nfatc1 signalling Search Results


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  • 93
    Thermo Fisher nfat1
    Nfat1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore polyvinylidene difluoride membranes
    Polyvinylidene Difluoride Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex gapdh
    Effects of DZNep and GSK343 on the anticancer activity of sorafenib in HepG2 cells. (a) HepG2 cells were treated with 2.5–10 μmol/l sorafenib for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against EZH2, <t>LC3B,</t> or <t>GAPDH.</t> (b) HepG2 cells were treated with various doses of sorafenib in the absence and presence of 10 μmol/l DZNep or GSK343 for 72 h. Cell viability was analyzed using an MTT assay. * P
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    99
    Cell Signaling Technology Inc akt
    Effects of ITKi on signalling pathways, calcium flux and cytokine production in Jurkat cells. ( A ) Jurkat cells were stimulated with anti-CD3 and anti-CD28 antibodies as indicated. Westerns showing expression of <t>PLCγ1</t> and phosphorylated-PLCγ1 (p-PLCγ1), MEK1/2 and phosphorylated-MEK1/2 (p-MEK1/2) and <t>AKT</t> and phosphorylated-AKT (p-AKT). GAPDH is a loading control. Molecular weight is indicated to the left of the western. ( B ) Jurkat cells were pre-treated with 1 µM ONO00779050 (1 µM) and Ibrutinib (1 µM) for 30 minutes, and then stimulated with anti-CD3/CD28 antibodies for 6 hours. Cytoplasmic and nuclear extracts were analysed by western blot. Arrowheads indicate NFATc1. GAPDH is a loading control. Molecular weight is indicated to the left of the western. ( C ) Calcium flux changes in response to ITKi. Left hand panel shows effect of PF-06465469 (dashed line) and ibrutinib (dotted line). Right hand panel shows effect of ONO7790500 (dashed line) and BMS509744 (dotted line). For each panel the no drug control is indicated by a solid line. The time of addition of anti-CD3 and anti-CD28 antibodies and ionomycin are shown. ( D ) IL-2 (left-hand panel) and IL-21 (right-hand panel) production in response to phytohaemagglutinin and phorbol myristate (PHA/PMA). Results are mean ± SEM. n = 4. IL-2 and IL-21 production were significantly (two-tailed unpaired t-test) repressed by ibrutinib, PF-6465469, BMS509744 and ONO7790500.
    Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 48747 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex β actin
    Expression level and number of motoneurons in wild-type and homozygous shaky mice. (A) Western blot analysis of GlyR α1 subunit protein expression in whole brain and spinal cord homogenates. Normalization with <t>β-actin</t> showed no significant differences of GlyR α1 protein levels between the two genotypes ( n = 3), right image shows an example of the Western blot from two different animals of each genotype ( Glra1 +/+ and Glra1 sh / sh ). (B) Quantitative analysis of motoneuron numbers. The quantification was made using 3–4 animals of each genotype ( Glra1 +/+ , n = 4; Glra1 sh / sh , n = 3). Right images are examples of brain stem sections from Glra1 +/+ and Glra1 sh / sh with arrow heads pointing to motoneurons. Error bars represent standard deviations (S.D.).
    β Actin, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 771 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex anti gapdh
    <t>LDOC1</t> mediates the in vitro malignancy progression in lung cancer cell lines. ( A ) Expression of LDOC1 mRNA and protein were stably suppressed in the A549-derived cell lines. The A549 cell line was transducing lentivirus carrying either scramble control vector (A549-shCtrl) or siRNA targeting LDOC1 (A549-shLDOC1). ( B ) Restoration of LDOC1 expression in A549-shLDOC1-derived cells. A549-shLDOC1 was only transducing GFP-tagged lentivirus vectors (A549-sh-rCtrl) or those fused with an LDOC1 open reading frame (A549-sh-rLDOC1). Expression of LDOC1 mRNA (left) and protein (right) was measured using qPCR and Western blotting analysis, respectively. ( C – F ) Cell proliferation ( C , by MTT), cell cycle progression ( D , by BrdU incorporation assay), and cell invasion ( E , F by Matrigel coated trans-well invasiveness assay) were assessed in the parental A549 cells and the generated A549-derived cell lines ( C – E ) or H1299 ectopic-expressing LDOC1 ( F ). H1299 cells were transduced with lentivirus carrying either open reading frame (ORF) of LDOC1 or empty vector (V, controls). Expression of LDOC1 and <t>GAPDH</t> proteins were measured using Western blotting analysis ( F , upper panels). The number of invading cells was the average of four independent experiments; data were analyzed using a Student’s t -test., where * p
    Anti Gapdh, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc c fos
    PPD inhibited osteoclast differentiation by the NF-κB pathway. (A–C) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Nuclear protein was isolated for western blot analysis. Cell lysates were analyzed using western blotting with specific antibodies against NF-κB and p-NF-κB were quantified and were normalized to <t>H3</t> using ImageJ software. (D) RAW264.7 cells that had been stably transfected with an NF-κB luciferase reporter construct were treated with the indicated concentrations of PPD for 1 h, followed by incubation in the absence or presence of RANKL for 8 h. Luciferase activity was measured using the Promega Luciferase Assay System. All experiments were performed at least 3 times; (E) BMDMs were cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL with or without 5 μM PPD for 0–5 days. Cell lysates were prepared and analyzed using western blotting. (F,G) The gray levels corresponding to the indicated proteins were quantified and normalized relative to β-actin using Image J for <t>c-fos</t> and NFATc1. Significant differences between the groups were determined by paired Student’s t -test. ∗ P
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    92
    GeneTex rad51
    HR is increased in T4-2 malignant breast epithelial cells compared to that in S1 cells. (A and B) T4-2 and S1 cells were irradiated with 6 Gy X rays, fixed post-IR, and coimmunostained with 53BP1 and BRCA1 antibodies. (A) 53BP1 and BRCA1 staining; (B) histogram of BRCA1 foci. (C and E) Cells were treated with 6 Gy X rays, fixed post-IR, and immunostained for RPA70 (C) and <t>Rad51</t> (E) antibodies. (D and F) Quantification of focus formation. (D) Histogram of > 20 RPA70 foci; (F) histogram of > 10 Rad51 foci. (B, D, and F) Columns represent the means ( n = 3), and bars represent the SDs; *, P
    Rad51, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc β actin
    HR is increased in T4-2 malignant breast epithelial cells compared to that in S1 cells. (A and B) T4-2 and S1 cells were irradiated with 6 Gy X rays, fixed post-IR, and coimmunostained with 53BP1 and BRCA1 antibodies. (A) 53BP1 and BRCA1 staining; (B) histogram of BRCA1 foci. (C and E) Cells were treated with 6 Gy X rays, fixed post-IR, and immunostained for RPA70 (C) and <t>Rad51</t> (E) antibodies. (D and F) Quantification of focus formation. (D) Histogram of > 20 RPA70 foci; (F) histogram of > 10 Rad51 foci. (B, D, and F) Columns represent the means ( n = 3), and bars represent the SDs; *, P
    β Actin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 38293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex α tubulin
    MPT0G211 sensitized MOLT-4 cells to vincristine-mediated mitotic arrest. a Cell cycle distributions of cells exposed to MPT0G211, tubastatin A (TBA), vincristine (VCR), or the indicated combination therapy for 24 h. A statistical analysis of the proportions of cells in the G2/M phase is shown in the right panel. b The levels of the apoptotic proteins caspase 3 and poly-ADP ribose polymerase (PARP) and the pro-survival proteins BCL-XL, BCL-2, and survivin were determined in cells treated with MPT0G211 (3 μM) or TBA (3 μM) in combination with VCR (1 nM) for 24 h. c The protein levels of cyclin B1, aurora B, p-CDC2, p-PLK, p-Histone 3, and MPM2 were evaluated following treatment with a combination of MPT0G211 or TBA with VCR for 24 h. d Cells were co-treated with MPT0G211 or TBA with VCR for 24 h and incubated with an <t>α-tubulin</t> antibody or DAPI. Microtubule dynamics were evaluated using a ZEISS LS 510META confocal microscope (magnification × 630). Scale bar = 20 μM. e Antitumor activity of MPT0G211 plus vincristine in a MOLT-4 xenograft model. When the tumor size reached 200 mm 3 , mice were injected with vehicle, vincristine (1 mg/kg, i.p., qwk), and MPT0G211 (30 mg/kg, i.p., qd) alone or a combination of both. The curves of tumor growth volume were expressed as mean ± SEM. f Changes of body weight after treatment. Data are shown as means ± standard errors of the means. * p
    α Tubulin, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 259 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc iκbα
    RA specifically inhibited SRC/AKT signaling pathways during osteoclastogenesis. a RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 10, or 30 min, respectively. Western blotting for p-AKT was analyzed with the cell lysates. b RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 1, 3, or 5 days respectively. Western blotting for SRC was analyzed with the cell lysates. c RAW264.7 cells were treated with or without RA (0.8 μM) with the addition of RANKL (50 ng/mL) for 0, 10 or 30 min, respectively. Western blotting for MAPK and <t>IκBα</t> signaling pathways was analyzed with the cell lysates
    Iκbα, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p84  (GeneTex)
    91
    GeneTex p84
    H3K36me3 increases across the early region of the HPV31 genome. ). To control for differences in viral copy number, PCR data was normalized to input values quantified in parallel for each experiment. The average fold change is graphed and the enrichment is expressed as percent input relative to the first primer set, which is set to one. Averages shown are representative of three independent experiments. Error bars represent means +/- standard error. (B) Western blot analysis was performed using antibodies to SETD2 and to involucrin and K10 as differentiation controls. <t>p84</t> served as a loading control. Ca = calcium.
    P84, supplied by GeneTex, used in various techniques. Bioz Stars score: 91/100, based on 202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho akt
    RA specifically inhibited <t>SRC/AKT</t> signaling pathways during osteoclastogenesis. a RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 10, or 30 min, respectively. Western blotting for p-AKT was analyzed with the cell lysates. b RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 1, 3, or 5 days respectively. Western blotting for SRC was analyzed with the cell lysates. c RAW264.7 cells were treated with or without RA (0.8 μM) with the addition of RANKL (50 ng/mL) for 0, 10 or 30 min, respectively. Western blotting for MAPK and IκBα signaling pathways was analyzed with the cell lysates
    Phospho Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 16290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex tsg101
    Expression levels of <t>TSG101</t> and Rab35 are lower in aged APOE4 mouse brains compared with age-matched APOE3 mice. ( A ) Representative western blot analyses of mouse hemibrain homogenates are shown for ALIX, TSG101, and Rab35, with β-actin used as a loading control. ( B – D ) Quantification of bands normalized to levels of β-actin is shown for levels of ALIX ( B ) , TSG101 ( C ), and Rab35 ( D ) . ( E ) Quantitative PCR analysis of 12-month-old mouse hemibrains shows Pdcd6ip (encodes ALIX), Tsg101 , and Rab35 transcript levels normalized to ddCT levels of the housekeeping gene Sdha . Data are expressed as the mean ± SEM of the ratios of APOE4 to APOE3 . * P
    Tsg101, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 480 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc nfatc2
    <t>NFATC2</t> is hypermethylated in patient fibroblasts. NFATC2 methylation in cells from patients with severe radiotherapy side-effects (n = 16) and controls (n = 8) were investigated on genome-wide methylation analysis. CpG sites in bold are differentially methylated between control and patient cells. CpG sites in red are part of promoter-associated regions defined by the ENCODE consortium.
    Nfatc2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc nf κb p65
    <t>NFATC2</t> is hypermethylated in patient fibroblasts. NFATC2 methylation in cells from patients with severe radiotherapy side-effects (n = 16) and controls (n = 8) were investigated on genome-wide methylation analysis. CpG sites in bold are differentially methylated between control and patient cells. CpG sites in red are part of promoter-associated regions defined by the ENCODE consortium.
    Nf κb P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5030 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex anti β actin
    Micro-Western Array to screen for signaling molecules involved in H . capsulatum -induced macrophage response. Macrophages from WT mice were stimulated with or without (0 min) HK H . capsulatum for 15, 30, 60, 90, and 120 min. (A) Heat map chart shows MWA results. Protein abundance was normalized against the mean of <t>β-actin</t> and GAPDH. Black color indicates no change, while red and green indicate increase and decrease, respectively, of the levels of protein compared to unstimulated control. Proteins below the level of detection are in grey. (B) Cell lysates were subjected to Western blotting. Beta-actin was used as an internal control. Data shown are representative of 3 independent experiments with similar results.
    Anti β Actin, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex e cadherin
    Knockdown of IQGAP1 inhibited proliferation and EMT of thyroid cancer cells. Notes: SW579 and TPC-1 cells transfected with si-control, si-IQGAP1-1 or si-IQGAP1-2 were cultured for 48 h. ( A ) qRT-PCR analysis of IQGAP1 mRNA level in transfected cells. ( B ) Western blot analysis of IQGAP1 protein in transfected cells. ( C ) MTT assay was performed to detect viability in transfected cells. ( D and E ) Western blot analysis of <t>E-cadherin,</t> N-cadherin, Vimentin and Twist in transfected cells. * P
    E Cadherin, supplied by GeneTex, used in various techniques. Bioz Stars score: 92/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p38
    PPD inhibited osteoclast differentiation by specifically impairing RANKL-induced MAPK cascades and the NF-κB pathway. (A,E) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Cell lysates were analyzed using western blotting with specific antibodies against <t>phospho-p38,</t> p38, phospho-ERK1/2, ERK1/2, phospho-JNK1/2, JNK1/2, phospho-IκBα, IκBα, phospho-TAK1, TAK1, and β-actin. (B–D) The gray levels corresponding to phosphorylation of the indicated proteins were quantified and normalized relative to β-actin using Image J for p-p38, p-JNK, and p-ERK, (F,G) The gray levels corresponding to phosphorylation of the indicated proteins were quantified and normalized relative to β-actin using Image J for p-TAK1 and p-IκBα. Significant differences between the groups were determined by paired Student’s t -test. ∗ P
    P38, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 14453 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GeneTex tet1
    H19 regulates TGFBR2 and TSP1 expression in a <t>TET1‐dependent</t> manner. A, HUVECs were transfected with siCon, siH19, or siH19 plus pTET1 (human TET1‐expressing plasmid). RNA and protein were extracted 48 and 72 hours later, respectively, and analyzed by RT‐qPCR (A) and Western blot (B). * P
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    GeneTex usp19
    Ubiquitin‐specific protease 19 <t>(USP19)</t> expression is increased in murine hypertrophic hearts and cardiomyocytes. A, Left, Western blot bands of USP19 in mice subjected to sham or transverse aortic constriction (TAC) surgery at 4 wk. Right, protein expression levels were normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and compared between indicated groups (n = 3, ** P < 0.01 vs sham). B, Left, Western blot analysis of USP19 in neonatal rat cardiomyocytes treated with phosphate buffered saline (PBS) or phenylephrine (PE) at 24 h. Right, protein expression levels were normalized to GAPDH and compared between indicated groups (n = 3, ** P < 0.01 vs PBS)
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    94
    GeneTex immunostaining
    Ubiquitin‐specific protease 19 <t>(USP19)</t> expression is increased in murine hypertrophic hearts and cardiomyocytes. A, Left, Western blot bands of USP19 in mice subjected to sham or transverse aortic constriction (TAC) surgery at 4 wk. Right, protein expression levels were normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and compared between indicated groups (n = 3, ** P < 0.01 vs sham). B, Left, Western blot analysis of USP19 in neonatal rat cardiomyocytes treated with phosphate buffered saline (PBS) or phenylephrine (PE) at 24 h. Right, protein expression levels were normalized to GAPDH and compared between indicated groups (n = 3, ** P < 0.01 vs PBS)
    Immunostaining, supplied by GeneTex, used in various techniques. Bioz Stars score: 94/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam nfatc1
    PPD inhibited osteoclast differentiation by the NF-κB pathway. (A–C) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Nuclear protein was isolated for western blot analysis. Cell lysates were analyzed using western blotting with specific antibodies against NF-κB and p-NF-κB were quantified and were normalized to H3 using ImageJ software. (D) RAW264.7 cells that had been stably transfected with an NF-κB luciferase reporter construct were treated with the indicated concentrations of PPD for 1 h, followed by incubation in the absence or presence of RANKL for 8 h. Luciferase activity was measured using the Promega Luciferase Assay System. All experiments were performed at least 3 times; (E) BMDMs were cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL with or without 5 μM PPD for 0–5 days. Cell lysates were prepared and analyzed using western blotting. (F,G) The gray levels corresponding to the indicated proteins were quantified and normalized relative to β-actin using Image J for c-fos and <t>NFATc1.</t> Significant differences between the groups were determined by paired Student’s t -test. ∗ P
    Nfatc1, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of DZNep and GSK343 on the anticancer activity of sorafenib in HepG2 cells. (a) HepG2 cells were treated with 2.5–10 μmol/l sorafenib for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against EZH2, LC3B, or GAPDH. (b) HepG2 cells were treated with various doses of sorafenib in the absence and presence of 10 μmol/l DZNep or GSK343 for 72 h. Cell viability was analyzed using an MTT assay. * P

    Journal: Anti-Cancer Drugs

    Article Title: S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells

    doi: 10.1097/CAD.0000000000000166

    Figure Lengend Snippet: Effects of DZNep and GSK343 on the anticancer activity of sorafenib in HepG2 cells. (a) HepG2 cells were treated with 2.5–10 μmol/l sorafenib for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against EZH2, LC3B, or GAPDH. (b) HepG2 cells were treated with various doses of sorafenib in the absence and presence of 10 μmol/l DZNep or GSK343 for 72 h. Cell viability was analyzed using an MTT assay. * P

    Article Snippet: Materials LC3B, p62, LAMP2, trimethylated histone H3K27, GAPDH, and β-actin antibodies were purchased from GeneTex (Hsinchu, Taiwan).

