μm (original photo), 10 μm (zoomed-in photo). ( F ) Correlation analysis of serum CCL3 levels with BV/TV and Ad.Ar/Ma.Ar of femur. ( G ) Correlation analysis of CCL3 levels in bone marrow with BV/TV and Ad.Ar/Ma.Ar. ( H ) CCL3 secretion levels in in vitro 24-hour culture supernatant of HSCs, B cells, BMSCs, and BMAT ( n = 12). CCL3 mRNA and protein expression in HSCs ( I ), BMAT ( J ), B cells ( K ), and BMSCs ( L ) from young and aged mice ( n = 12). All data were obtained from 3 independent experiments. The images and numerical data are representative. Data are presented as mean ± SD; 1-way ANOVA in H ; Pearson’s correlation was used to obtain r values in D , F , and G ; Student’s t test was used in the other experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For various purposes, BMSCs were treated in vitro with neu CCL3 antibody (0.5 μg/mL; R&D Systems, Bio-Techne), recombinant mouse CCL3 protein (5 ng/mL; R&D Systems, Bio-Techne), AG490 (50 μM; MilliporeSigma), 5′-aza (10 μM; MilliporeSigma), or DKK1 neutralization antibody (10 μg/mL; AF1096, R&D Systems, Bio-Techne).

Techniques: Ab Array, Expressing, In Vitro

Journal: JCI Insight

Article Title: CCL3 in the bone marrow microenvironment causes bone loss and bone marrow adiposity in aged mice

doi: 10.1172/jci.insight.159107

Figure Lengend Snippet: ( A ) Femur length of young and aged wild-type (WT) and Ccl3 –/– mice ( n = 12). ( B ) BV/TV, Tb.N, Tb.Th, and Tb.Sp of distal femur from young and aged WT and Ccl3 –/– mice ( n = 12). ( C ) Representative micro-CT 3D reconstruction images. Scale bar: 500 μm. ( D ) Ct.Ar/Tt.Ar and Ct.Th of femur midshaft from young and aged WT and Ccl3 –/– mice ( n = 12). ( E ) Representative micro-CT 3D reconstruction images. Scale bar: 500 μm. ( F ) Double calcein labeling images and BFR quantification in femur of young and aged WT and Ccl3 –/– mice ( n = 12). Scale bar: 50 μm. ( G ) Quantification of maximum load and stiffness in 3-point bending test in femur of young and aged WT and Ccl3 –/– mice ( n = 12). ( H ) Quantification of Ad.Ar/Ma.Ar and Ad.N/Ma.Ar on the basis of Oil Red O staining of young and aged WT and Ccl3 –/– mice femurs ( n = 12). Scale bar: 500 μm. All data were obtained from 3 independent experiments. The images and numerical data are representative. Data are presented as mean ± SD; 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For various purposes, BMSCs were treated in vitro with neu CCL3 antibody (0.5 μg/mL; R&D Systems, Bio-Techne), recombinant mouse CCL3 protein (5 ng/mL; R&D Systems, Bio-Techne), AG490 (50 μM; MilliporeSigma), 5′-aza (10 μM; MilliporeSigma), or DKK1 neutralization antibody (10 μg/mL; AF1096, R&D Systems, Bio-Techne).

Techniques: Micro-CT, Labeling, Staining

Journal: JCI Insight

Article Title: CCL3 in the bone marrow microenvironment causes bone loss and bone marrow adiposity in aged mice

doi: 10.1172/jci.insight.159107

Figure Lengend Snippet: ( A ) CCL3 mRNA and protein expression when BMSCs undergo adipogenic differentiation ( n = 12). ( B ) Luciferase activity of CCL3 promoter deletion mutant–driven luciferase reporter gene vector in BMSCs undergoing adipogenic differentiation ( n = 12). ( C ) ChIP assay using C/EBPα antibody against the putative C/EBPα binding site within CCL3 promoter when BMSCs undergo adipogenic differentiation ( n = 12). ( D ) DNA methylation percentage of 11 CG sites within proximal CCL3 promoter in BMSCs ( n = 12). ( E ) DNA methylation percentage of 11 CG sites within proximal CCL3 promoter in BMSCs with adipogenesis induction ( n = 12). ( F ) Luciferase activity of methylated and unmethylated CCL3 promoter–driven luciferase reporter gene vector in BMSCs undergo adipogenic differentiation ( n = 12). ( G ) ChIP assay using C/EBPα antibody against the putative C/EBPα binding site within the CCL3 promoter in BMSCs treated with 5′-aza. ( H ) Ccl3 mRNA expression in response to 5′-aza in young BMSCs ( n = 12). ( I ) Oil Red O staining of BMSCs undergoing adipogenic differentiation with 5′-aza treatment. ( J ) mRNA expression when BMSCs undergo adipogenic differentiation with 5′-aza treatment ( n = 10). ( K ) ChIP assay using C/EBPα antibody against the putative C/EBPα binding site within the CCL3 promoter of aged BMSCs when Dnmt3a was overexpressed. ( L ) Ccl3 mRNA expression in response to Dnmt3a overexpression in aged BMSCs ( n = 12). (M) Oil Red O staining of aged BMSCs undergoing adipogenic differentiation with Dnmt3a overexpression. ( N ) mRNA expression of Pparγ, C/ebpα, aP2, and Glut4 when aged BMSCs undergo adipogenic differentiation with Dnmt3a overexpression ( n = 10). All data were obtained from 3 independent experiments. Statistics, Student’s t test ( D ); 1-way ANOVA ( F ); 2-way ANOVA ( A , B , E , H , J , L , and N ). * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bar: 10 μm ( I and M ).

