Journal: The Journal of Biological Chemistry

Article Title: Secreted Hsp90 Is a Novel Regulator of the Epithelial to Mesenchymal Transition (EMT) in Prostate Cancer *

doi: 10.1074/jbc.M112.389015

Figure Lengend Snippet: eHsp90 induces molecular and morphological changes consistent with an epithelial to mesenchymal transition. A, ARCaPE cells treated for the indicated times (1, 3, or 5 day (d)) with exogenous Hsp90 protein, and immunoblot analysis of epithelial E-cadherin (E-cad) and mesenchymal proteins N-cadherin (N-cad) and Twist. Phase-contrast images of cell morphology. B, effects of NPGA treatment of ARCaPM upon E- and N-cadherin expression and corresponding cell morphology. C, analysis of eHsp90 treatment of P69 nontumorigenic cells as in A. D, analysis of NPGA treatment of M12 as in B.

Article Snippet: Antibodies for N-cadherin (ab12221), Slug (ab27568), Snail (ab63371), and Vimentin (ab8978) were purchased from Abcam.

Techniques: Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Secreted Hsp90 Is a Novel Regulator of the Epithelial to Mesenchymal Transition (EMT) in Prostate Cancer *

doi: 10.1074/jbc.M112.389015

Figure Lengend Snippet: Modest elevation of eHsp90 is sufficient to suppress E-cadherin function and promote cell motility. A, upper panel, ELISA analysis of secreted eHsp90 protein detected from conditioned media collected from parental ARCaPE cells stably transduced with control (lacZ) or V5-tagged eHsp90α lentivirus. Lower panel, immunoblot detection of total (endogenous and exogenous) eHsp90α, or V5 detection of transduced eHsp90 protein. B, immunoblot analysis of cell lysates from ARCaPE-LacZ or ARCaPE-eHsp90 confirmed consistent levels of intracellular eHsp90α (IC Hsp90). Indicated analysis of E- and N-cadherin and ERK activity. C, representative morphology of the indicated ARCaPE cells. Analysis of cell motility of ARCaPE-eHsp90 either untreated or treated with NPGA. D, effect of NPGA upon E-cadherin expression in ARCaPE-eHsp90. E, analysis of E-cadherin localization in ARCaPE-LacZ and ARCaPE-eHsp90 untreated cells, or treated for the indicated times with NPGA. F, membrane localization of ZO1 in ARCaPE-LacZ and ARCaPE-eHsp90 untreated cells, or treated with NPGA for 3 days. Asterisks (*) indicate significance of p value ≤0.05. Scale bar is 50 μm.

Article Snippet: Antibodies for N-cadherin (ab12221), Slug (ab27568), Snail (ab63371), and Vimentin (ab8978) were purchased from Abcam.

Techniques: Enzyme-linked Immunosorbent Assay, Stable Transfection, Transduction, Western Blot, Activity Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Secreted Hsp90 Is a Novel Regulator of the Epithelial to Mesenchymal Transition (EMT) in Prostate Cancer *

doi: 10.1074/jbc.M112.389015

Figure Lengend Snippet: eHsp90 modulates the expression of multiple genes associated with EMT activation. A, a focused EMT qRT-PCR array was utilized to assess EMT-regulated genes modulated by eHsp90 in ARCaPE cells. Samples for array data were derived from two identical biological replicate experiments. B, transcript expression of E-cadherin and the indicated EMT transcriptional effectors from the array were validated by qRT-PCR in untreated (UT) ARCaPE or cells treated with eHsp90 protein for 1, 3, or 5 days (upper panel), whereas a similar analysis was performed for control or ARCaPE-eHsp90 genetically modified cells (lower panel). C, increased expression of proteolytic MMP transcripts was also validated from both protein-treated (upper panel) and ARCaPE-eHsp90-modified cells (lower panel). Quantitative PCR levels were normalized to GAPDH expression. UT refers to untreated vehicle control. Asterisks (*) indicate significance of p value ≤0.05.

Article Snippet: Antibodies for N-cadherin (ab12221), Slug (ab27568), Snail (ab63371), and Vimentin (ab8978) were purchased from Abcam.