    Techniques: Activity Assay, Western Blot, MTT Assay

    Effects of DZNep and GSK343 on the cell viability of MDA-MB-231 cells. (a) Chemical structures of DZNep and GSK343. (b) MDA-MB-231 cells were treated with different doses of DZNep or GSK343 for 72 h, and cell viability was analyzed using an MTT assay. (c) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against H3K27-me3 or GAPDH. (d) MDA-MB-231 cells were treated with 10 and 20 μmol/l DZNep or GSK343, or 0.75 mol/l doxorubicin (DOXO) for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against PARP1, caspase-3, LC3B, or GAPDH. DZNep, 3-deazaneplanocin A; EZH2, enhancer of zeste homolog 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SAH, S -adenosyl- l -homocysteine; SAM, S -adenosyl- l -methionine.

    Journal: Anti-Cancer Drugs

    Article Title: S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells

    doi: 10.1097/CAD.0000000000000166

    Figure Lengend Snippet: Effects of DZNep and GSK343 on the cell viability of MDA-MB-231 cells. (a) Chemical structures of DZNep and GSK343. (b) MDA-MB-231 cells were treated with different doses of DZNep or GSK343 for 72 h, and cell viability was analyzed using an MTT assay. (c) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against H3K27-me3 or GAPDH. (d) MDA-MB-231 cells were treated with 10 and 20 μmol/l DZNep or GSK343, or 0.75 mol/l doxorubicin (DOXO) for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against PARP1, caspase-3, LC3B, or GAPDH. DZNep, 3-deazaneplanocin A; EZH2, enhancer of zeste homolog 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SAH, S -adenosyl- l -homocysteine; SAM, S -adenosyl- l -methionine.

    Article Snippet: Materials LC3B, p62, LAMP2, trimethylated histone H3K27, GAPDH, and β-actin antibodies were purchased from GeneTex (Hsinchu, Taiwan).

    Techniques: Multiple Displacement Amplification, MTT Assay, Western Blot

    Effects of DZNep and GSK343 on autophagy of MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with 10 and 20 μmol/l DZNep or GSK343 for 24 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or GAPDH. (b) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and 20 nmol/l bafilomycin was added 4 h before cells were harvested. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, p62, LAMP2, or GAPDH. (c) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and then stained by Cyto-ID autophagy detection reagent. The Cyto-ID fluorescence was analyzed by fluorescence microscopy. (d) MDA-MB-231 cells were treated with 10 μmol/l berberine (BBR), 2 μmol/l SAHA, 1 μmol/l doxorubicin (DOXO), 1 μmol/l taxol, 10 μmol/l VP-16, 10 μmol/l GSK343 (GSK), or 10 μmol/l UNC1999 (UNC) for 48 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or β-actin. (e) The chemical structure of UNC1999. DZNep, 3-deazaneplanocin A.

    Journal: Anti-Cancer Drugs

    Article Title: S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells

    doi: 10.1097/CAD.0000000000000166

    Figure Lengend Snippet: Effects of DZNep and GSK343 on autophagy of MDA-MB-231 cells. (a) MDA-MB-231 cells were treated with 10 and 20 μmol/l DZNep or GSK343 for 24 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or GAPDH. (b) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and 20 nmol/l bafilomycin was added 4 h before cells were harvested. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, p62, LAMP2, or GAPDH. (c) MDA-MB-231 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and then stained by Cyto-ID autophagy detection reagent. The Cyto-ID fluorescence was analyzed by fluorescence microscopy. (d) MDA-MB-231 cells were treated with 10 μmol/l berberine (BBR), 2 μmol/l SAHA, 1 μmol/l doxorubicin (DOXO), 1 μmol/l taxol, 10 μmol/l VP-16, 10 μmol/l GSK343 (GSK), or 10 μmol/l UNC1999 (UNC) for 48 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or β-actin. (e) The chemical structure of UNC1999. DZNep, 3-deazaneplanocin A.

    Article Snippet: Materials LC3B, p62, LAMP2, trimethylated histone H3K27, GAPDH, and β-actin antibodies were purchased from GeneTex (Hsinchu, Taiwan).

    Techniques: Multiple Displacement Amplification, Western Blot, Staining, Fluorescence, Microscopy

    Effects of DZNep and GSK343 on the cell viability and autophagy of HepG2 and A549 cells. (a) HepG2 and A549 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against H3K27-me3 or GAPDH. (b) HepG2 and A549 cells were treated with different doses of DZNep or GSK343 for 72 h, and the cell viability was analyzed using an MTT assay. (c) HepG2 and A549 cells were treated with 10 and 20 μmol/l DZNep or GSK343 for 24 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, GAPDH, or β-actin. (d) HepG2 and A549 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and 20 nmol/l bafilomycin was added 4 h before cells were harvested. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, GAPDH, or β-actin. DZNep, 3-deazaneplanocin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

    Journal: Anti-Cancer Drugs

    Article Title: S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells

    doi: 10.1097/CAD.0000000000000166

    Figure Lengend Snippet: Effects of DZNep and GSK343 on the cell viability and autophagy of HepG2 and A549 cells. (a) HepG2 and A549 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 72 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against H3K27-me3 or GAPDH. (b) HepG2 and A549 cells were treated with different doses of DZNep or GSK343 for 72 h, and the cell viability was analyzed using an MTT assay. (c) HepG2 and A549 cells were treated with 10 and 20 μmol/l DZNep or GSK343 for 24 h, and whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, GAPDH, or β-actin. (d) HepG2 and A549 cells were treated with 20 μmol/l DZNep or 10 μmol/l GSK343 for 24 h, and 20 nmol/l bafilomycin was added 4 h before cells were harvested. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, GAPDH, or β-actin. DZNep, 3-deazaneplanocin A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

    Article Snippet: Materials LC3B, p62, LAMP2, trimethylated histone H3K27, GAPDH, and β-actin antibodies were purchased from GeneTex (Hsinchu, Taiwan).

    Techniques: Western Blot, MTT Assay

    Effects of EZH2 knockdown on autophagy of cancer cells. MDA-MB-231, HepG2, and A549 cells were transiently transfected with EZH2 siRNA for 72 h, and then whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, p62, or GAPDH. EZH2, enhancer of zeste homolog 2; siRNA, small interfering RNA.

    Journal: Anti-Cancer Drugs

    Article Title: S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells

    doi: 10.1097/CAD.0000000000000166

    Figure Lengend Snippet: Effects of EZH2 knockdown on autophagy of cancer cells. MDA-MB-231, HepG2, and A549 cells were transiently transfected with EZH2 siRNA for 72 h, and then whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B, p62, or GAPDH. EZH2, enhancer of zeste homolog 2; siRNA, small interfering RNA.

    Article Snippet: Materials LC3B, p62, LAMP2, trimethylated histone H3K27, GAPDH, and β-actin antibodies were purchased from GeneTex (Hsinchu, Taiwan).

    Techniques: Multiple Displacement Amplification, Transfection, Western Blot, Small Interfering RNA

    Effect of 3-MA on GSK343-induced autophagy and cytotoxicity of MDA-MB-231 cells. (a) MDA-MB-231 cells were pretreated with 2 and 5 mmol/l 3-MA for 1 h and then exposed to 10 μmol/l GSK343 for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or GAPDH. (b) MDA-MB-231 cells were pretreated with 2 mmol/l 3-MA for 1 h and then exposed to various doses of GSK343 for 72 h. Cell viability was analyzed using an MTT assay. Cell viability was analyzed using an MTT assay. * P

    Journal: Anti-Cancer Drugs

    Article Title: S-Adenosyl-l-methionine-competitive inhibitors of the histone methyltransferase EZH2 induce autophagy and enhance drug sensitivity in cancer cells

    doi: 10.1097/CAD.0000000000000166

    Figure Lengend Snippet: Effect of 3-MA on GSK343-induced autophagy and cytotoxicity of MDA-MB-231 cells. (a) MDA-MB-231 cells were pretreated with 2 and 5 mmol/l 3-MA for 1 h and then exposed to 10 μmol/l GSK343 for 24 h. Whole-cell lysates were subjected to a western blot analysis using antibodies against LC3B or GAPDH. (b) MDA-MB-231 cells were pretreated with 2 mmol/l 3-MA for 1 h and then exposed to various doses of GSK343 for 72 h. Cell viability was analyzed using an MTT assay. Cell viability was analyzed using an MTT assay. * P

    Article Snippet: Materials LC3B, p62, LAMP2, trimethylated histone H3K27, GAPDH, and β-actin antibodies were purchased from GeneTex (Hsinchu, Taiwan).

    Techniques: Multiple Displacement Amplification, Western Blot, MTT Assay

    Effects of ITKi on signalling pathways, calcium flux and cytokine production in Jurkat cells. ( A ) Jurkat cells were stimulated with anti-CD3 and anti-CD28 antibodies as indicated. Westerns showing expression of PLCγ1 and phosphorylated-PLCγ1 (p-PLCγ1), MEK1/2 and phosphorylated-MEK1/2 (p-MEK1/2) and AKT and phosphorylated-AKT (p-AKT). GAPDH is a loading control. Molecular weight is indicated to the left of the western. ( B ) Jurkat cells were pre-treated with 1 µM ONO00779050 (1 µM) and Ibrutinib (1 µM) for 30 minutes, and then stimulated with anti-CD3/CD28 antibodies for 6 hours. Cytoplasmic and nuclear extracts were analysed by western blot. Arrowheads indicate NFATc1. GAPDH is a loading control. Molecular weight is indicated to the left of the western. ( C ) Calcium flux changes in response to ITKi. Left hand panel shows effect of PF-06465469 (dashed line) and ibrutinib (dotted line). Right hand panel shows effect of ONO7790500 (dashed line) and BMS509744 (dotted line). For each panel the no drug control is indicated by a solid line. The time of addition of anti-CD3 and anti-CD28 antibodies and ionomycin are shown. ( D ) IL-2 (left-hand panel) and IL-21 (right-hand panel) production in response to phytohaemagglutinin and phorbol myristate (PHA/PMA). Results are mean ± SEM. n = 4. IL-2 and IL-21 production were significantly (two-tailed unpaired t-test) repressed by ibrutinib, PF-6465469, BMS509744 and ONO7790500.

    Journal: Scientific Reports

    Article Title: Comparison of interleukin-2-inducible kinase (ITK) inhibitors and potential for combination therapies for T-cell lymphoma

    doi: 10.1038/s41598-018-32634-5

    Figure Lengend Snippet: Effects of ITKi on signalling pathways, calcium flux and cytokine production in Jurkat cells. ( A ) Jurkat cells were stimulated with anti-CD3 and anti-CD28 antibodies as indicated. Westerns showing expression of PLCγ1 and phosphorylated-PLCγ1 (p-PLCγ1), MEK1/2 and phosphorylated-MEK1/2 (p-MEK1/2) and AKT and phosphorylated-AKT (p-AKT). GAPDH is a loading control. Molecular weight is indicated to the left of the western. ( B ) Jurkat cells were pre-treated with 1 µM ONO00779050 (1 µM) and Ibrutinib (1 µM) for 30 minutes, and then stimulated with anti-CD3/CD28 antibodies for 6 hours. Cytoplasmic and nuclear extracts were analysed by western blot. Arrowheads indicate NFATc1. GAPDH is a loading control. Molecular weight is indicated to the left of the western. ( C ) Calcium flux changes in response to ITKi. Left hand panel shows effect of PF-06465469 (dashed line) and ibrutinib (dotted line). Right hand panel shows effect of ONO7790500 (dashed line) and BMS509744 (dotted line). For each panel the no drug control is indicated by a solid line. The time of addition of anti-CD3 and anti-CD28 antibodies and ionomycin are shown. ( D ) IL-2 (left-hand panel) and IL-21 (right-hand panel) production in response to phytohaemagglutinin and phorbol myristate (PHA/PMA). Results are mean ± SEM. n = 4. IL-2 and IL-21 production were significantly (two-tailed unpaired t-test) repressed by ibrutinib, PF-6465469, BMS509744 and ONO7790500.

    Article Snippet: Primary antibodies used (all at 1:1000 unless otherwise stated) were anti-ITK (ab32039, Abcam, Cambridge, UK), anti-phospho-BTK/ITK (14-9015-82, eBioscience, San Diego, CA, USA), anti-PLCγ1 (#5690, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-PLCγ1 (#2821, Cell Signaling Technology), anti-AKT (#4691, Cell Signaling Technology), anti-phospho-AKT (#4060, Cell Signaling Technology), anti-MEK (#8727, Cell Signaling Technology). anti-phospho-MEK (#9154, Cell Signaling Technology) and anti-NFAT2 (NFATc) (1 µg/ml) (ab2796, Abcam).

    Techniques: Expressing, Molecular Weight, Western Blot, Two Tailed Test

    Expression level and number of motoneurons in wild-type and homozygous shaky mice. (A) Western blot analysis of GlyR α1 subunit protein expression in whole brain and spinal cord homogenates. Normalization with β-actin showed no significant differences of GlyR α1 protein levels between the two genotypes ( n = 3), right image shows an example of the Western blot from two different animals of each genotype ( Glra1 +/+ and Glra1 sh / sh ). (B) Quantitative analysis of motoneuron numbers. The quantification was made using 3–4 animals of each genotype ( Glra1 +/+ , n = 4; Glra1 sh / sh , n = 3). Right images are examples of brain stem sections from Glra1 +/+ and Glra1 sh / sh with arrow heads pointing to motoneurons. Error bars represent standard deviations (S.D.).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease

    doi: 10.3389/fnmol.2018.00167

    Figure Lengend Snippet: Expression level and number of motoneurons in wild-type and homozygous shaky mice. (A) Western blot analysis of GlyR α1 subunit protein expression in whole brain and spinal cord homogenates. Normalization with β-actin showed no significant differences of GlyR α1 protein levels between the two genotypes ( n = 3), right image shows an example of the Western blot from two different animals of each genotype ( Glra1 +/+ and Glra1 sh / sh ). (B) Quantitative analysis of motoneuron numbers. The quantification was made using 3–4 animals of each genotype ( Glra1 +/+ , n = 4; Glra1 sh / sh , n = 3). Right images are examples of brain stem sections from Glra1 +/+ and Glra1 sh / sh with arrow heads pointing to motoneurons. Error bars represent standard deviations (S.D.).