Article Snippet: For various purposes, BMSCs were treated in vitro with neu CCL3 antibody (0.5 μg/mL; R&D Systems, Bio-Techne), recombinant mouse CCL3 protein (5 ng/mL; R&D Systems, Bio-Techne), AG490 (50 μM; MilliporeSigma), 5′-aza (10 μM; MilliporeSigma), or DKK1 neutralization antibody (10 μg/mL; AF1096, R&D Systems, Bio-Techne).

Techniques: Expressing, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Binding Assay, DNA Methylation Assay, Methylation, Staining, Over Expression

Journal: JCI Insight

Article Title: CCL3 in the bone marrow microenvironment causes bone loss and bone marrow adiposity in aged mice

doi: 10.1172/jci.insight.159107

Figure Lengend Snippet: ( A ) Quantification of Ad.Ar/Ma.Ar and Ad.N/Ma.Ar via Oil Red O staining of femurs of 6-, 9-, 12-, 15-, and 18-month-old mice ( n = 8). Scale bar: 500 μm. ( B ) BV/TV, Tb.N, Tb.Th, and Tb.Sp of distal femur from aged mice with neu anti-CCL3 injection ( n = 8). ( C ) Representative micro-CT 3D reconstruction images. Scale bar: 500 μm. ( D ) Quantification of maximum load and stiffness in 3-point bending test in femur of aged mice with neu anti-CCL3 injection ( n = 8). ( E ) Quantification of Ad.Ar/Ma.Ar and Ad.N/Ma.Ar in femurs of aged mice with neu anti-CCL3 injection ( n = 8). Scale bar: 500 μm. All data were obtained from 3 independent experiments. The images and numerical data are representative. Data are presented as mean ± SD; 1-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For various purposes, BMSCs were treated in vitro with neu CCL3 antibody (0.5 μg/mL; R&D Systems, Bio-Techne), recombinant mouse CCL3 protein (5 ng/mL; R&D Systems, Bio-Techne), AG490 (50 μM; MilliporeSigma), 5′-aza (10 μM; MilliporeSigma), or DKK1 neutralization antibody (10 μg/mL; AF1096, R&D Systems, Bio-Techne).

Techniques: Staining, Injection, Micro-CT

Journal: JCI Insight

Article Title: CCL3 in the bone marrow microenvironment causes bone loss and bone marrow adiposity in aged mice

doi: 10.1172/jci.insight.159107

Figure Lengend Snippet: ( A ) Imbalance between osteogenic and adipogenic differentiation of aged BMSCs. ( B ) Positive feedback loop between CCL3 and C/EBPα during adipogenic differentiation of BMSCs. ( C ) DNA hypomethylation of CCL3 promoter in aged BMSCs to facilitate C/EBPα binding. ( D ) Inhibition of osteogenic differentiation of aged BMSCs by CCL3 through ERK-mediated DKK1 upregulation. ( E ) Comparison of young and aged bone. Pink bubbles indicate hematopoietic cells, blue bubbles indicate BMSCs, and yellow bubbles indicate adipocytes in bone marrow.

Article Snippet: For various purposes, BMSCs were treated in vitro with neu CCL3 antibody (0.5 μg/mL; R&D Systems, Bio-Techne), recombinant mouse CCL3 protein (5 ng/mL; R&D Systems, Bio-Techne), AG490 (50 μM; MilliporeSigma), 5′-aza (10 μM; MilliporeSigma), or DKK1 neutralization antibody (10 μg/mL; AF1096, R&D Systems, Bio-Techne).

Techniques: Binding Assay, Inhibition, Comparison