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Derivative Assay, Genetically Modified, Modification, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Secreted Hsp90 Is a Novel Regulator of the Epithelial to Mesenchymal Transition (EMT) in Prostate Cancer *

doi: 10.1074/jbc.M112.389015

Figure Lengend Snippet: MMP and ERK activity are required for eHsp90-mediated motility and EMT events. A, a gelatin zymography assay was utilized to assay MMP-2/9 activity in control or ARCaPE-Hsp90 cells. For the indicated inhibitors, cells were treated for 2 days prior to media collection. Cells were treated with ERK inhibitor (UO126, 10 μm), MMP-2/9 inhibitor (SB-3CT, 1 μm), or NPGA (1 μm). B, transcript expression of E-cadherin was evaluated in ARCaPE-eHsp90 following a 3-day treatment with the following: NPGA (1 μm), pan-MMP inhibitor (GM6001, 1 μm), MMP-2/9 inhibitor (SB-3CT, 1 μm), MMP-3 inhibitor (inhibitor IV, 5 μm), or ERK inhibitor (UO126, 10 μm). C, immunoblot analysis of E-cadherin and ERK proteins in ARCaPE-eHsp90 following the time-dependent inhibition of the following: MMP-2/9, MMP-3, or ERK. D, the effect of 3 days of MMP and ERK inhibition upon E-cadherin and ZO-1 localization in ARCaPE-LacZ and ARCaPE-eHsp90 was assessed by confocal microscopy. Cells were treated as in A, with inclusion of the pan-MMP inhibitor (GM6001, 1 μm) and the MMP-3 inhibitor (inhibitor IV, 5 μm). E, evaluation of MMP and ERK in directing eHsp90 cell motility following a scratch wound assay. Scale bar is 50 μm. UT refers to untreated vehicle control. Asterisks (*) indicate significance p value ≤0.05.

Article Snippet: Antibodies for N-cadherin (ab12221), Slug (ab27568), Snail (ab63371), and Vimentin (ab8978) were purchased from Abcam.

Techniques: Activity Assay, Zymography Assay, Expressing, Western Blot, Inhibition, Confocal Microscopy, Scratch Wound Assay Assay

Journal: The Journal of Biological Chemistry

Article Title: Secreted Hsp90 Is a Novel Regulator of the Epithelial to Mesenchymal Transition (EMT) in Prostate Cancer *

doi: 10.1074/jbc.M112.389015

Figure Lengend Snippet: Detection of eHsp90 protein and regulated transcripts in human prostatectomy tumor specimens. A, prostate tissue from 2 patients was FACS sorted for eHsp90low and eHsp90high populations using a phycoerythrin-conjugated antibody specific for Hsp90α. In each instance, the subpopulation of eHsp90high cells represented ∼5% of the cell population. Patient 1 was identified as Gleason 3 + 4 (stage T3aNO), whereas Patient 2 was Gleason 4 + 5 (stage T3bNO). B, RNA was harvested from these subpopulations and transcripts for MMP-2, MMP-3, and MMP-9 were evaluated via qRT-PCR. C, proposed mechanism for eHsp90-mediated regulation of cell motility and EMT events. Tumor-secreted eHsp90 functions in an autocrine manner via its receptor LRP1 to transduce ERK phosphorylation. eHsp90-LRP1-ERK signaling subsequently initiates transcription of several pro-EMT transcription factors (Snail/Zeb/Twist), as well as MMPs. MMP activation serves to reinforce sustained ERK activation and E-cadherin suppression through several potential mechanisms (dotted arrows, see text for details). These concurrent processes deregulate junctional complexes (E-cadherin and ZO-1), resulting in a loss of cell polarity, increased migratory potential, and initiation of a subset of EMT events.

Article Snippet: Antibodies for N-cadherin (ab12221), Slug (ab27568), Snail (ab63371), and Vimentin (ab8978) were purchased from Abcam.

Techniques: Quantitative RT-PCR, Transduction, Activation Assay

Journal: Science Advances

Article Title: Cytoskeletal remodeling promotes tunneling nanotube formation and drives cardiac resident cell mitochondrial transfer in sepsis