    Article Snippet: GlyR proteins were detected with the GlyR α1 specific antibody mAb2b (cat. no. 146003 1:1,500, Synaptic Systems, Göttingen, Germany). β-Actin (cat. no. GTX26276, WB 1:5,000, GeneTex/Biozol, Eching, Germany) served as loading control.

    Techniques: Expressing, Mouse Assay, Western Blot

    Backcross of shaky mouse line into the spasmodic mouse line. Developmental expression of the GlyR α1 subunit in shaky mice and after backcross into the mouse line spasmodic (A–D) . After backcross of shaky into the spasmodic line, the expression profile was determined in spinal cord (sc), brain stem (bs) and cortex (cx) for stages P0, P7, P14, P28, P56, P84, and P100. (A) Glra1 +/+ , (B) Glra1 sh / spd , (C) heterozygous Glra1 +/ spd , and (D) heterozygous Glra1 +/ sh . Cortex (cx) served as negative control for GlyR α1. GlyR α1 subunit was stained with mAb2b (48 kDa), and β-Actin (46 kDa) served as a loading control (LC). Cortex samples were probed with a GlyR pan-α antibody (mAb4a) labeling other GlyR α subunits in the cortex (A,B) .

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease

    doi: 10.3389/fnmol.2018.00167

    Figure Lengend Snippet: Backcross of shaky mouse line into the spasmodic mouse line. Developmental expression of the GlyR α1 subunit in shaky mice and after backcross into the mouse line spasmodic (A–D) . After backcross of shaky into the spasmodic line, the expression profile was determined in spinal cord (sc), brain stem (bs) and cortex (cx) for stages P0, P7, P14, P28, P56, P84, and P100. (A) Glra1 +/+ , (B) Glra1 sh / spd , (C) heterozygous Glra1 +/ spd , and (D) heterozygous Glra1 +/ sh . Cortex (cx) served as negative control for GlyR α1. GlyR α1 subunit was stained with mAb2b (48 kDa), and β-Actin (46 kDa) served as a loading control (LC). Cortex samples were probed with a GlyR pan-α antibody (mAb4a) labeling other GlyR α subunits in the cortex (A,B) .

    Article Snippet: GlyR proteins were detected with the GlyR α1 specific antibody mAb2b (cat. no. 146003 1:1,500, Synaptic Systems, Göttingen, Germany). β-Actin (cat. no. GTX26276, WB 1:5,000, GeneTex/Biozol, Eching, Germany) served as loading control.

    Techniques: Expressing, Mouse Assay, Negative Control, Staining, Labeling

    Genotype of shaky mice. (A) The recognition site for the restriction enzyme HpyCH4V is disrupted by the shaky mouse mutation in exon 6; left panel. PCR genotyping of shaky ( Glra1 sh / sh ), wild-type ( Glra1 +/+ ) and heterozygous ( Glra1 +/ sh ) mice with subsequent HpyCH4V digest (right panel). (B) Sequencing chromatograms of wild-type strains C57BL6 and 129/SvJ, heterozygous Glra1 +/ sh , and homozygous Glra1 sh / sh showing a c.T198C transition in exon 3 and a c.C613A transition in exon 6. Both wild-type mouse strains are shown since the shaky mutation arose in a hybrid background of C57BL6 and 129/SvJ. (C) RT-PCR analysis of GlyR α1 subunit mRNA levels in spinal cord (sc) and brain stem (bs) of wild-type ( n = 4) and shaky mice ( n = 4). β-actin cDNA was amplified as a reference gene to ensure equal cDNA content in all samples.

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease

    doi: 10.3389/fnmol.2018.00167

    Figure Lengend Snippet: Genotype of shaky mice. (A) The recognition site for the restriction enzyme HpyCH4V is disrupted by the shaky mouse mutation in exon 6; left panel. PCR genotyping of shaky ( Glra1 sh / sh ), wild-type ( Glra1 +/+ ) and heterozygous ( Glra1 +/ sh ) mice with subsequent HpyCH4V digest (right panel). (B) Sequencing chromatograms of wild-type strains C57BL6 and 129/SvJ, heterozygous Glra1 +/ sh , and homozygous Glra1 sh / sh showing a c.T198C transition in exon 3 and a c.C613A transition in exon 6. Both wild-type mouse strains are shown since the shaky mutation arose in a hybrid background of C57BL6 and 129/SvJ. (C) RT-PCR analysis of GlyR α1 subunit mRNA levels in spinal cord (sc) and brain stem (bs) of wild-type ( n = 4) and shaky mice ( n = 4). β-actin cDNA was amplified as a reference gene to ensure equal cDNA content in all samples.

    Article Snippet: GlyR proteins were detected with the GlyR α1 specific antibody mAb2b (cat. no. 146003 1:1,500, Synaptic Systems, Göttingen, Germany). β-Actin (cat. no. GTX26276, WB 1:5,000, GeneTex/Biozol, Eching, Germany) served as loading control.

    Techniques: Mouse Assay, Mutagenesis, Polymerase Chain Reaction, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification

    LDOC1 mediates the in vitro malignancy progression in lung cancer cell lines. ( A ) Expression of LDOC1 mRNA and protein were stably suppressed in the A549-derived cell lines. The A549 cell line was transducing lentivirus carrying either scramble control vector (A549-shCtrl) or siRNA targeting LDOC1 (A549-shLDOC1). ( B ) Restoration of LDOC1 expression in A549-shLDOC1-derived cells. A549-shLDOC1 was only transducing GFP-tagged lentivirus vectors (A549-sh-rCtrl) or those fused with an LDOC1 open reading frame (A549-sh-rLDOC1). Expression of LDOC1 mRNA (left) and protein (right) was measured using qPCR and Western blotting analysis, respectively. ( C – F ) Cell proliferation ( C , by MTT), cell cycle progression ( D , by BrdU incorporation assay), and cell invasion ( E , F by Matrigel coated trans-well invasiveness assay) were assessed in the parental A549 cells and the generated A549-derived cell lines ( C – E ) or H1299 ectopic-expressing LDOC1 ( F ). H1299 cells were transduced with lentivirus carrying either open reading frame (ORF) of LDOC1 or empty vector (V, controls). Expression of LDOC1 and GAPDH proteins were measured using Western blotting analysis ( F , upper panels). The number of invading cells was the average of four independent experiments; data were analyzed using a Student’s t -test., where * p

    Journal: Cancers

    Article Title: Novel STAT3 Inhibitor LDOC1 Targets Phospho-JAK2 for Degradation by Interacting with LNX1 and Regulates the Aggressiveness of Lung Cancer

    doi: 10.3390/cancers11010063

    Figure Lengend Snippet: LDOC1 mediates the in vitro malignancy progression in lung cancer cell lines. ( A ) Expression of LDOC1 mRNA and protein were stably suppressed in the A549-derived cell lines. The A549 cell line was transducing lentivirus carrying either scramble control vector (A549-shCtrl) or siRNA targeting LDOC1 (A549-shLDOC1). ( B ) Restoration of LDOC1 expression in A549-shLDOC1-derived cells. A549-shLDOC1 was only transducing GFP-tagged lentivirus vectors (A549-sh-rCtrl) or those fused with an LDOC1 open reading frame (A549-sh-rLDOC1). Expression of LDOC1 mRNA (left) and protein (right) was measured using qPCR and Western blotting analysis, respectively. ( C – F ) Cell proliferation ( C , by MTT), cell cycle progression ( D , by BrdU incorporation assay), and cell invasion ( E , F by Matrigel coated trans-well invasiveness assay) were assessed in the parental A549 cells and the generated A549-derived cell lines ( C – E ) or H1299 ectopic-expressing LDOC1 ( F ). H1299 cells were transduced with lentivirus carrying either open reading frame (ORF) of LDOC1 or empty vector (V, controls). Expression of LDOC1 and GAPDH proteins were measured using Western blotting analysis ( F , upper panels). The number of invading cells was the average of four independent experiments; data were analyzed using a Student’s t -test., where * p

    Article Snippet: Antibodies against STAT3 (79D7), pSTAT3Y705 (D3A7), JAK2 (D2E12), pJAK2 (3771), Src (36D10), and α-tubulin (2144) were purchased from Cell Signaling; anti-LNX1 (NBP1-49975, Novus, Littleton, CO, USA); anti-pSrc (9A6, Millipore, Billerica, MA, USA); anti-pJAK2 (PJAK2-240AP, FabGennix, Frisco, TX, USA); anti-GAPDH (GTX100118, GeneTex, Hsinchu, Taiwan); anti-LDOC1 (2507C1a, Santa Cruz, Dallas, TX, USA) for immunoprecipitation and immunofluorescent assay (IFA); anti-LDOC1 (LS-B3527, LifeSpan, Providence, RI, USA) for immunohistochemistry (IHC) study, and anti-β-actin (AC-15, Novus).

    Techniques: In Vitro, Expressing, Stable Transfection, Derivative Assay, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, BrdU Incorporation Assay, Generated, Transduction

    PPD inhibited osteoclast differentiation by the NF-κB pathway. (A–C) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Nuclear protein was isolated for western blot analysis. Cell lysates were analyzed using western blotting with specific antibodies against NF-κB and p-NF-κB were quantified and were normalized to H3 using ImageJ software. (D) RAW264.7 cells that had been stably transfected with an NF-κB luciferase reporter construct were treated with the indicated concentrations of PPD for 1 h, followed by incubation in the absence or presence of RANKL for 8 h. Luciferase activity was measured using the Promega Luciferase Assay System. All experiments were performed at least 3 times; (E) BMDMs were cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL with or without 5 μM PPD for 0–5 days. Cell lysates were prepared and analyzed using western blotting. (F,G) The gray levels corresponding to the indicated proteins were quantified and normalized relative to β-actin using Image J for c-fos and NFATc1. Significant differences between the groups were determined by paired Student’s t -test. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: 20(S)-Protopanaxadiol Inhibits Titanium Particle-Induced Inflammatory Osteolysis and RANKL-Mediated Osteoclastogenesis via MAPK and NF-κB Signaling Pathways

    doi: 10.3389/fphar.2018.01538

    Figure Lengend Snippet: PPD inhibited osteoclast differentiation by the NF-κB pathway. (A–C) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Nuclear protein was isolated for western blot analysis. Cell lysates were analyzed using western blotting with specific antibodies against NF-κB and p-NF-κB were quantified and were normalized to H3 using ImageJ software. (D) RAW264.7 cells that had been stably transfected with an NF-κB luciferase reporter construct were treated with the indicated concentrations of PPD for 1 h, followed by incubation in the absence or presence of RANKL for 8 h. Luciferase activity was measured using the Promega Luciferase Assay System. All experiments were performed at least 3 times; (E) BMDMs were cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL with or without 5 μM PPD for 0–5 days. Cell lysates were prepared and analyzed using western blotting. (F,G) The gray levels corresponding to the indicated proteins were quantified and normalized relative to β-actin using Image J for c-fos and NFATc1. Significant differences between the groups were determined by paired Student’s t -test. ∗ P

    Article Snippet: Specific primary antibodies against NF-κB inhibitor alpha (IκBα) (#4814), p-IκBα (#9246), NF-κB p65 (#8242), phospho-NF-κB p-p65 (#3033), ERK1/2 (#4695), phospho-ERK1/2 (#4370), SAPK/JNK (#9252), phospho-SAPK/JNK (#4668), p38 (#8690), phospho-p38 (#4511), histone H3 (#4499), β-actin(#4967) and c-Fos (#4384) were purchased from Cell Signaling Technology (MA, United States).

    Techniques: Isolation, Western Blot, Software, Stable Transfection, Transfection, Luciferase, Construct, Incubation, Activity Assay, Cell Culture

    HR is increased in T4-2 malignant breast epithelial cells compared to that in S1 cells. (A and B) T4-2 and S1 cells were irradiated with 6 Gy X rays, fixed post-IR, and coimmunostained with 53BP1 and BRCA1 antibodies. (A) 53BP1 and BRCA1 staining; (B) histogram of BRCA1 foci. (C and E) Cells were treated with 6 Gy X rays, fixed post-IR, and immunostained for RPA70 (C) and Rad51 (E) antibodies. (D and F) Quantification of focus formation. (D) Histogram of > 20 RPA70 foci; (F) histogram of > 10 Rad51 foci. (B, D, and F) Columns represent the means ( n = 3), and bars represent the SDs; *, P

    Journal: Molecular and Cellular Biology

    Article Title: β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination

    doi: 10.1128/MCB.00672-17

    Figure Lengend Snippet: HR is increased in T4-2 malignant breast epithelial cells compared to that in S1 cells. (A and B) T4-2 and S1 cells were irradiated with 6 Gy X rays, fixed post-IR, and coimmunostained with 53BP1 and BRCA1 antibodies. (A) 53BP1 and BRCA1 staining; (B) histogram of BRCA1 foci. (C and E) Cells were treated with 6 Gy X rays, fixed post-IR, and immunostained for RPA70 (C) and Rad51 (E) antibodies. (D and F) Quantification of focus formation. (D) Histogram of > 20 RPA70 foci; (F) histogram of > 10 Rad51 foci. (B, D, and F) Columns represent the means ( n = 3), and bars represent the SDs; *, P

    Article Snippet: Primary antibodies against the following were used in this study: from BD Transduction Laboratories, β1-integrin (clone 18); from Cell Signaling, H2AX (2595), γ-H2AX (Ser139; 9718), ATR (2790), pATR (Ser428; 2853), and pATM (Ser1981; 13050); from Santa Cruz, ATM (Sc-53173), 53BP1 (Sc-22760), BRCA1 (Sc-642), and α-tubulin (Sc-5286); from Abcam, Rad51 (ab63801), RPA70 (ab79398), and RING1 (ab32644); from GeneTex, MRE11 (GTX70212); from Bethyl Laboratories, RIF1 (A300-5671); from Proteintech, GAPDH (HRP-60004); from MP Biomedicals, β-actin (691001).

    Techniques: Irradiation, Staining

    Inhibition of Rad51 increased malignant breast cancer T4-2 cell radiosensitivity. (A and B) Western analysis of Rad51, BRCA1, and RPA70 using whole-cell lysates prepared from T4-2 and S1 cells sham irradiated or exposed to 5 Gy X rays (β-actin as a loading control). (C) Radiosensitivity assay of T4-2 and S1 cells treated with control siRNA or T4-2 cells treated with Rad51 siRNA before exposure to 1, 2, 4, or 8 Gy X rays. Clonogenic survival was measured 14 days after IR. Colonies consisting of more than 50 cells were scored as surviving colonies and normalized against nonirradiated clones ( n = 3, mean ± SD; **, P

    Journal: Molecular and Cellular Biology

    Article Title: β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination

    doi: 10.1128/MCB.00672-17

    Figure Lengend Snippet: Inhibition of Rad51 increased malignant breast cancer T4-2 cell radiosensitivity. (A and B) Western analysis of Rad51, BRCA1, and RPA70 using whole-cell lysates prepared from T4-2 and S1 cells sham irradiated or exposed to 5 Gy X rays (β-actin as a loading control). (C) Radiosensitivity assay of T4-2 and S1 cells treated with control siRNA or T4-2 cells treated with Rad51 siRNA before exposure to 1, 2, 4, or 8 Gy X rays. Clonogenic survival was measured 14 days after IR. Colonies consisting of more than 50 cells were scored as surviving colonies and normalized against nonirradiated clones ( n = 3, mean ± SD; **, P

    Article Snippet: Primary antibodies against the following were used in this study: from BD Transduction Laboratories, β1-integrin (clone 18); from Cell Signaling, H2AX (2595), γ-H2AX (Ser139; 9718), ATR (2790), pATR (Ser428; 2853), and pATM (Ser1981; 13050); from Santa Cruz, ATM (Sc-53173), 53BP1 (Sc-22760), BRCA1 (Sc-642), and α-tubulin (Sc-5286); from Abcam, Rad51 (ab63801), RPA70 (ab79398), and RING1 (ab32644); from GeneTex, MRE11 (GTX70212); from Bethyl Laboratories, RIF1 (A300-5671); from Proteintech, GAPDH (HRP-60004); from MP Biomedicals, β-actin (691001).