doi: 10.1126/sciadv.adz3266

Figure Lengend Snippet: ( A ) UMAP of four subclusters of CFs in the Sham, CLP 12h, and CLP 24h groups with integrated transcriptomes. ( B ) Distribution of four subclusters of CFs with prolonged duration after sepsis UMAP. ( C ) Histogram of the proportion of four subclusters of CFs in each group. ( D ) mIHC staining images showing the expression and distribution of DAPI, Gsn, Fmo2, and Lcn2 in CFs after sepsis; n = 8 per group. Scale bars, 25 μm. ( E ) Pseudotime trajectory analysis of Fmo2+ and Lcn2+ CF subclusters (right) with significant changes after sepsis and their changes with sepsis duration (left). ( F ) HALLMARK functional analysis heatmap of Lcn2+ and Fmo2+ CFs with significant changes after sepsis. ( G ) TEM images showing the morphology of CFs in mice of the Sham or sepsis groups; n = 8 per group. Scale bars, 2 μm. ( H ) ROS detection of Fmo2+ and Lcn2+ CFs; n = 3 per group. Scale bars, 50 μm. ( I ) Mitochondrial fatty acid metabolism capacity assay of Fmo2+ and Lcn2+ CFs after a Seahorse XF24 assay and flow cytometry; n = 3 per group. ( J ) GO enrichment analysis of Lcn2+ CFs after sepsis. ( K ) Top five TFs with the largest differences in expression estimated in the postsepsis Fmo2+ and Lcn2+ CFs. Fibro, fibroblasts.

Article Snippet: After blocking with goat serum, sections were incubated overnight at 4°C with primary antibodies [Cldn5 (1:200; Cell Signaling, #66879), Metal (:200; Abcam, ab12228), Cxcl2 (1:200; Abcam, ab317569), Gsn (1:200; Proteintech, 11644-2-AP), Fmo2 (1:200; Proteintech, 67019-1-Ig), Lcn2 (1:200; Proteintech, 26991-1-AP), CD74 (1:200; Abcam, ab289885), troponin (1:200; Abcam, ab209813), CD31 (1:200; Abcam, ab182981), PDGFR (1:200; Abcam, ab203491), CD68 (1:200; Abcam, ab283654), and actin (1:200; Abcam, ab8227)].

Techniques: Staining, Expressing, Functional Assay, Flow Cytometry

Journal: Science Advances

Article Title: Cytoskeletal remodeling promotes tunneling nanotube formation and drives cardiac resident cell mitochondrial transfer in sepsis

doi: 10.1126/sciadv.adz3266

Figure Lengend Snippet: ( A ) UMAP of four subclusters of cardiac monocytes-macrophages in the Sham, CLP 12h, and CLP 24h group integrated transcriptomes. ( B ) UMAP distribution of four subclusters of cardiac monocytes-macrophages with the duration of sepsis. ( C ) Histogram of the proportion of four subclusters of cardiac monocytes-macrophages in each group. ( D ) mIHC staining images showing the expression and distribution of DAPI, CD74, Metal, and Ccr2 in cardiac monocytes-macrophages after sepsis; n = 8 per group. Scale bars, 25 μm. ( E ) Pseudotime trajectory of the occurrence and development of Ccr2+, Metal+, and Hpgd+ cardiac macrophage subclusters under normal conditions (left); pseudotime trajectory of the occurrence and development of cardiac macrophages with the prolongation of sepsis duration (right); and pseudotime trajectory of Ccr2+, Metal+, and Hpgd+ cardiac macrophage subclusters with the duration of sepsis (bottom). ( F ) HALLMARK functional analysis heatmap of Hpgd+, Metal+, and Ccr2+ cardiac macrophages with significant changes after sepsis. ( G ) Mitochondrial respiration assay of Hpgd+, Metal+, and Ccr2+ cardiac macrophages; n = 3 per group. ( H ) TEM images showing the morphology of cardiac macrophages in mice of the Sham or sepsis group; n = 8 per group. Scale bars, 2 μm. ( I ) ROS detection of Hpgd+, Metal+, and Ccr2+ cardiac macrophages; n = 3 per group. Scale bars, 50 μm. ( J ) GO enrichment analysis of Metal+ and Ccr2+ cardiac macrophages after sepsis. ( K ) Top five TFs with the largest differences in expression estimated in the postsepsis Hpgd+ and Metal+ cardiac macrophages and Lcn2+ cardiac monocytes. Macro, macrophages; Mono, monocytes.

Article Snippet: After blocking with goat serum, sections were incubated overnight at 4°C with primary antibodies [Cldn5 (1:200; Cell Signaling, #66879), Metal (:200; Abcam, ab12228), Cxcl2 (1:200; Abcam, ab317569), Gsn (1:200; Proteintech, 11644-2-AP), Fmo2 (1:200; Proteintech, 67019-1-Ig), Lcn2 (1:200; Proteintech, 26991-1-AP), CD74 (1:200; Abcam, ab289885), troponin (1:200; Abcam, ab209813), CD31 (1:200; Abcam, ab182981), PDGFR (1:200; Abcam, ab203491), CD68 (1:200; Abcam, ab283654), and actin (1:200; Abcam, ab8227)].

Techniques: Staining, Expressing, Functional Assay, Respiration Assay

Journal: Science Advances

Article Title: Cytoskeletal remodeling promotes tunneling nanotube formation and drives cardiac resident cell mitochondrial transfer in sepsis

doi: 10.1126/sciadv.adz3266

Figure Lengend Snippet: ( A ) TEM images showing the morphology of cardiomyocytes, endothelial cells, fibroblasts, and macrophages in mouse heart tissue from the Drp1 WT-CLP and Drp1 eCKO-CLP mice; n = 8 per group. Scale bars, 2 μm. ( B ) ROS detection in primary endothelial cells, fibroblasts, and macrophages from the Drp1 WT-CLP and Drp1 eCKO-CLP mice after treated with LPS; n = 3 per group. Scale bars, 50 μm. ( C ) scRNA-seq analysis of Cxcl2+ endothelial cells, Metal+ endothelial cells, Lcn2+ fibroblasts, and Metal+ macrophages in Drp1 WT-CLP and Drp1 eCKO-CLP mice with cell distribution density, UMAP, and violin plots. ( D ) GO enrichment analysis of endothelial cells, fibroblasts, and macrophages after sepsis. ( E ) Mitochondrial respiration capacity assays and basal and maximal respiratory profiling of primary endothelial cells, fibroblasts, and macrophages from Drp1 WT-CLP and Drp1 eCKO-CLP mice; n = 8 per group. ( F ) Representative images of O 2 concentration changes and O 2 flux per mass and bar graphs from Drp1 WT-CLP and Drp1 eCKO-CLP mice. Oxygen consumption capacity measured by high-resolution respirometry in complexes I and II, including CI leak, CI OXPHOS, CI + CII OXPHOS, CI + CII ET, CII ET, and CIV OXPHOS; n = 8 per group. ( G ) Ultrasound and endocardial/epicardial strain coefficients from Drp1 WT-CLP and Drp1 eCKO-CLP mice; n = 8 per group. ET, electron transfer system.

Article Snippet: After blocking with goat serum, sections were incubated overnight at 4°C with primary antibodies [Cldn5 (1:200; Cell Signaling, #66879), Metal (:200; Abcam, ab12228), Cxcl2 (1:200; Abcam, ab317569), Gsn (1:200; Proteintech, 11644-2-AP), Fmo2 (1:200; Proteintech, 67019-1-Ig), Lcn2 (1:200; Proteintech, 26991-1-AP), CD74 (1:200; Abcam, ab289885), troponin (1:200; Abcam, ab209813), CD31 (1:200; Abcam, ab182981), PDGFR (1:200; Abcam, ab203491), CD68 (1:200; Abcam, ab283654), and actin (1:200; Abcam, ab8227)].

Techniques: Concentration Assay

Journal: Biomedicines

Article Title: Implications of a Multi-Step Trigger of Retinal Regeneration in the Adult Newt

doi: 10.3390/biomedicines5020025

Figure Lengend Snippet: Change in the expression pattern of N-cadherin in RPE cells after retinectomy in vivo. ( A ) Representative N-cadherin immunoreactivity along the RPE layer in the intact eye ( n = 3). Intense immunoreactivity was observed in the region of cell–cell contact in the RPE (arrows). Lower panel: merge of triple stain. RPE65 (red): RPE cells. TO-PRO-3 (TP3; blue): nuclei. ONL: outer nuclear layer; ( B ) Representative N-cadherin immunoreactivity in the RPE sheet of the eye immediately after retinectomy (day 0) ( n = 3). Intense immunoreactivity was observed along the cell membrane which was in contact with neighboring cells. Right hand panel: merge of the triple stain; ( C ) Representative N-cadherin immunoreactivity in RPE cells at 1 day after retinectomy ( n = 5). Lower panel: merge of the triple stain. At this stage, RPE cells still lined along Bruch’s membrane. N-cadherin immunoreactivity was recognized in the region of cell–cell contact; ( D ) Representative N-cadherin immunoreactivity in RPE cells/RPE-derived mesenchymal-like cells at 3 days after retinectomy ( n = 5). Lower panel: merge of the triple stain. At this stage, cell–cell attachment in the RPE became loose but most of the cells still lay on Bruch’s membrane. In those cells, N-cadherin immunoreactivity was recognized along the cell membrane but in most cells the signal was low. Scale = 50 μm.