    Techniques: Inhibition, Western Blot, Irradiation, Clone Assay

    Regulation of Rad51 protein levels is predominantly through the ubiquitin proteasomal pathway. (A) Quantitative reverse transcription-PCR analysis of β1-integrin mRNA expression in T4-2 and S1 cells exposed to 5 Gy X rays. GAPDH served as an internal control. (B) Proteasomal inhibitor MG-132 (carbobenzoxy-Leu-Leu-leucinal) blocks the degradation of Rad51. Western blot analysis of the expression of Rad51 was done using whole-cell lysate prepared from S1 cells treated with MG-132 (20 μM) or vehicle (dimethyl sulfoxide [DMSO]; GAPDH as a loading control and whole-cell lysate of T4-2 cells as a control for the expression level of Rad51). (C and D) A specific synthetic inhibitor of calpain, PD150606 (PD), blocks the degradation of Rad51. (C) Western blot analysis of Rad51 expression was performed using whole-cell lysate prepared from S1 cells treated with cycloheximide (CHX; 50 μg/ml) to inhibit protein synthesis or not treated with CHX and then treated with PD150606 or vehicle (DMSO; α-tubulin as a loading control and p53 as a negative or system control). (D) Relative expression levels of Rad51 normalized to the expression levels of α-tubulin. (E and F) Half-lives ( t 1/2 ) of Rad51 in T4-2 (E) and S1 (F) cells. Western blot analysis of Rad51 expression was performed using whole-cell lysate prepared from S1 cells treated with CHX (50 μg/ml) or not treated with CHX before exposure to 5 Gy X rays (α-tubulin as the loading control). Bottom panels, densitometric analysis of Rad51 normalized with α-tubulin.

    Journal: Molecular and Cellular Biology

    Article Title: β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination

    doi: 10.1128/MCB.00672-17

    Figure Lengend Snippet: Regulation of Rad51 protein levels is predominantly through the ubiquitin proteasomal pathway. (A) Quantitative reverse transcription-PCR analysis of β1-integrin mRNA expression in T4-2 and S1 cells exposed to 5 Gy X rays. GAPDH served as an internal control. (B) Proteasomal inhibitor MG-132 (carbobenzoxy-Leu-Leu-leucinal) blocks the degradation of Rad51. Western blot analysis of the expression of Rad51 was done using whole-cell lysate prepared from S1 cells treated with MG-132 (20 μM) or vehicle (dimethyl sulfoxide [DMSO]; GAPDH as a loading control and whole-cell lysate of T4-2 cells as a control for the expression level of Rad51). (C and D) A specific synthetic inhibitor of calpain, PD150606 (PD), blocks the degradation of Rad51. (C) Western blot analysis of Rad51 expression was performed using whole-cell lysate prepared from S1 cells treated with cycloheximide (CHX; 50 μg/ml) to inhibit protein synthesis or not treated with CHX and then treated with PD150606 or vehicle (DMSO; α-tubulin as a loading control and p53 as a negative or system control). (D) Relative expression levels of Rad51 normalized to the expression levels of α-tubulin. (E and F) Half-lives ( t 1/2 ) of Rad51 in T4-2 (E) and S1 (F) cells. Western blot analysis of Rad51 expression was performed using whole-cell lysate prepared from S1 cells treated with CHX (50 μg/ml) or not treated with CHX before exposure to 5 Gy X rays (α-tubulin as the loading control). Bottom panels, densitometric analysis of Rad51 normalized with α-tubulin.

    Article Snippet: Primary antibodies against the following were used in this study: from BD Transduction Laboratories, β1-integrin (clone 18); from Cell Signaling, H2AX (2595), γ-H2AX (Ser139; 9718), ATR (2790), pATR (Ser428; 2853), and pATM (Ser1981; 13050); from Santa Cruz, ATM (Sc-53173), 53BP1 (Sc-22760), BRCA1 (Sc-642), and α-tubulin (Sc-5286); from Abcam, Rad51 (ab63801), RPA70 (ab79398), and RING1 (ab32644); from GeneTex, MRE11 (GTX70212); from Bethyl Laboratories, RIF1 (A300-5671); from Proteintech, GAPDH (HRP-60004); from MP Biomedicals, β-actin (691001).

    Techniques: Polymerase Chain Reaction, Expressing, Western Blot

    β1-Integrin regulates Rad51 protein levels and recruitment to DSB sites and promotes HR. (A) T4-2 and S1 cells were electroporated with mammalian expression vectors encoding Flag-tagged β1-integrin or with GFP as a control. Cells were lysed 48 h following electroporation and subjected to immunoblotting with β1-integrin and Rad51 antibodies. Tubulin served as an internal loading control. (B and C) Western blot analysis of Rad51 in whole-cell lysates prepared from malignant breast cancer T4-2 cells treated with the β1-integrin inhibitory monoclonal antibody AIIB2 (0.1 μg/μl) or nonspecific rat IgG before exposure to IR (tubulin served as a loading control). (D) HR was assessed using a DR-GFP reporter assay, as described in Materials and Methods, using T4-2 cells electroporated with control siRNA or Rad51 siRNA or treated with AIIB2 or IgG (“control” represents both control siRNA and IgG). (E) DR95hyg-xt cells were electroporated with either control siRNA or β1-integrin siRNA and harvested after 48 h. Cell lysates were immunoblotted for the indicated proteins. (F and G) ChIP analysis of Rad51 (F) and γ-H2AX (G) on a unique DSB induced by I-SceI in vivo in DR95hyg-xt cells treated with control siRNA (siCon) or β1-integrin siRNA (siβ1), before and after DSB induction by I-SceI transfection. Real-time PCR on ChIP samples used primers directed at nucleotides 94 to 378 from the DSB. The enrichment of Rad51 and γ-H2AX after induction of the DSB was compared with that of an IgG control. (D, F, and G) Columns represent the means ( n = 3), and bars represent the SDs; **, P

    Journal: Molecular and Cellular Biology

    Article Title: β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination

    doi: 10.1128/MCB.00672-17

    Figure Lengend Snippet: β1-Integrin regulates Rad51 protein levels and recruitment to DSB sites and promotes HR. (A) T4-2 and S1 cells were electroporated with mammalian expression vectors encoding Flag-tagged β1-integrin or with GFP as a control. Cells were lysed 48 h following electroporation and subjected to immunoblotting with β1-integrin and Rad51 antibodies. Tubulin served as an internal loading control. (B and C) Western blot analysis of Rad51 in whole-cell lysates prepared from malignant breast cancer T4-2 cells treated with the β1-integrin inhibitory monoclonal antibody AIIB2 (0.1 μg/μl) or nonspecific rat IgG before exposure to IR (tubulin served as a loading control). (D) HR was assessed using a DR-GFP reporter assay, as described in Materials and Methods, using T4-2 cells electroporated with control siRNA or Rad51 siRNA or treated with AIIB2 or IgG (“control” represents both control siRNA and IgG). (E) DR95hyg-xt cells were electroporated with either control siRNA or β1-integrin siRNA and harvested after 48 h. Cell lysates were immunoblotted for the indicated proteins. (F and G) ChIP analysis of Rad51 (F) and γ-H2AX (G) on a unique DSB induced by I-SceI in vivo in DR95hyg-xt cells treated with control siRNA (siCon) or β1-integrin siRNA (siβ1), before and after DSB induction by I-SceI transfection. Real-time PCR on ChIP samples used primers directed at nucleotides 94 to 378 from the DSB. The enrichment of Rad51 and γ-H2AX after induction of the DSB was compared with that of an IgG control. (D, F, and G) Columns represent the means ( n = 3), and bars represent the SDs; **, P

    Article Snippet: Primary antibodies against the following were used in this study: from BD Transduction Laboratories, β1-integrin (clone 18); from Cell Signaling, H2AX (2595), γ-H2AX (Ser139; 9718), ATR (2790), pATR (Ser428; 2853), and pATM (Ser1981; 13050); from Santa Cruz, ATM (Sc-53173), 53BP1 (Sc-22760), BRCA1 (Sc-642), and α-tubulin (Sc-5286); from Abcam, Rad51 (ab63801), RPA70 (ab79398), and RING1 (ab32644); from GeneTex, MRE11 (GTX70212); from Bethyl Laboratories, RIF1 (A300-5671); from Proteintech, GAPDH (HRP-60004); from MP Biomedicals, β-actin (691001).

    Techniques: Expressing, Electroporation, Western Blot, Reporter Assay, Chromatin Immunoprecipitation, In Vivo, Transfection, Real-time Polymerase Chain Reaction

    β1-Integrin regulates Rad51 ubiquitination and Rad51 protein levels by E3 ubiquitin-protein ligase RING1. (A and B) T4-2 and S1 cells were left untreated or treated with 5 Gy of IR. Cells were lysed under denaturing conditions. Rad51 was immunoprecipitated (IP), and immunoblots (IB) were probed with panubiquitin (A) and K48-Ub (B) antibodies. Positions and sizes in kilodaltons of marker proteins are shown on the left. (A) Lower panels show the Rad51 before immunoprecipitation (input) and Rad51 in the supernatant postimmunoprecipitation (sup). Neg. con, negative control. (C and D) Western blot analysis of RING1 and Rad51 expression using whole-cell lysate prepared from T4-2 and S1 cells (C) and from S1 cells electroporated with control siRNA (siC) or Rad51 siRNA (siR) (D). Nonspecific bands (NS) and α-tubulin served as loading controls. (E) Inhibition of RING1 increased radioresistance in S1 cells. Malignant breast T4-2 cells and the nonmalignant S1 breast epithelial cell counterparts were treated with control siRNA or the S1 cells were treated with RING1 siRNA before exposure to 1, 2, 4, or 8 Gy X rays. Clonogenic survival was measured 14 days post-IR. Colonies consisting of more than 50 cells were scored as surviving colonies and normalized against nonirradiated clones ( n = 3, mean ± SD; **, P

    Journal: Molecular and Cellular Biology

    Article Title: β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination

    doi: 10.1128/MCB.00672-17

    Figure Lengend Snippet: β1-Integrin regulates Rad51 ubiquitination and Rad51 protein levels by E3 ubiquitin-protein ligase RING1. (A and B) T4-2 and S1 cells were left untreated or treated with 5 Gy of IR. Cells were lysed under denaturing conditions. Rad51 was immunoprecipitated (IP), and immunoblots (IB) were probed with panubiquitin (A) and K48-Ub (B) antibodies. Positions and sizes in kilodaltons of marker proteins are shown on the left. (A) Lower panels show the Rad51 before immunoprecipitation (input) and Rad51 in the supernatant postimmunoprecipitation (sup). Neg. con, negative control. (C and D) Western blot analysis of RING1 and Rad51 expression using whole-cell lysate prepared from T4-2 and S1 cells (C) and from S1 cells electroporated with control siRNA (siC) or Rad51 siRNA (siR) (D). Nonspecific bands (NS) and α-tubulin served as loading controls. (E) Inhibition of RING1 increased radioresistance in S1 cells. Malignant breast T4-2 cells and the nonmalignant S1 breast epithelial cell counterparts were treated with control siRNA or the S1 cells were treated with RING1 siRNA before exposure to 1, 2, 4, or 8 Gy X rays. Clonogenic survival was measured 14 days post-IR. Colonies consisting of more than 50 cells were scored as surviving colonies and normalized against nonirradiated clones ( n = 3, mean ± SD; **, P

    Article Snippet: Primary antibodies against the following were used in this study: from BD Transduction Laboratories, β1-integrin (clone 18); from Cell Signaling, H2AX (2595), γ-H2AX (Ser139; 9718), ATR (2790), pATR (Ser428; 2853), and pATM (Ser1981; 13050); from Santa Cruz, ATM (Sc-53173), 53BP1 (Sc-22760), BRCA1 (Sc-642), and α-tubulin (Sc-5286); from Abcam, Rad51 (ab63801), RPA70 (ab79398), and RING1 (ab32644); from GeneTex, MRE11 (GTX70212); from Bethyl Laboratories, RIF1 (A300-5671); from Proteintech, GAPDH (HRP-60004); from MP Biomedicals, β-actin (691001).

    Techniques: Immunoprecipitation, Western Blot, Marker, Negative Control, Expressing, Inhibition, Clone Assay

    β1-Integrin promotes IR-induced nuclear accumulation of Rad51 and focus formation in irradiated cells. (A and B) Increased nuclear Rad51 levels in irradiated cells. (A) Western blotting was performed with nuclear and cytosolic Rad51 of T4-2 cells treated with AIIB2 for 16 h before exposure to 5 Gy X rays (Mre11 and β-actin are markers for nuclear and cytosolic fractions). (B) Relative amounts of nuclear Rad51 shown in panel A were estimated by densitometry. (C and E) Decreases in Rad51 foci by AIIB2 treatment were associated with an increase in γ-H2AX foci in irradiated cells. T4-2 cells were treated with AIIB2 before exposure to 6 Gy X rays, fixed post-IR, and immunostained for Rad51 (C) and γ-H2AX (E) antibodies. (D and F) The percentages of cells with more than 10 Rad51 (D) or γ-H2AX (F) foci are represented. (B, D, and F) Columns represent the means ( n = 3), and bars represent the SDs; *, P

    Journal: Molecular and Cellular Biology

    Article Title: β1-Integrin Impacts Rad51 Stability and DNA Double-Strand Break Repair by Homologous Recombination

    doi: 10.1128/MCB.00672-17

    Figure Lengend Snippet: β1-Integrin promotes IR-induced nuclear accumulation of Rad51 and focus formation in irradiated cells. (A and B) Increased nuclear Rad51 levels in irradiated cells. (A) Western blotting was performed with nuclear and cytosolic Rad51 of T4-2 cells treated with AIIB2 for 16 h before exposure to 5 Gy X rays (Mre11 and β-actin are markers for nuclear and cytosolic fractions). (B) Relative amounts of nuclear Rad51 shown in panel A were estimated by densitometry. (C and E) Decreases in Rad51 foci by AIIB2 treatment were associated with an increase in γ-H2AX foci in irradiated cells. T4-2 cells were treated with AIIB2 before exposure to 6 Gy X rays, fixed post-IR, and immunostained for Rad51 (C) and γ-H2AX (E) antibodies. (D and F) The percentages of cells with more than 10 Rad51 (D) or γ-H2AX (F) foci are represented. (B, D, and F) Columns represent the means ( n = 3), and bars represent the SDs; *, P

    Article Snippet: Primary antibodies against the following were used in this study: from BD Transduction Laboratories, β1-integrin (clone 18); from Cell Signaling, H2AX (2595), γ-H2AX (Ser139; 9718), ATR (2790), pATR (Ser428; 2853), and pATM (Ser1981; 13050); from Santa Cruz, ATM (Sc-53173), 53BP1 (Sc-22760), BRCA1 (Sc-642), and α-tubulin (Sc-5286); from Abcam, Rad51 (ab63801), RPA70 (ab79398), and RING1 (ab32644); from GeneTex, MRE11 (GTX70212); from Bethyl Laboratories, RIF1 (A300-5671); from Proteintech, GAPDH (HRP-60004); from MP Biomedicals, β-actin (691001).