Article Snippet: The primary antibodies used were rabbit polyclonal anti-phospho-ERK1/2 antibody (1:150; Phospho-p44/42 MAP kinase antibody, 9101S, Cell Signaling Technology, Danvers, MA, USA; [ , ]), rabbit polyclonal anti-N-cadherin antibody (1/200; ab12221, Abcam, Cambridge, UK; [ ], mouse monoclonal anti-β-catenin antibody (1:1000; C7207, Sigma-Aldrich; Saint Louis, MO, USA, [ ]) and mouse monoclonal anti-RPE65 antibody (1:1000; MAB5428, Millipore; Billerica, MA, USA, [ ]).

Techniques: Expressing, In Vivo, Staining, Membrane, Derivative Assay, Cell Attachment Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KDM3A histone demethylase functions as an essential factor for activation of JAK2−STAT3 signaling pathway

doi: 10.1073/pnas.1805662115

Figure Lengend Snippet: KDM3A phosphorylation by JAK2 increases its demethylase activity. (A) The level of H3K9me2 was analyzed in HeLa and HEK293T cells after IL-6 treatment. (B) The level of H3K9me2 was analyzed after knockdown of JAK2 by shRNA in the absence or presence of IL-6. (C) Representative images of cells knocked down by JAK2 shRNA in the absence or presence of IL-6. The cells were stained with anti-H3K9me2 or anti-JAK2 antibodies. Nuclei were counterstained with DAPI. Arrows indicate cells expressing JAK2 and the level of H3K9me2. (Scale bar, 10 μm.) (D) Demethylation of H3K9me2 was induced by JAK2, and knockdown of KDM3A abolished JAK2-dependent demethylation of H3K9me2 in the presence of IL-6. (E) HeLa cells were knocked down by KDM3A shRNA and reconstituted with shRNA-resistant form of KDM3A WT (WTR) or YA (YAR). Transfected cells were fixed and stained for anti-H3K9me2 and anti-Flag antibodies. The cells were counterstained with DAPI to visualize cell nuclei. Arrows indicate cells expressing KDM3A and the level of H3K9me2. (Scale bar, 10 μm.) (F) Demethylation of H3K9me2 induced by IL-6 is dependent on KDM3A. Protein extracts from HeLa cells were knocked down by KDM3A shRNA and reconstituted with KDM3A WTR or YAR. Transfected cells were collected to determine the H3K9me2 levels in the absence or presence of IL-6 by immunoblot analysis. (G and H) The level of H3K9me2 was analyzed after treatment of (G) OSM (20 ng/mL) or (H) IL-11 (5 ng/mL), which share a gp130-mediated signaling molecule in HeLa cells, serum-starved for 24 h.

Article Snippet: The following antibodies were used: anti-JAK1 (#3332), anti-JAK2 (#3230), anti−phospho-STAT3 (#9131), and anti-H3K9me2 (#9753, #4658, ab1220) (Cell Signaling Technology, Abcam); anti-STAT3 (sc-8019) (Santa Cruz); anti-KDM3A (nb100-77282) (Novus); anti−phospho-Tyrosine 4G10 (05-1050) (Millipore); anti-HA (MMS-101R) (Covance); and anti-FLAG (F3165) (Sigma).

Techniques: Activity Assay, shRNA, Staining, Expressing, Transfection, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KDM3A histone demethylase functions as an essential factor for activation of JAK2−STAT3 signaling pathway

doi: 10.1073/pnas.1805662115

Figure Lengend Snippet: KDM3A functions as a transcriptional coactivator of STAT3 in JAK−STAT signaling pathway. (A) Co-IP assay was performed to detect interaction between STAT3 and KDM3A in HeLa cells with or without IL-6 treatment for 2 h. (B) ChIP assays were performed using anti-JAK2, anti−phospho-STAT3, anti−phospho-KDM3A, and anti-H3K9me2 antibodies on the MYC promoters after IL-6 treatment in HeLa cells. **P < 0.01; ***P < 0.001 (Student’s t test). (C) Quantitative RT-PCR analysis of MYC mRNA levels after knockdown of KDM3A by shRNA in HeLa cells following IL-6 treatment. ***P < 0.001 (Student’s t test). (D) Immunoblot analysis of MYC protein levels after knockdown of KDM3A by shRNA following IL-6 treatment in HeLa cells. (E) MYC mRNA levels were measured by quantitative RT-PCR in HeLa cells after rescuing resistant forms of KDM3A WTR or YAR in shRNA-mediated KDM3A knockdown cells following IL-6 treatment. *P < 0.05 (Student’s t test). (F) Immunoblot analysis in HeLa cells after rescuing resistant forms of KDM3A WTR or YAR in shRNA-mediated KDM3A knockdown cells following IL-6 treatment. (G–I) STAT-responsive M67 promoter luciferase reporter was transfected into HEK293T cells with indicated plasmids. Luciferase reporter activity was measured at 48 h after transfection and normalized by β-galactosidase activity. Values are expressed as mean ± SD for three independent experiments. *P < 0.05; **P < 0.01 (Student’s t test).

Article Snippet: The following antibodies were used: anti-JAK1 (#3332), anti-JAK2 (#3230), anti−phospho-STAT3 (#9131), and anti-H3K9me2 (#9753, #4658, ab1220) (Cell Signaling Technology, Abcam); anti-STAT3 (sc-8019) (Santa Cruz); anti-KDM3A (nb100-77282) (Novus); anti−phospho-Tyrosine 4G10 (05-1050) (Millipore); anti-HA (MMS-101R) (Covance); and anti-FLAG (F3165) (Sigma).

Techniques: Co-Immunoprecipitation Assay, Quantitative RT-PCR, shRNA, Western Blot, Luciferase, Transfection, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: KDM3A histone demethylase functions as an essential factor for activation of JAK2−STAT3 signaling pathway

doi: 10.1073/pnas.1805662115

Figure Lengend Snippet: KDM3A phosphorylation is responsible for increased cell proliferation and motility. (A) (Left) Photomicrographs from the scratch-motility assay of HeLa cells expressing KDM3A shRNA with or without IL-6 treatment. Wound closure was monitored at 24-h intervals for 48 h in HeLa cells. (Right) Cell migration (percent) was quantified by calculating the wound width. ***P < 0.001 (Student’s t test). (B) Proliferation was monitored at 6-h intervals in HeLa cells ectopically expressing either control shRNA or KDM3A shRNA. Proliferation efficiency (percent) was quantified by calculating areas of cell population as shown in the graph. ***P < 0.001 (Student’s t test). (C) Confocal images of cells stained with BrdU. The fraction of increased BrdU-positive (BrdU+) cells after IL-6 treatment decreased following knockdown of KDM3A by shRNA. HeLa cells were knocked down by KDM3A shRNA and reconstituted with an shRNA-resistant form of KDM3A WT (WTR) or YA (YAR). Nuclei were counterstained with DAPI. (Scale bar, 10 μm.) ***P < 0.001 (Student’s t test). (D) (Top) Colony formation assay of HeLa cells transfected with either control shRNA or KDM3A shRNA in combination with MYC with or without IL-6 treatment. Cells were fixed and stained with crystal violet solution. (Bottom) Colony number was quantified as shown in the graph. ***P < 0.001 (Student’s t test). (E) Schematic model depicting JAK2−KDM3A−STAT3 signaling axis. Modulation of H3K9 methylation signature by JAK2-dependent phosphorylation of KDM3A is one of the predominant epigenetic events in transcriptional regulation of STAT3 target genes.

Article Snippet: The following antibodies were used: anti-JAK1 (#3332), anti-JAK2 (#3230), anti−phospho-STAT3 (#9131), and anti-H3K9me2 (#9753, #4658, ab1220) (Cell Signaling Technology, Abcam); anti-STAT3 (sc-8019) (Santa Cruz); anti-KDM3A (nb100-77282) (Novus); anti−phospho-Tyrosine 4G10 (05-1050) (Millipore); anti-HA (MMS-101R) (Covance); and anti-FLAG (F3165) (Sigma).