    Techniques: Irradiation, Western Blot

    MPT0G211 sensitized MOLT-4 cells to vincristine-mediated mitotic arrest. a Cell cycle distributions of cells exposed to MPT0G211, tubastatin A (TBA), vincristine (VCR), or the indicated combination therapy for 24 h. A statistical analysis of the proportions of cells in the G2/M phase is shown in the right panel. b The levels of the apoptotic proteins caspase 3 and poly-ADP ribose polymerase (PARP) and the pro-survival proteins BCL-XL, BCL-2, and survivin were determined in cells treated with MPT0G211 (3 μM) or TBA (3 μM) in combination with VCR (1 nM) for 24 h. c The protein levels of cyclin B1, aurora B, p-CDC2, p-PLK, p-Histone 3, and MPM2 were evaluated following treatment with a combination of MPT0G211 or TBA with VCR for 24 h. d Cells were co-treated with MPT0G211 or TBA with VCR for 24 h and incubated with an α-tubulin antibody or DAPI. Microtubule dynamics were evaluated using a ZEISS LS 510META confocal microscope (magnification × 630). Scale bar = 20 μM. e Antitumor activity of MPT0G211 plus vincristine in a MOLT-4 xenograft model. When the tumor size reached 200 mm 3 , mice were injected with vehicle, vincristine (1 mg/kg, i.p., qwk), and MPT0G211 (30 mg/kg, i.p., qd) alone or a combination of both. The curves of tumor growth volume were expressed as mean ± SEM. f Changes of body weight after treatment. Data are shown as means ± standard errors of the means. * p

    Journal: Clinical Epigenetics

    Article Title: The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

    doi: 10.1186/s13148-018-0595-8

    Figure Lengend Snippet: MPT0G211 sensitized MOLT-4 cells to vincristine-mediated mitotic arrest. a Cell cycle distributions of cells exposed to MPT0G211, tubastatin A (TBA), vincristine (VCR), or the indicated combination therapy for 24 h. A statistical analysis of the proportions of cells in the G2/M phase is shown in the right panel. b The levels of the apoptotic proteins caspase 3 and poly-ADP ribose polymerase (PARP) and the pro-survival proteins BCL-XL, BCL-2, and survivin were determined in cells treated with MPT0G211 (3 μM) or TBA (3 μM) in combination with VCR (1 nM) for 24 h. c The protein levels of cyclin B1, aurora B, p-CDC2, p-PLK, p-Histone 3, and MPM2 were evaluated following treatment with a combination of MPT0G211 or TBA with VCR for 24 h. d Cells were co-treated with MPT0G211 or TBA with VCR for 24 h and incubated with an α-tubulin antibody or DAPI. Microtubule dynamics were evaluated using a ZEISS LS 510META confocal microscope (magnification × 630). Scale bar = 20 μM. e Antitumor activity of MPT0G211 plus vincristine in a MOLT-4 xenograft model. When the tumor size reached 200 mm 3 , mice were injected with vehicle, vincristine (1 mg/kg, i.p., qwk), and MPT0G211 (30 mg/kg, i.p., qd) alone or a combination of both. The curves of tumor growth volume were expressed as mean ± SEM. f Changes of body weight after treatment. Data are shown as means ± standard errors of the means. * p

    Article Snippet: Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA).

    Techniques: Incubation, Microscopy, Activity Assay, Mouse Assay, Injection

    The effects of MPT0G211 on HL-60 and MOLT-4 cell growth and cell cycle progression. a Acetyl-α-tubulin and acetyl-histone 3 were detected in cells treated with MPT0G211 or tubastatin A (TBA) for 24 h. b HL-60, MOLT-4, and HUVECs were incubated with different concentrations of MPT0G211 or TBA for 48 h. Cell viability was evaluated using an MTT assay. c HL-60 and d MOLT-4 cells were treated with MPT0G211 or TBA for 48 h and analyzed by flow cytometry to assess cell cycle distribution. Data are shown as means ± standard errors of the means. * p

    Journal: Clinical Epigenetics

    Article Title: The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

    doi: 10.1186/s13148-018-0595-8

    Figure Lengend Snippet: The effects of MPT0G211 on HL-60 and MOLT-4 cell growth and cell cycle progression. a Acetyl-α-tubulin and acetyl-histone 3 were detected in cells treated with MPT0G211 or tubastatin A (TBA) for 24 h. b HL-60, MOLT-4, and HUVECs were incubated with different concentrations of MPT0G211 or TBA for 48 h. Cell viability was evaluated using an MTT assay. c HL-60 and d MOLT-4 cells were treated with MPT0G211 or TBA for 48 h and analyzed by flow cytometry to assess cell cycle distribution. Data are shown as means ± standard errors of the means. * p

    Article Snippet: Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA).

    Techniques: Incubation, MTT Assay, Flow Cytometry, Cytometry

    Ectopic expression of HDAC6 rescues cells from apoptosis induced by MPT0G211/doxorubicin combination. a HDAC6 levels were measured 18 h after transfection with an HDAC6-expression plasmid. b Following co-treatment with MPT0G211 and doxorubicin (DOXO) for 24 h, the levels of the apoptotic proteins poly-ADP ribose polymerase (PARP), caspase 3, γ-H2AX, and acetyl-α-tubulin were measured in HDAC6-overexpressing cells. c Cell cycle distribution was measured in HDAC6-transfected HL-60 cells following treatment with a combination of MPT0G211 and DOXO. A quantitative analysis of the proportions of cells in the sub-G1 phase of cell cycle is shown in d . Data are shown as means ± standard errors of the means. * p

    Journal: Clinical Epigenetics

    Article Title: The anticancer effects of MPT0G211, a novel HDAC6 inhibitor, combined with chemotherapeutic agents in human acute leukemia cells

    doi: 10.1186/s13148-018-0595-8

    Figure Lengend Snippet: Ectopic expression of HDAC6 rescues cells from apoptosis induced by MPT0G211/doxorubicin combination. a HDAC6 levels were measured 18 h after transfection with an HDAC6-expression plasmid. b Following co-treatment with MPT0G211 and doxorubicin (DOXO) for 24 h, the levels of the apoptotic proteins poly-ADP ribose polymerase (PARP), caspase 3, γ-H2AX, and acetyl-α-tubulin were measured in HDAC6-overexpressing cells. c Cell cycle distribution was measured in HDAC6-transfected HL-60 cells following treatment with a combination of MPT0G211 and DOXO. A quantitative analysis of the proportions of cells in the sub-G1 phase of cell cycle is shown in d . Data are shown as means ± standard errors of the means. * p

    Article Snippet: Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation

    RA specifically inhibited SRC/AKT signaling pathways during osteoclastogenesis. a RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 10, or 30 min, respectively. Western blotting for p-AKT was analyzed with the cell lysates. b RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 1, 3, or 5 days respectively. Western blotting for SRC was analyzed with the cell lysates. c RAW264.7 cells were treated with or without RA (0.8 μM) with the addition of RANKL (50 ng/mL) for 0, 10 or 30 min, respectively. Western blotting for MAPK and IκBα signaling pathways was analyzed with the cell lysates

    Journal: Cell Death & Disease

    Article Title: Raddeanin A suppresses breast cancer-associated osteolysis through inhibiting osteoclasts and breast cancer cells

    doi: 10.1038/s41419-018-0417-0

    Figure Lengend Snippet: RA specifically inhibited SRC/AKT signaling pathways during osteoclastogenesis. a RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 10, or 30 min, respectively. Western blotting for p-AKT was analyzed with the cell lysates. b RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 1, 3, or 5 days respectively. Western blotting for SRC was analyzed with the cell lysates. c RAW264.7 cells were treated with or without RA (0.8 μM) with the addition of RANKL (50 ng/mL) for 0, 10 or 30 min, respectively. Western blotting for MAPK and IκBα signaling pathways was analyzed with the cell lysates

    Article Snippet: Specific antibodies targeting SRC, ERK, JNK, p38, IκBα, phospho-IκBα, phospho-ERK, phospho-JNK, phospho-p38, phospho-AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signalling Technology (Cambridge, MA, USA).

    Techniques: Western Blot

    H3K36me3 increases across the early region of the HPV31 genome. ). To control for differences in viral copy number, PCR data was normalized to input values quantified in parallel for each experiment. The average fold change is graphed and the enrichment is expressed as percent input relative to the first primer set, which is set to one. Averages shown are representative of three independent experiments. Error bars represent means +/- standard error. (B) Western blot analysis was performed using antibodies to SETD2 and to involucrin and K10 as differentiation controls. p84 served as a loading control. Ca = calcium.

    Journal: PLoS Pathogens

    Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

    doi: 10.1371/journal.ppat.1007367

    Figure Lengend Snippet: H3K36me3 increases across the early region of the HPV31 genome. ). To control for differences in viral copy number, PCR data was normalized to input values quantified in parallel for each experiment. The average fold change is graphed and the enrichment is expressed as percent input relative to the first primer set, which is set to one. Averages shown are representative of three independent experiments. Error bars represent means +/- standard error. (B) Western blot analysis was performed using antibodies to SETD2 and to involucrin and K10 as differentiation controls. p84 served as a loading control. Ca = calcium.

    Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

    Techniques: Polymerase Chain Reaction, Western Blot

    SETD2 is increased post-transcriptionally in HPV31 positive cells in an E7-dependent manner. Whole cell lysates were harvested from (A) uninfected HFKs, HFKs retrovirally transduced and stably expressing either wild-type E7 or E7 containing a deletion of the Rb binding domain ( Δ LHCYE) as well as (B) HFKs, HFKs stably maintaining wild-type HPV31 genomes (HFK-31) or genomes containing the E7 Δ LHCYE mutation (HFK-31 Δ LHCYE). Western blot analysis was performed using antibodies to SETD2, (A) p53 and (B) pRb, with p84 as a loading control. Shown is a representative image of three independent experiments. (C) HFKs, HFKs containing wild-type HPV31 genomes (HFK-31) or genomes containing a mutation in E7’s Rb binding domain ( Δ LHCYE), as well as (D) HFKs and CIN612 were treated with 50 ug/ml cycloheximide over an 8hr time course. Lysates were harvested at the indicated time points and western blot analysis was performed using an antibody to SETD2, as well as p84 as a loading control. Graphed are the relative protein levels at each time point normalized to GAPDH, with T0 for each cell line set to 100. Densitometry was performed using Biorad ImageLab 5.0 software. Data shown is representative of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

    doi: 10.1371/journal.ppat.1007367

    Figure Lengend Snippet: SETD2 is increased post-transcriptionally in HPV31 positive cells in an E7-dependent manner. Whole cell lysates were harvested from (A) uninfected HFKs, HFKs retrovirally transduced and stably expressing either wild-type E7 or E7 containing a deletion of the Rb binding domain ( Δ LHCYE) as well as (B) HFKs, HFKs stably maintaining wild-type HPV31 genomes (HFK-31) or genomes containing the E7 Δ LHCYE mutation (HFK-31 Δ LHCYE). Western blot analysis was performed using antibodies to SETD2, (A) p53 and (B) pRb, with p84 as a loading control. Shown is a representative image of three independent experiments. (C) HFKs, HFKs containing wild-type HPV31 genomes (HFK-31) or genomes containing a mutation in E7’s Rb binding domain ( Δ LHCYE), as well as (D) HFKs and CIN612 were treated with 50 ug/ml cycloheximide over an 8hr time course. Lysates were harvested at the indicated time points and western blot analysis was performed using an antibody to SETD2, as well as p84 as a loading control. Graphed are the relative protein levels at each time point normalized to GAPDH, with T0 for each cell line set to 100. Densitometry was performed using Biorad ImageLab 5.0 software. Data shown is representative of three independent experiments.

    Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

    Techniques: Stable Transfection, Expressing, Binding Assay, Mutagenesis, Western Blot, Software

    SETD2 is necessary to maintain H3K36me3 on HPV31 chromatin. ). Data of ChIP signals from three independent experiments were normalized to 1% of input used. Shown in the fold change in H3K36me3 or H3.1 binding relative to the first primer set, which is set to one. Error bars represent means ± standard error. Statistics were assayed using a student’s t test. * p≤ .05 and ** p≤ .01. (D) Western blot analysis was performed using antibodies to SETD2, to involucrin and K10 as differentiation controls and p84 as a loading control. Ca = calcium.

    Journal: PLoS Pathogens

    Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

    doi: 10.1371/journal.ppat.1007367

    Figure Lengend Snippet: SETD2 is necessary to maintain H3K36me3 on HPV31 chromatin. ). Data of ChIP signals from three independent experiments were normalized to 1% of input used. Shown in the fold change in H3K36me3 or H3.1 binding relative to the first primer set, which is set to one. Error bars represent means ± standard error. Statistics were assayed using a student’s t test. * p≤ .05 and ** p≤ .01. (D) Western blot analysis was performed using antibodies to SETD2, to involucrin and K10 as differentiation controls and p84 as a loading control. Ca = calcium.

    Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Western Blot

    SETD2 protein levels are increased in HPV positive cells. (A) Lysates were harvested from undifferentiated human foreskin keratinocytes (HFKs), HFKs stably maintaining HPV16 (HFK-16) or HPV31 (HFK-31) genomes as well as HPV31 positive CIN612 cells, and western blot analysis was performed using an antibody to SETD2. GAPDH served as loading control. (B) Total RNA was extracted from undifferentiated HFKs, HFK-31, HFK-16 and CIN612 cells and quantitative RT-PCR was performed using primers specific to SETD2. Fold change was calculated using 2 −ΔΔCT method. Shown is the fold-change relative to HFKs, which is set to 1. The values represent the average of three independent experiments. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. (C) Lysates were harvested from undifferentiated (T0) HFKs and CIN612 cells, as well as after differentiation in high calcium medium (48, 96hr), and western blot analysis was performed using an antibodies to SETD2 as well as involucrin and keratin 10 (K10) as differentiation controls. p84 serving as a loading control,. (A, C) Shown is a representative image of three independent experiments. Ca = calcium.

    Journal: PLoS Pathogens

    Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

    doi: 10.1371/journal.ppat.1007367

    Figure Lengend Snippet: SETD2 protein levels are increased in HPV positive cells. (A) Lysates were harvested from undifferentiated human foreskin keratinocytes (HFKs), HFKs stably maintaining HPV16 (HFK-16) or HPV31 (HFK-31) genomes as well as HPV31 positive CIN612 cells, and western blot analysis was performed using an antibody to SETD2. GAPDH served as loading control. (B) Total RNA was extracted from undifferentiated HFKs, HFK-31, HFK-16 and CIN612 cells and quantitative RT-PCR was performed using primers specific to SETD2. Fold change was calculated using 2 −ΔΔCT method. Shown is the fold-change relative to HFKs, which is set to 1. The values represent the average of three independent experiments. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. (C) Lysates were harvested from undifferentiated (T0) HFKs and CIN612 cells, as well as after differentiation in high calcium medium (48, 96hr), and western blot analysis was performed using an antibodies to SETD2 as well as involucrin and keratin 10 (K10) as differentiation controls. p84 serving as a loading control,. (A, C) Shown is a representative image of three independent experiments. Ca = calcium.

    Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

    Techniques: Stable Transfection, Western Blot, Quantitative RT-PCR

    SETD2 is necessary for productive viral replication. (A) CIN612 cells were transiently transduced with either a scramble control shRNA (shScram) or one of two SETD2 shRNAs (shSetd2#1 and shSetd2#2) for 72hrs. At this time, DNA and protein were either harvested as an undifferentiated (T0) sample, or cells were grown in high calcium medium to induce differentiation (72hr). DNA was digested with BamHI, which does not cut the viral genome, and Southern blot analysis was performed using the HPV31 genome as a probe. Lysates harvested at the indicated time points were analyzed by immunoblotting to demonstrate the decrease in SETD2 and H3K36me3 upon shRNA-mediated knockdown. Involucrin and K10 were used as markers of differentiation, and p84 and histone H3.1 (H3.1) served as loading controls. (B) CIN612 cells were transduced with lentivirus expressing either control guide RNAs (sgCTR) or guide RNAs targeting SETD2 (sgSETD2 #1 and sgSETD2 #2) and selected with puromycin. Following selection, DNA and protein were harvested from the heterogenous population of cells. DNA was digested with BamHI (non-cutter) and Southern blot analysis performed using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of SETD2, involucrin and K10 as differentiation controls, with GAPDH as a loading control. (A, B) Fold change in episome copy number for SETD2 knockdown using shRNAs as well as guide RNAs was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Shown is the fold change relative to shScram T0 (A) and sgCTR T0 (B), which are set to one. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. WB = western blot. Ca = calcium.