Techniques: Motility Assay, Expressing, shRNA, Migration, Staining, Colony Assay, Transfection, Methylation

Journal: PLoS ONE

Article Title: Treatment with 4-Methylpyrazole Modulated Stellate Cells and Natural Killer Cells and Ameliorated Liver Fibrosis in Mice

doi: 10.1371/journal.pone.0127946

Figure Lengend Snippet: A single dose of CCl 4 (20% CCl 4 in olive oil, 2 ml/kg body weight) was administered to wild-type mice at 12 or 24 h after the treatment with 10 μg 4-MP/g of body weight. (A) Serum levels of ALT and AST. (B) Western blotting for CYP2E1 and ADH1 and the quantified data. (C) FACS analyses were performed on liver mononuclear cells from poly I:C and/or 4-MP-treated mice using antibodies against NK1.1, CD3, CD45, NKG2D and IFN-γ. (D) Cytotoxicity assays on 4 days old HSCs (D4 HSC). (E) Gene expression analyses on freshly isolated liver NK cells. The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 compared with the respective controls.

Article Snippet: Western blotting was performed with 30–50 μg protein using antibodies for β-actin (#A1978), α-SMA (#A2547) (Sigma-Aldrich, St. Louis, MO), TGF-ß1 (#3709S), phospho-pSMAD3 (#9520) (Cell Signaling, Danvers, MA), Class III ADH (#AB59134, Abcam, Cambridge, UK), ADH1 (#SC-22750, SantaCruz, Dallas, TX), and cytochrome P4502E1 (CYP2E1) (#AB1252, Merck Millipore, Darmstadt, Germany).

Techniques: Western Blot, Expressing, Isolation

Journal: PLoS ONE

Article Title: Treatment with 4-Methylpyrazole Modulated Stellate Cells and Natural Killer Cells and Ameliorated Liver Fibrosis in Mice

doi: 10.1371/journal.pone.0127946

Figure Lengend Snippet: Fibrosis was induced with serial administrations of CCl 4 and the animals were concurrently treated with 4-MP for 2 weeks. (A) Serum levels of ALT, IL-6, MCP-1, and TNF-α. (B) Sirius red staining for collagen (scale bar = 500 μm). (C) Immunohistochemical staining for α-SMA demonstrating activated HSCs (scale bar = 500 μm). (D) Western blotting for α-SMA, TGF-β1, ADH3, and CYP2E1 and the respective quantification of proteins. The data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 compared with the respective controls.

Article Snippet: Western blotting was performed with 30–50 μg protein using antibodies for β-actin (#A1978), α-SMA (#A2547) (Sigma-Aldrich, St. Louis, MO), TGF-ß1 (#3709S), phospho-pSMAD3 (#9520) (Cell Signaling, Danvers, MA), Class III ADH (#AB59134, Abcam, Cambridge, UK), ADH1 (#SC-22750, SantaCruz, Dallas, TX), and cytochrome P4502E1 (CYP2E1) (#AB1252, Merck Millipore, Darmstadt, Germany).

Techniques: Staining, Immunohistochemical staining, Western Blot

Journal: Molecular Cancer Research

Article Title: Stem Cell Marker Nestin Is Critical for TGF-β1-Mediated Tumor Progression in Pancreatic Cancer

doi: 10.1158/1541-7786.mcr-12-0511

Figure Lengend Snippet: Figure 2. Nestin increased PDAC cell migration and induced EMT in PDAC. A, representative image of nestin induced increases in cells migration of the indicated cell lines by wound-healing assays. Original magnification 400. B, transmembrane invasion assays for GFP control and nestin-overexpressing MiaPaCa-2 cells, or for PANC-1 nestin shRNA cells and eGFP shRNA control. The y-axis represents the fold change in a number of stable pool cells invaded and migrated, compared with control cells. Bars represent SD. , P < 0.01 versus control, x2 test. C, qPCR analysis of the relative mRNA levels of various EMT markers in a GFP control versus various nestin-overexpressing MiaPaCa-2 cells, or in PANC-1 shRNA control and PANC-1 shNestin cells (mean SD; n ¼3; P < 0.01). D, Western blot analysis comparing various EMT-related marker expressions either in GFP controls versus nestin-overexpressing BxPC-3, AsPC-1 (b) and MiaPaCa-2 cells, or in an shRNA eGFP versus PANC-1 nestin shRNA stable transfected cells (a). Total lysates were analyzed for protein levels of nestin, E-cadherin, N-cadherin, vimentin, SMA, and b-actin.

Article Snippet: Primary antibodies used were nestin antibodies (Sigma); smooth muscle actin (SMA, ab5698); N-cadherin (ab12221); TGF-b1 (ab66043; Abcam); E-cadherin (sc-8426); vimentin (sc32322); Smad4 (sc-7966); CD44 (sc-18849; Santa Cruz Biotechnology); and mouse anti-b-actin (Sigma).

Techniques: Migration, Control, shRNA, Western Blot, Marker, Transfection

Journal: Molecular Cancer Research

Article Title: Stem Cell Marker Nestin Is Critical for TGF-β1-Mediated Tumor Progression in Pancreatic Cancer

doi: 10.1158/1541-7786.mcr-12-0511

Figure Lengend Snippet: Figure 5. Nestin regulates Wnt signaling and is crucial for TGF-b1/Smad4–induced EMT. A, reporter assays were conducted using TOP/FOP reporters (Wnt signaling responsive reporter system) and CD44-luc reporter in the eGFP control and PANC-1-shNestin RNA cells. Data shown represent the mean SD for 3 independent experiments (, P < 0.05). B, analysis of EMT markers by Western blot analysis using vimentin and E-cadherin (E-cad) antibodies in PANC-1 shNestin knockdown and control cells, in the presence or absence of TGF-b (5 ng/mL) treatment overnight. C, morphologic and cytochemical properties of PANC-1 shNestin knockdown (iii and iv) and control cells (i and ii). PANC-1 shRNA controls cells show more epithelial cobblestone appearance (i). PANC-1 nestin shRNA knockdown cells exhibit a sheet-like appearance, and also tend to pile up to form irregular-shaped colonies (iii). E-cadherin (green) staining is increased at the cellular junctions in PANC-1 shNestin knockdown cells (16) compared with control cells (ii). Images were taken at 100 (i and iii) and 1,000 magnifications (ii and iv). D, immunofluorescent staining was conducted for E-cadherin (green) and vimentin (red) expression in PANC-1 nestin shRNA and control cells that treated with or without TGF-b1 for 24 hours. Nuclei were visualized with 40,6-diamidino-2-phenylindole (DAPI; blue). Images were taken at 400 magnifications.

Article Snippet: Primary antibodies used were nestin antibodies (Sigma); smooth muscle actin (SMA, ab5698); N-cadherin (ab12221); TGF-b1 (ab66043; Abcam); E-cadherin (sc-8426); vimentin (sc32322); Smad4 (sc-7966); CD44 (sc-18849; Santa Cruz Biotechnology); and mouse anti-b-actin (Sigma).

Techniques: Control, Western Blot, Knockdown, shRNA, Staining, Expressing

Journal: Molecular Cancer Research

Article Title: Stem Cell Marker Nestin Is Critical for TGF-β1-Mediated Tumor Progression in Pancreatic Cancer

doi: 10.1158/1541-7786.mcr-12-0511

Figure Lengend Snippet: Figure 6. Effect of nestin knockdown on in vivo tumorigenesis. A, nestin knockdown significantly decreased in vivo tumorigenesis in PANC-1 cells. Tumor weights and volumes were measured after autopsy. Mean SEM (n ¼ 6). , P < 0.01; x2 test. B, H&E and IHC staining analyses of the in vivo effects of nestin knockdown on expression patterns of EMT- related markers (E-cadherin, vimentin, and CD44) and TGF-b1 signaling molecules (Smad4 and TGF-b1). H&E images taken at 100 magnification (small inserts are 400). The photographs show representative sections of nestin, Smad4, TGF-b1, E-cadherin, vimentin, and CD44 expression levels in PANC-1 nestin shRNA and control groups, respectively. Images were taken at 400 magnifications.

Article Snippet: Primary antibodies used were nestin antibodies (Sigma); smooth muscle actin (SMA, ab5698); N-cadherin (ab12221); TGF-b1 (ab66043; Abcam); E-cadherin (sc-8426); vimentin (sc32322); Smad4 (sc-7966); CD44 (sc-18849; Santa Cruz Biotechnology); and mouse anti-b-actin (Sigma).

Techniques: Knockdown, In Vivo, Immunohistochemistry, Expressing, shRNA, Control