    Journal: PLoS Pathogens

    Article Title: SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

    doi: 10.1371/journal.ppat.1007367

    Figure Lengend Snippet: SETD2 is necessary for productive viral replication. (A) CIN612 cells were transiently transduced with either a scramble control shRNA (shScram) or one of two SETD2 shRNAs (shSetd2#1 and shSetd2#2) for 72hrs. At this time, DNA and protein were either harvested as an undifferentiated (T0) sample, or cells were grown in high calcium medium to induce differentiation (72hr). DNA was digested with BamHI, which does not cut the viral genome, and Southern blot analysis was performed using the HPV31 genome as a probe. Lysates harvested at the indicated time points were analyzed by immunoblotting to demonstrate the decrease in SETD2 and H3K36me3 upon shRNA-mediated knockdown. Involucrin and K10 were used as markers of differentiation, and p84 and histone H3.1 (H3.1) served as loading controls. (B) CIN612 cells were transduced with lentivirus expressing either control guide RNAs (sgCTR) or guide RNAs targeting SETD2 (sgSETD2 #1 and sgSETD2 #2) and selected with puromycin. Following selection, DNA and protein were harvested from the heterogenous population of cells. DNA was digested with BamHI (non-cutter) and Southern blot analysis performed using the HPV31 genome as a probe. Western blot analysis was performed to examine the levels of SETD2, involucrin and K10 as differentiation controls, with GAPDH as a loading control. (A, B) Fold change in episome copy number for SETD2 knockdown using shRNAs as well as guide RNAs was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Shown is the fold change relative to shScram T0 (A) and sgCTR T0 (B), which are set to one. Error bars represent means +/- standard error. Statistics were assayed using a student’s t test. *p≤ .05. WB = western blot. Ca = calcium.

    Article Snippet: Primary antibodies used: anti-SETD2 (Kind gift from Dr. Brian Strahl, Epicypher), anti-H3K36me3, anti-cyclin B, anti-CDK2 (Abcam), anti-H3.1 (Active Motif), p84 (GeneTex), anti-Involucrin, anti-keratin 10, anti-CDC25C, anti-cyclin A, anti-cyclin E and anti-GAPDH (Santa Cruz), anti-CDK1, anti-RPA32 (Bethyl laboratories) and anti-Flag (Sigma).

    Techniques: Transduction, shRNA, Southern Blot, Expressing, Selection, Western Blot, Software

    RA specifically inhibited SRC/AKT signaling pathways during osteoclastogenesis. a RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 10, or 30 min, respectively. Western blotting for p-AKT was analyzed with the cell lysates. b RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 1, 3, or 5 days respectively. Western blotting for SRC was analyzed with the cell lysates. c RAW264.7 cells were treated with or without RA (0.8 μM) with the addition of RANKL (50 ng/mL) for 0, 10 or 30 min, respectively. Western blotting for MAPK and IκBα signaling pathways was analyzed with the cell lysates

    Journal: Cell Death & Disease

    Article Title: Raddeanin A suppresses breast cancer-associated osteolysis through inhibiting osteoclasts and breast cancer cells

    doi: 10.1038/s41419-018-0417-0

    Figure Lengend Snippet: RA specifically inhibited SRC/AKT signaling pathways during osteoclastogenesis. a RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 10, or 30 min, respectively. Western blotting for p-AKT was analyzed with the cell lysates. b RAW264.7 cells were treated with or without RANKL (50 ng/mL), RA (0.8 μM), or SC79 (25 μM) for 0, 1, 3, or 5 days respectively. Western blotting for SRC was analyzed with the cell lysates. c RAW264.7 cells were treated with or without RA (0.8 μM) with the addition of RANKL (50 ng/mL) for 0, 10 or 30 min, respectively. Western blotting for MAPK and IκBα signaling pathways was analyzed with the cell lysates

    Article Snippet: Specific antibodies targeting SRC, ERK, JNK, p38, IκBα, phospho-IκBα, phospho-ERK, phospho-JNK, phospho-p38, phospho-AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signalling Technology (Cambridge, MA, USA).

    Techniques: Western Blot

    Expression levels of TSG101 and Rab35 are lower in aged APOE4 mouse brains compared with age-matched APOE3 mice. ( A ) Representative western blot analyses of mouse hemibrain homogenates are shown for ALIX, TSG101, and Rab35, with β-actin used as a loading control. ( B – D ) Quantification of bands normalized to levels of β-actin is shown for levels of ALIX ( B ) , TSG101 ( C ), and Rab35 ( D ) . ( E ) Quantitative PCR analysis of 12-month-old mouse hemibrains shows Pdcd6ip (encodes ALIX), Tsg101 , and Rab35 transcript levels normalized to ddCT levels of the housekeeping gene Sdha . Data are expressed as the mean ± SEM of the ratios of APOE4 to APOE3 . * P

    Journal: Brain

    Article Title: Apolipoprotein E4 genotype compromises brain exosome production

    doi: 10.1093/brain/awy289

    Figure Lengend Snippet: Expression levels of TSG101 and Rab35 are lower in aged APOE4 mouse brains compared with age-matched APOE3 mice. ( A ) Representative western blot analyses of mouse hemibrain homogenates are shown for ALIX, TSG101, and Rab35, with β-actin used as a loading control. ( B – D ) Quantification of bands normalized to levels of β-actin is shown for levels of ALIX ( B ) , TSG101 ( C ), and Rab35 ( D ) . ( E ) Quantitative PCR analysis of 12-month-old mouse hemibrains shows Pdcd6ip (encodes ALIX), Tsg101 , and Rab35 transcript levels normalized to ddCT levels of the housekeeping gene Sdha . Data are expressed as the mean ± SEM of the ratios of APOE4 to APOE3 . * P

    Article Snippet: Membranes were incubated with antibodies against the exosomal markers ALIX (1:500, Millipore Sigma #ABC40), TSG101 (1:350, GeneTex Inc #GTX70255), Rab35 (1:1000, Cell Signaling #9690S), or β-actin (1:2500, Abcam #ab8226) overnight, and horseradish peroxidase-coupled secondary antibodies for 1 h. The membranes were incubated in chemiluminescent fluid (Pierce) for 5 min, and chemiluminescence was visualized on reflection autoradiography film.

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

    APOE4 -driven effects on exosome levels are gene dose dependent in 12-month-old mice. ( A ) Protein levels in sucrose gradient fractions b–d are shown for extracellular vesicles isolated from mouse hemibrains normalized to hemibrain weight. ( B ) ALIX, TSG101, and flotillin-1 were analysed by western blotting. Quantification of band intensities normalized to initial hemibrain weight are shown for levels of ALIX ( c ), TSG101 ( D ), and flotillin-1 ( E ). Data are expressed as the mean ± SEM of the ratios when compared with APOE3 . * P

    Journal: Brain

    Article Title: Apolipoprotein E4 genotype compromises brain exosome production

    doi: 10.1093/brain/awy289

    Figure Lengend Snippet: APOE4 -driven effects on exosome levels are gene dose dependent in 12-month-old mice. ( A ) Protein levels in sucrose gradient fractions b–d are shown for extracellular vesicles isolated from mouse hemibrains normalized to hemibrain weight. ( B ) ALIX, TSG101, and flotillin-1 were analysed by western blotting. Quantification of band intensities normalized to initial hemibrain weight are shown for levels of ALIX ( c ), TSG101 ( D ), and flotillin-1 ( E ). Data are expressed as the mean ± SEM of the ratios when compared with APOE3 . * P

    Article Snippet: Membranes were incubated with antibodies against the exosomal markers ALIX (1:500, Millipore Sigma #ABC40), TSG101 (1:350, GeneTex Inc #GTX70255), Rab35 (1:1000, Cell Signaling #9690S), or β-actin (1:2500, Abcam #ab8226) overnight, and horseradish peroxidase-coupled secondary antibodies for 1 h. The membranes were incubated in chemiluminescent fluid (Pierce) for 5 min, and chemiluminescence was visualized on reflection autoradiography film.

    Techniques: Mouse Assay, Isolation, Western Blot

    Brain exosome levels are lower in human APOE4 -carriers compared with homozygous APOE3 individuals. ( A ) Extracellular vesicle protein levels from human brains normalized to brain tissue weight. ( B ) A representative western blot showing ALIX and TSG101 (exosomal proteins), and flotillin-1 (a lipid-raft enriched protein) in the extracellular vesicles. ( C ) Quantification of band intensities normalized to brain tissue weight is shown for levels of ALIX, TSG101, and flotillin-1. Data are expressed as the mean ± SEM of the ratios of APOE4 carriers to APOE3 . * P

    Journal: Brain

    Article Title: Apolipoprotein E4 genotype compromises brain exosome production

    doi: 10.1093/brain/awy289

    Figure Lengend Snippet: Brain exosome levels are lower in human APOE4 -carriers compared with homozygous APOE3 individuals. ( A ) Extracellular vesicle protein levels from human brains normalized to brain tissue weight. ( B ) A representative western blot showing ALIX and TSG101 (exosomal proteins), and flotillin-1 (a lipid-raft enriched protein) in the extracellular vesicles. ( C ) Quantification of band intensities normalized to brain tissue weight is shown for levels of ALIX, TSG101, and flotillin-1. Data are expressed as the mean ± SEM of the ratios of APOE4 carriers to APOE3 . * P

    Article Snippet: Membranes were incubated with antibodies against the exosomal markers ALIX (1:500, Millipore Sigma #ABC40), TSG101 (1:350, GeneTex Inc #GTX70255), Rab35 (1:1000, Cell Signaling #9690S), or β-actin (1:2500, Abcam #ab8226) overnight, and horseradish peroxidase-coupled secondary antibodies for 1 h. The membranes were incubated in chemiluminescent fluid (Pierce) for 5 min, and chemiluminescence was visualized on reflection autoradiography film.

    Techniques: Western Blot

    Brain exosome levels are lower in APOE4 compared with APOE3 mice in an age-dependent manner. ( A ) Protein levels in sucrose gradient fractions b–d from each age group are shown for extracellular vesicles isolated from mouse hemibrains normalized to hemibrain weight. ( B ) ALIX, TSG101, and flotillin-1 were analysed by western blotting. Quantification of band intensities normalized to initial hemibrain weight are shown for levels of ALIX ( C ), TSG101 ( D ), and flotillin-1 ( E ) . Data are expressed as the mean ± SEM of the ratios of APOE4 to APOE3 . * P

    Journal: Brain

    Article Title: Apolipoprotein E4 genotype compromises brain exosome production

    doi: 10.1093/brain/awy289

    Figure Lengend Snippet: Brain exosome levels are lower in APOE4 compared with APOE3 mice in an age-dependent manner. ( A ) Protein levels in sucrose gradient fractions b–d from each age group are shown for extracellular vesicles isolated from mouse hemibrains normalized to hemibrain weight. ( B ) ALIX, TSG101, and flotillin-1 were analysed by western blotting. Quantification of band intensities normalized to initial hemibrain weight are shown for levels of ALIX ( C ), TSG101 ( D ), and flotillin-1 ( E ) . Data are expressed as the mean ± SEM of the ratios of APOE4 to APOE3 . * P

    Article Snippet: Membranes were incubated with antibodies against the exosomal markers ALIX (1:500, Millipore Sigma #ABC40), TSG101 (1:350, GeneTex Inc #GTX70255), Rab35 (1:1000, Cell Signaling #9690S), or β-actin (1:2500, Abcam #ab8226) overnight, and horseradish peroxidase-coupled secondary antibodies for 1 h. The membranes were incubated in chemiluminescent fluid (Pierce) for 5 min, and chemiluminescence was visualized on reflection autoradiography film.

    Techniques: Mouse Assay, Isolation, Western Blot

    NFATC2 is hypermethylated in patient fibroblasts. NFATC2 methylation in cells from patients with severe radiotherapy side-effects (n = 16) and controls (n = 8) were investigated on genome-wide methylation analysis. CpG sites in bold are differentially methylated between control and patient cells. CpG sites in red are part of promoter-associated regions defined by the ENCODE consortium.

    Journal: Frontiers in Oncology

    Article Title: NFATC2 Modulates Radiation Sensitivity in Dermal Fibroblasts From Patients With Severe Side Effects of Radiotherapy

    doi: 10.3389/fonc.2020.589168

    Figure Lengend Snippet: NFATC2 is hypermethylated in patient fibroblasts. NFATC2 methylation in cells from patients with severe radiotherapy side-effects (n = 16) and controls (n = 8) were investigated on genome-wide methylation analysis. CpG sites in bold are differentially methylated between control and patient cells. CpG sites in red are part of promoter-associated regions defined by the ENCODE consortium.

    Article Snippet: Interestingly, the authors detected these associations between actors of DNA damage response and NFATC2, but not with NFATC1, which suggests a specific role for NFATC2 in the DNA damage response ( ).

    Techniques: Methylation, Genome Wide

    NFATC2 downregulation in fibroblasts from overreacting patients. (A) NFATC2 mRNA levels were measured by RTqPCR in control cells (n = 8) and in fibroblasts from patients with severe radiotherapy side-effects (n = 16). Results are mean +/− SD. The p-value was calculated by Student’s t-test. Significant at *** P

    Journal: Frontiers in Oncology

    Article Title: NFATC2 Modulates Radiation Sensitivity in Dermal Fibroblasts From Patients With Severe Side Effects of Radiotherapy

    doi: 10.3389/fonc.2020.589168

    Figure Lengend Snippet: NFATC2 downregulation in fibroblasts from overreacting patients. (A) NFATC2 mRNA levels were measured by RTqPCR in control cells (n = 8) and in fibroblasts from patients with severe radiotherapy side-effects (n = 16). Results are mean +/− SD. The p-value was calculated by Student’s t-test. Significant at *** P

    Article Snippet: Interestingly, the authors detected these associations between actors of DNA damage response and NFATC2, but not with NFATC1, which suggests a specific role for NFATC2 in the DNA damage response ( ).

    Techniques:

    NFATC2 downregulation leads to cellular radiosensitivity. (A) NFATC2 mRNA levels were measured by RT-qPCR in three control fibroblast strains (HNF) infected with a lentiviral vector carrying either a shRNA scramble (sh- SCR ) sequence or a shRNA targeting NFATC2 (sh- NFATC2 ). Results are mean +/− SD. The p-value was calculated using a Student’s t-test. Significant at * P

    Journal: Frontiers in Oncology

    Article Title: NFATC2 Modulates Radiation Sensitivity in Dermal Fibroblasts From Patients With Severe Side Effects of Radiotherapy

    doi: 10.3389/fonc.2020.589168

    Figure Lengend Snippet: NFATC2 downregulation leads to cellular radiosensitivity. (A) NFATC2 mRNA levels were measured by RT-qPCR in three control fibroblast strains (HNF) infected with a lentiviral vector carrying either a shRNA scramble (sh- SCR ) sequence or a shRNA targeting NFATC2 (sh- NFATC2 ). Results are mean +/− SD. The p-value was calculated using a Student’s t-test. Significant at * P

    Article Snippet: Interestingly, the authors detected these associations between actors of DNA damage response and NFATC2, but not with NFATC1, which suggests a specific role for NFATC2 in the DNA damage response ( ).

    Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation, shRNA, Sequencing

    Micro-Western Array to screen for signaling molecules involved in H . capsulatum -induced macrophage response. Macrophages from WT mice were stimulated with or without (0 min) HK H . capsulatum for 15, 30, 60, 90, and 120 min. (A) Heat map chart shows MWA results. Protein abundance was normalized against the mean of β-actin and GAPDH. Black color indicates no change, while red and green indicate increase and decrease, respectively, of the levels of protein compared to unstimulated control. Proteins below the level of detection are in grey. (B) Cell lysates were subjected to Western blotting. Beta-actin was used as an internal control. Data shown are representative of 3 independent experiments with similar results.

    Journal: PLoS Pathogens

    Article Title: CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway

    doi: 10.1371/journal.ppat.1004985

    Figure Lengend Snippet: Micro-Western Array to screen for signaling molecules involved in H . capsulatum -induced macrophage response. Macrophages from WT mice were stimulated with or without (0 min) HK H . capsulatum for 15, 30, 60, 90, and 120 min. (A) Heat map chart shows MWA results. Protein abundance was normalized against the mean of β-actin and GAPDH. Black color indicates no change, while red and green indicate increase and decrease, respectively, of the levels of protein compared to unstimulated control. Proteins below the level of detection are in grey. (B) Cell lysates were subjected to Western blotting. Beta-actin was used as an internal control. Data shown are representative of 3 independent experiments with similar results.

    Article Snippet: Anti-Syk, anti-β-actin, HRP-conjugated anti-rabbit IgG, and HRP-conjugated anti-mouse IgG antibodies were purchased from GeneTex Inc. (Irvine, CA, USA).

    Techniques: Western Blot, Mouse Assay

    Clustering of CR3 and Dectin-1 on lipid rafts is required for their collaboration in cytokine production and signaling activation. (A and C) Macrophages were stimulated with or without (0 min) HK H . capsulatum for 15 or 30 min. Cells were fixed and stained for CR3 (red) and Dectin-1 (green) (A), or for p-Syk (green) (C). Cells were viewed under confocal microscope. Lipid raft was identified by staining with cholera toxin B (CTB) (violet). Nuclear compartment was stained by DAPI (blue). Arrowheads in the DIC/DAPI fields point to H . capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged images is shown in the histogram on the right. (B) Macrophages from WT mice were stimulated with or without (control) HK H . capsulatum for 30 min. Cell lysates were subjected to sucrose gradient ultracentrifugation. The fractions were collected and subjected to Western blotting and probed with anti-CD11b, anti-Dectin-1 and anti-flotillin-1 antibodies. (D and E) Macrophages were untreated or treated with methyl-β-cyclodextrin (MβCD, 10 mM) for 30 min. To reconstitute cholesterol, MβCD-treated cells were cultured in medium containing water-soluble cholesterol (MβCD-CHO, 400 μg/ml) for 1 h. After washing, macrophages were stimulated with HK H . capsulatum for 6 h (D), or 30 min (E). The concentrations of TNF and IL-6 in culture supernatants were analyzed by ELISA. Mean ± SD are shown (n = 10 from 4 independent experiments) (D). Cell lysates were analyzed by Western blotting by using antibodies against p-Syk, Syk, and β-actin. Data are representative of 2 (A and C) and 3 (B and E) independent experiments with similar results. ** p ≦ 0.01, *** p ≦ 0.001. NS, not significant [one-way ANOVA with Tukey post-hoc analysis (D)].

    Journal: PLoS Pathogens

    Article Title: CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway

    doi: 10.1371/journal.ppat.1004985

    Figure Lengend Snippet: Clustering of CR3 and Dectin-1 on lipid rafts is required for their collaboration in cytokine production and signaling activation. (A and C) Macrophages were stimulated with or without (0 min) HK H . capsulatum for 15 or 30 min. Cells were fixed and stained for CR3 (red) and Dectin-1 (green) (A), or for p-Syk (green) (C). Cells were viewed under confocal microscope. Lipid raft was identified by staining with cholera toxin B (CTB) (violet). Nuclear compartment was stained by DAPI (blue). Arrowheads in the DIC/DAPI fields point to H . capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged images is shown in the histogram on the right. (B) Macrophages from WT mice were stimulated with or without (control) HK H . capsulatum for 30 min. Cell lysates were subjected to sucrose gradient ultracentrifugation. The fractions were collected and subjected to Western blotting and probed with anti-CD11b, anti-Dectin-1 and anti-flotillin-1 antibodies. (D and E) Macrophages were untreated or treated with methyl-β-cyclodextrin (MβCD, 10 mM) for 30 min. To reconstitute cholesterol, MβCD-treated cells were cultured in medium containing water-soluble cholesterol (MβCD-CHO, 400 μg/ml) for 1 h. After washing, macrophages were stimulated with HK H . capsulatum for 6 h (D), or 30 min (E). The concentrations of TNF and IL-6 in culture supernatants were analyzed by ELISA. Mean ± SD are shown (n = 10 from 4 independent experiments) (D). Cell lysates were analyzed by Western blotting by using antibodies against p-Syk, Syk, and β-actin. Data are representative of 2 (A and C) and 3 (B and E) independent experiments with similar results. ** p ≦ 0.01, *** p ≦ 0.001. NS, not significant [one-way ANOVA with Tukey post-hoc analysis (D)].

    Article Snippet: Anti-Syk, anti-β-actin, HRP-conjugated anti-rabbit IgG, and HRP-conjugated anti-mouse IgG antibodies were purchased from GeneTex Inc. (Irvine, CA, USA).

    Techniques: Activation Assay, Staining, Microscopy, CtB Assay, Mouse Assay, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

    Knockdown of IQGAP1 inhibited proliferation and EMT of thyroid cancer cells. Notes: SW579 and TPC-1 cells transfected with si-control, si-IQGAP1-1 or si-IQGAP1-2 were cultured for 48 h. ( A ) qRT-PCR analysis of IQGAP1 mRNA level in transfected cells. ( B ) Western blot analysis of IQGAP1 protein in transfected cells. ( C ) MTT assay was performed to detect viability in transfected cells. ( D and E ) Western blot analysis of E-cadherin, N-cadherin, Vimentin and Twist in transfected cells. * P

    Journal: OncoTargets and therapy

    Article Title: Knockdown of IQGAP1 inhibits proliferation and epithelial–mesenchymal transition by Wnt/β-catenin pathway in thyroid cancer

    doi: 10.2147/OTT.S128564

    Figure Lengend Snippet: Knockdown of IQGAP1 inhibited proliferation and EMT of thyroid cancer cells. Notes: SW579 and TPC-1 cells transfected with si-control, si-IQGAP1-1 or si-IQGAP1-2 were cultured for 48 h. ( A ) qRT-PCR analysis of IQGAP1 mRNA level in transfected cells. ( B ) Western blot analysis of IQGAP1 protein in transfected cells. ( C ) MTT assay was performed to detect viability in transfected cells. ( D and E ) Western blot analysis of E-cadherin, N-cadherin, Vimentin and Twist in transfected cells. * P

    Article Snippet: When the Wnt/β-catenin pathway is activated, Wnt molecules bind to frizzled receptors and lead to destruction of the complex, and then β-catenin transfers into the nucleus and accumulate, finally inducing the loss of E-cadherin., The Wnt/β-catenin signaling pathway is involved in EMT in gastrulation, cardiac valve formation and cancer.

    Techniques: Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, MTT Assay

    Downregulation of IQGAP1 suppressed the growth of SW579 tumor xenografts in vivo. Notes: When the tumor volume reached the required size (50–100 mm 3 ), 5 μg siRNA (si-control or si-IQGAP1-2) was injected into tumor cavity daily for 21 days. The mice were euthanized at day 21. ( A ) Effects of IQGAP1 knockdown on the growth of SW579 xenografts. ( B ) Tumor weights were measured at day 21. ( C and D ) Western blot analysis of β-catenin, cyclin D1 and c-myc in resected tumor tissues. ( E and F ) Western blot analysis of E-cadherin, N-cadherin, Vimentin and Twist excised in tumor tissues. n=5 and * P

    Journal: OncoTargets and therapy

    Article Title: Knockdown of IQGAP1 inhibits proliferation and epithelial–mesenchymal transition by Wnt/β-catenin pathway in thyroid cancer

    doi: 10.2147/OTT.S128564

    Figure Lengend Snippet: Downregulation of IQGAP1 suppressed the growth of SW579 tumor xenografts in vivo. Notes: When the tumor volume reached the required size (50–100 mm 3 ), 5 μg siRNA (si-control or si-IQGAP1-2) was injected into tumor cavity daily for 21 days. The mice were euthanized at day 21. ( A ) Effects of IQGAP1 knockdown on the growth of SW579 xenografts. ( B ) Tumor weights were measured at day 21. ( C and D ) Western blot analysis of β-catenin, cyclin D1 and c-myc in resected tumor tissues. ( E and F ) Western blot analysis of E-cadherin, N-cadherin, Vimentin and Twist excised in tumor tissues. n=5 and * P

    Article Snippet: When the Wnt/β-catenin pathway is activated, Wnt molecules bind to frizzled receptors and lead to destruction of the complex, and then β-catenin transfers into the nucleus and accumulate, finally inducing the loss of E-cadherin., The Wnt/β-catenin signaling pathway is involved in EMT in gastrulation, cardiac valve formation and cancer.

    Techniques: In Vivo, Injection, Mouse Assay, Western Blot

    Inactivation of the Wnt/β-catenin pathway reversed the effects of IQGAP1 overexpression on thyroid cancer cells. Notes: ( A and B ) Effects of combination of pcDNA-IQGAP1 and XAV939 on E-cadherin, N-cadherin, Vimentin and Twist in SW579 cells. ( C ) MTT assay was used to detect the effects of combination of pcDNA-IQGAP1 and XAV939 on proliferation in SW579 cells. ( D and E ) Effects of co-transfection with pcDNA-IQGAP1 and XAV939 on E-cadherin, N-cadherin, Vimentin and Twist in TCP-1 cells. ( F ) MTT assay was used to measure the effects of co-transfection with pcDNA-IQGAP1 and XAV939 on proliferation in TPC-1 cells. * P

    Journal: OncoTargets and therapy

    Article Title: Knockdown of IQGAP1 inhibits proliferation and epithelial–mesenchymal transition by Wnt/β-catenin pathway in thyroid cancer

    doi: 10.2147/OTT.S128564

    Figure Lengend Snippet: Inactivation of the Wnt/β-catenin pathway reversed the effects of IQGAP1 overexpression on thyroid cancer cells. Notes: ( A and B ) Effects of combination of pcDNA-IQGAP1 and XAV939 on E-cadherin, N-cadherin, Vimentin and Twist in SW579 cells. ( C ) MTT assay was used to detect the effects of combination of pcDNA-IQGAP1 and XAV939 on proliferation in SW579 cells. ( D and E ) Effects of co-transfection with pcDNA-IQGAP1 and XAV939 on E-cadherin, N-cadherin, Vimentin and Twist in TCP-1 cells. ( F ) MTT assay was used to measure the effects of co-transfection with pcDNA-IQGAP1 and XAV939 on proliferation in TPC-1 cells. * P

    Article Snippet: When the Wnt/β-catenin pathway is activated, Wnt molecules bind to frizzled receptors and lead to destruction of the complex, and then β-catenin transfers into the nucleus and accumulate, finally inducing the loss of E-cadherin., The Wnt/β-catenin signaling pathway is involved in EMT in gastrulation, cardiac valve formation and cancer.

    Techniques: Over Expression, MTT Assay, Cotransfection

    Inactivation of the Wnt/β-catenin pathway suppressed cell proliferation and EMT in thyroid cancer cells. Notes: SW579 cells were treated with different concentrations of Wnt/β-catenin signaling inhibitor XAV939 (5, 10 and 20 μM), and TPC-1 cells were transfected with si-β-catenin-1 or si-β-catenin-2. ( A and B ) Western blot analysis of β-catenin, cyclin D1 and c-myc in SW579 cells at 48 h. ( C and D ) Western blot analysis of E-cadherin, N-cadherin, Vimentin and Twist in SW579 cells at 48 h. ( E and F ) Western blot analysis was performed to test the expression of β-catenin, cyclin D1 and c-myc in TPC-1 cells at 48 h. ( G and H ) Western blot analysis was applied to determine the level of E-cadherin, N-cadherin, Vimentin and Twist in TPC-1 cells at 48 h. ( I ) MTT assay was applied to examine the viability of SW579 cells at 48 h. ( J ) MTT assay was carried out to measure the viability of TPC-1 cells at 48 h. * P

    Journal: OncoTargets and therapy

    Article Title: Knockdown of IQGAP1 inhibits proliferation and epithelial–mesenchymal transition by Wnt/β-catenin pathway in thyroid cancer

    doi: 10.2147/OTT.S128564

    Figure Lengend Snippet: Inactivation of the Wnt/β-catenin pathway suppressed cell proliferation and EMT in thyroid cancer cells. Notes: SW579 cells were treated with different concentrations of Wnt/β-catenin signaling inhibitor XAV939 (5, 10 and 20 μM), and TPC-1 cells were transfected with si-β-catenin-1 or si-β-catenin-2. ( A and B ) Western blot analysis of β-catenin, cyclin D1 and c-myc in SW579 cells at 48 h. ( C and D ) Western blot analysis of E-cadherin, N-cadherin, Vimentin and Twist in SW579 cells at 48 h. ( E and F ) Western blot analysis was performed to test the expression of β-catenin, cyclin D1 and c-myc in TPC-1 cells at 48 h. ( G and H ) Western blot analysis was applied to determine the level of E-cadherin, N-cadherin, Vimentin and Twist in TPC-1 cells at 48 h. ( I ) MTT assay was applied to examine the viability of SW579 cells at 48 h. ( J ) MTT assay was carried out to measure the viability of TPC-1 cells at 48 h. * P

    Article Snippet: When the Wnt/β-catenin pathway is activated, Wnt molecules bind to frizzled receptors and lead to destruction of the complex, and then β-catenin transfers into the nucleus and accumulate, finally inducing the loss of E-cadherin., The Wnt/β-catenin signaling pathway is involved in EMT in gastrulation, cardiac valve formation and cancer.

    Techniques: Transfection, Western Blot, Expressing, MTT Assay

    PPD inhibited osteoclast differentiation by specifically impairing RANKL-induced MAPK cascades and the NF-κB pathway. (A,E) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Cell lysates were analyzed using western blotting with specific antibodies against phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-JNK1/2, JNK1/2, phospho-IκBα, IκBα, phospho-TAK1, TAK1, and β-actin. (B–D) The gray levels corresponding to phosphorylation of the indicated proteins were quantified and normalized relative to β-actin using Image J for p-p38, p-JNK, and p-ERK, (F,G) The gray levels corresponding to phosphorylation of the indicated proteins were quantified and normalized relative to β-actin using Image J for p-TAK1 and p-IκBα. Significant differences between the groups were determined by paired Student’s t -test. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: 20(S)-Protopanaxadiol Inhibits Titanium Particle-Induced Inflammatory Osteolysis and RANKL-Mediated Osteoclastogenesis via MAPK and NF-κB Signaling Pathways

    doi: 10.3389/fphar.2018.01538

    Figure Lengend Snippet: PPD inhibited osteoclast differentiation by specifically impairing RANKL-induced MAPK cascades and the NF-κB pathway. (A,E) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Cell lysates were analyzed using western blotting with specific antibodies against phospho-p38, p38, phospho-ERK1/2, ERK1/2, phospho-JNK1/2, JNK1/2, phospho-IκBα, IκBα, phospho-TAK1, TAK1, and β-actin. (B–D) The gray levels corresponding to phosphorylation of the indicated proteins were quantified and normalized relative to β-actin using Image J for p-p38, p-JNK, and p-ERK, (F,G) The gray levels corresponding to phosphorylation of the indicated proteins were quantified and normalized relative to β-actin using Image J for p-TAK1 and p-IκBα. Significant differences between the groups were determined by paired Student’s t -test. ∗ P

    Article Snippet: Specific primary antibodies against NF-κB inhibitor alpha (IκBα) (#4814), p-IκBα (#9246), NF-κB p65 (#8242), phospho-NF-κB p-p65 (#3033), ERK1/2 (#4695), phospho-ERK1/2 (#4370), SAPK/JNK (#9252), phospho-SAPK/JNK (#4668), p38 (#8690), phospho-p38 (#4511), histone H3 (#4499), β-actin(#4967) and c-Fos (#4384) were purchased from Cell Signaling Technology (MA, United States).

    Techniques: Western Blot

    H19 regulates TGFBR2 and TSP1 expression in a TET1‐dependent manner. A, HUVECs were transfected with siCon, siH19, or siH19 plus pTET1 (human TET1‐expressing plasmid). RNA and protein were extracted 48 and 72 hours later, respectively, and analyzed by RT‐qPCR (A) and Western blot (B). * P

    Journal: The FASEB Journal

    Article Title: H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition, et al. H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition

    doi: 10.1096/fj.202000073RRRRR

    Figure Lengend Snippet: H19 regulates TGFBR2 and TSP1 expression in a TET1‐dependent manner. A, HUVECs were transfected with siCon, siH19, or siH19 plus pTET1 (human TET1‐expressing plasmid). RNA and protein were extracted 48 and 72 hours later, respectively, and analyzed by RT‐qPCR (A) and Western blot (B). * P

    Article Snippet: Moreover, there was a strong positive linear correlation between luminal EC expression of H19 and FN1, H19 and VIM, TET1 and FN1, and TET1 and VIM (Figure ).

    Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    Overexpression of H19 or TET1 promotes TGF‐β signaling and EndMT marker expression in vitro. HUVECs (A and B) or HAoECs (C and D) were transfected with empty vector, pH19, or pTET1. Proteins were extracted 48 hours post‐transfection and analyzed by Western blot, with quantification of indicated proteins shown on the right. * P

    Journal: The FASEB Journal

    Article Title: H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition, et al. H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition

    doi: 10.1096/fj.202000073RRRRR

    Figure Lengend Snippet: Overexpression of H19 or TET1 promotes TGF‐β signaling and EndMT marker expression in vitro. HUVECs (A and B) or HAoECs (C and D) were transfected with empty vector, pH19, or pTET1. Proteins were extracted 48 hours post‐transfection and analyzed by Western blot, with quantification of indicated proteins shown on the right. * P

    Article Snippet: Moreover, there was a strong positive linear correlation between luminal EC expression of H19 and FN1, H19 and VIM, TET1 and FN1, and TET1 and VIM (Figure ).

    Techniques: Over Expression, Marker, Expressing, In Vitro, Transfection, Plasmid Preparation, Western Blot

    H19 regulates TET1 expression posttranscriptionally via reducing the bioavailability of let‐7. A, Schematics of human and mouse TET1 mRNAs, with numbers on top depicting positions of let‐7‐binding sites relative to the transcriptional start sites. Figures are not drawn to scale. B and C, HUVECs were transfected with a mixture of siCon and iCon, siH19 and iCon, or siH19 and iLet7. RNA and protein were isolated 48 hours later and analyzed by RT‐qPCR (B) and Western blot (C). In C, protein samples were loaded in triplicate, with quantification of TET1 protein shown on the right. GAPDH was used as a loading control. D, Top, reporter constructs; bottom, the indicated constructs were each transfected into U‐2 OS cells, together with control miRNA (Con), let‐7b, or miR‐133 at a final concentration of 15 or 30 nM. * P

    Journal: The FASEB Journal

    Article Title: H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition, et al. H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition

    doi: 10.1096/fj.202000073RRRRR

    Figure Lengend Snippet: H19 regulates TET1 expression posttranscriptionally via reducing the bioavailability of let‐7. A, Schematics of human and mouse TET1 mRNAs, with numbers on top depicting positions of let‐7‐binding sites relative to the transcriptional start sites. Figures are not drawn to scale. B and C, HUVECs were transfected with a mixture of siCon and iCon, siH19 and iCon, or siH19 and iLet7. RNA and protein were isolated 48 hours later and analyzed by RT‐qPCR (B) and Western blot (C). In C, protein samples were loaded in triplicate, with quantification of TET1 protein shown on the right. GAPDH was used as a loading control. D, Top, reporter constructs; bottom, the indicated constructs were each transfected into U‐2 OS cells, together with control miRNA (Con), let‐7b, or miR‐133 at a final concentration of 15 or 30 nM. * P

    Article Snippet: Moreover, there was a strong positive linear correlation between luminal EC expression of H19 and FN1, H19 and VIM, TET1 and FN1, and TET1 and VIM (Figure ).

    Techniques: Expressing, Binding Assay, Transfection, Isolation, Quantitative RT-PCR, Western Blot, Construct, Concentration Assay

    TET1 affects promoter methylation and histone modifications of TGFBR2 and TSP1. A and B, HUVECs were transfected with siCon or siTET1. RNA and protein were isolated 48 hours later and analyzed by RT‐qPCR (A) and Western blot (B). In B, protein samples were loaded in triplicate, with quantification of TGFBR2 and TSP1 proteins shown on the bottom. C, HUVECs were transfected with siCon or siTET1, followed by ChIP‐qPCR analysis. Data are presented as mean relative TET1 enrichment over input, with gray bars representing background IgG signal. Numbers indicate nucleotide positions relative to the transcriptional start sites, with PCR products depicted as red bars underneath. D, Sequences of critical transcription regulatory regions (CTRR) of TGFBR2 and TSP1. The differentially methylated cytosine residues are marked in red. The red numbers mark the positions of the indicated nucleotides relative to the transcriptional start sites. E, HUVECs were transfected with siCon or siTET1 for 48 hours, followed by QMSP analysis. F and G, HUVECs were transfected with siCon or siTET1 for 48 hours, followed by ChIP‐qPCR analysis. * P

    Journal: The FASEB Journal

    Article Title: H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition, et al. H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition

    doi: 10.1096/fj.202000073RRRRR

    Figure Lengend Snippet: TET1 affects promoter methylation and histone modifications of TGFBR2 and TSP1. A and B, HUVECs were transfected with siCon or siTET1. RNA and protein were isolated 48 hours later and analyzed by RT‐qPCR (A) and Western blot (B). In B, protein samples were loaded in triplicate, with quantification of TGFBR2 and TSP1 proteins shown on the bottom. C, HUVECs were transfected with siCon or siTET1, followed by ChIP‐qPCR analysis. Data are presented as mean relative TET1 enrichment over input, with gray bars representing background IgG signal. Numbers indicate nucleotide positions relative to the transcriptional start sites, with PCR products depicted as red bars underneath. D, Sequences of critical transcription regulatory regions (CTRR) of TGFBR2 and TSP1. The differentially methylated cytosine residues are marked in red. The red numbers mark the positions of the indicated nucleotides relative to the transcriptional start sites. E, HUVECs were transfected with siCon or siTET1 for 48 hours, followed by QMSP analysis. F and G, HUVECs were transfected with siCon or siTET1 for 48 hours, followed by ChIP‐qPCR analysis. * P

    Article Snippet: Moreover, there was a strong positive linear correlation between luminal EC expression of H19 and FN1, H19 and VIM, TET1 and FN1, and TET1 and VIM (Figure ).

    Techniques: Methylation, Transfection, Isolation, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    H19 depletion abrogates endothelial activation of TGF‐β signaling and EndMT marker expression in vivo. A, ELISA analysis of serum TNF‐α levels of WT and KO mice 2 weeks after initial injection of STZ (+) or vehicle (−), n =3‐ 4 animals per group. B, RT‐qPCR analysis of expression of H19, TET1, TGFBR2, and TSP1 in lung ECs collected from WT and KO mice 5 weeks following initial injection of STZ (+) or vehicle (−). n = 3 animals per group. C, Western blot analysis of indicated proteins in lung ECs collected from WT and KO mice 5 weeks following initial injection of STZ (+) or vehicle (−). n = 3 animals per group. D, Quantification of proteins in C. * P

    Journal: The FASEB Journal

    Article Title: H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition, et al. H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition

    doi: 10.1096/fj.202000073RRRRR

    Figure Lengend Snippet: H19 depletion abrogates endothelial activation of TGF‐β signaling and EndMT marker expression in vivo. A, ELISA analysis of serum TNF‐α levels of WT and KO mice 2 weeks after initial injection of STZ (+) or vehicle (−), n =3‐ 4 animals per group. B, RT‐qPCR analysis of expression of H19, TET1, TGFBR2, and TSP1 in lung ECs collected from WT and KO mice 5 weeks following initial injection of STZ (+) or vehicle (−). n = 3 animals per group. C, Western blot analysis of indicated proteins in lung ECs collected from WT and KO mice 5 weeks following initial injection of STZ (+) or vehicle (−). n = 3 animals per group. D, Quantification of proteins in C. * P

    Article Snippet: Moreover, there was a strong positive linear correlation between luminal EC expression of H19 and FN1, H19 and VIM, TET1 and FN1, and TET1 and VIM (Figure ).

    Techniques: Activation Assay, Marker, Expressing, In Vivo, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Quantitative RT-PCR, Western Blot

    H19 and TET1 expression in ECs in human coronary arteries. Left main coronary arteries from patients with no/mild, moderate, and severe CAD were assessed. n = 5 in each group. A, Representative images of RNA ISH staining of left main coronary arteries from patients for H19 (blue) and CD31 (red), with nuclei stained in purple. Note, a fraction of CD31 mRNA is also present in the nucleus which overlaps with the purple nuclear counterstaining. Scale bar: 100 μm. C, Representative images of immunofluorescence staining for TET1 (green) and CD31 (red), with nuclei stained with DAPI in blue. Scale bar: 100 μm. B and D, Left panels: percentage of H19 + ECs or TET1 + ECs in the lumen. ** P

    Journal: The FASEB Journal

    Article Title: H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition, et al. H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition

    doi: 10.1096/fj.202000073RRRRR

    Figure Lengend Snippet: H19 and TET1 expression in ECs in human coronary arteries. Left main coronary arteries from patients with no/mild, moderate, and severe CAD were assessed. n = 5 in each group. A, Representative images of RNA ISH staining of left main coronary arteries from patients for H19 (blue) and CD31 (red), with nuclei stained in purple. Note, a fraction of CD31 mRNA is also present in the nucleus which overlaps with the purple nuclear counterstaining. Scale bar: 100 μm. C, Representative images of immunofluorescence staining for TET1 (green) and CD31 (red), with nuclei stained with DAPI in blue. Scale bar: 100 μm. B and D, Left panels: percentage of H19 + ECs or TET1 + ECs in the lumen. ** P

    Article Snippet: Moreover, there was a strong positive linear correlation between luminal EC expression of H19 and FN1, H19 and VIM, TET1 and FN1, and TET1 and VIM (Figure ).

    Techniques: Expressing, In Situ Hybridization, Staining, Immunofluorescence

    TNF‐α upregulates endothelial expression of H19 and TGF‐β signaling genes in a H19‐dependent manner. RT‐qPCR analysis of H19 in TNF‐α‐treated (overnight) HUVECs (A) or HAoECs (B). One‐way ANOVA with Newman‐Keuls post hoc test. C, RT‐qPCR analysis of H19, TET1, TGFBR2, and TSP1 48 hours after transfection of HUVECs with control siRNA (siCon) or H19‐specific siRNA (siH19). D, RT‐qPCR analysis of the indicated genes 48 hours after transfection of HUVECs with empty vector (Vec) or human H19‐expressing plasmid (pH19). E and F, RT‐qPCR of H19, TET1, TGFBR2, and TSP1 following transfection of siCon (‐) or siH19 (+) for 24 hours and treatment with TNF‐α (10 ng/mL) (+) or vehicle (‐) for an additional 24 hours in HUVECs (E) or HAoECs (F). Two‐way ANOVA with Newman‐Keuls post hoc test. G, Western blots of TET1 following TNF‐α treatment of HUVECs or HAoECs. * P

    Journal: The FASEB Journal

    Article Title: H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition, et al. H19/TET1 axis promotes TGF‐β signaling linked to endothelial‐to‐mesenchymal transition

    doi: 10.1096/fj.202000073RRRRR

    Figure Lengend Snippet: TNF‐α upregulates endothelial expression of H19 and TGF‐β signaling genes in a H19‐dependent manner. RT‐qPCR analysis of H19 in TNF‐α‐treated (overnight) HUVECs (A) or HAoECs (B). One‐way ANOVA with Newman‐Keuls post hoc test. C, RT‐qPCR analysis of H19, TET1, TGFBR2, and TSP1 48 hours after transfection of HUVECs with control siRNA (siCon) or H19‐specific siRNA (siH19). D, RT‐qPCR analysis of the indicated genes 48 hours after transfection of HUVECs with empty vector (Vec) or human H19‐expressing plasmid (pH19). E and F, RT‐qPCR of H19, TET1, TGFBR2, and TSP1 following transfection of siCon (‐) or siH19 (+) for 24 hours and treatment with TNF‐α (10 ng/mL) (+) or vehicle (‐) for an additional 24 hours in HUVECs (E) or HAoECs (F). Two‐way ANOVA with Newman‐Keuls post hoc test. G, Western blots of TET1 following TNF‐α treatment of HUVECs or HAoECs. * P

    Article Snippet: Moreover, there was a strong positive linear correlation between luminal EC expression of H19 and FN1, H19 and VIM, TET1 and FN1, and TET1 and VIM (Figure ).

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot

    Ubiquitin‐specific protease 19 (USP19) expression is increased in murine hypertrophic hearts and cardiomyocytes. A, Left, Western blot bands of USP19 in mice subjected to sham or transverse aortic constriction (TAC) surgery at 4 wk. Right, protein expression levels were normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and compared between indicated groups (n = 3, ** P < 0.01 vs sham). B, Left, Western blot analysis of USP19 in neonatal rat cardiomyocytes treated with phosphate buffered saline (PBS) or phenylephrine (PE) at 24 h. Right, protein expression levels were normalized to GAPDH and compared between indicated groups (n = 3, ** P < 0.01 vs PBS)

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway. Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway

    doi: 10.1111/jcmm.15724

    Figure Lengend Snippet: Ubiquitin‐specific protease 19 (USP19) expression is increased in murine hypertrophic hearts and cardiomyocytes. A, Left, Western blot bands of USP19 in mice subjected to sham or transverse aortic constriction (TAC) surgery at 4 wk. Right, protein expression levels were normalized to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and compared between indicated groups (n = 3, ** P < 0.01 vs sham). B, Left, Western blot analysis of USP19 in neonatal rat cardiomyocytes treated with phosphate buffered saline (PBS) or phenylephrine (PE) at 24 h. Right, protein expression levels were normalized to GAPDH and compared between indicated groups (n = 3, ** P < 0.01 vs PBS)

    Article Snippet: 3.2 USP19 suppressed cardiac hypertrophy via blocking TAK1‐dependent pathwaySince the anti‐hypertrophy function of USP19 was confirmed, the investigators determined the underlying mechanism that mediated this effect.

    Techniques: Expressing, Western Blot, Mouse Assay

    Ubiquitin‐specific protease 19 (USP19) reduces TAK1‐p38/JNK1/2 phosphorylation levels in hypertrophic hearts and cardiomyocytes. A, Above, Western blots showing the phosphorylation and total protein levels of TAK1, ERK1/2, JNK1/2 and p38 in primary neonatal rat cardiomyocytes (NRCMs) infected with AdshRNA or short hairpin RNA (AdshUSP19) followed by treatment with phosphate buffered saline (PBS) or phenylephrine (PE). Bottom, Statistical analysis of the phosphorylation levels of TAK1, ERK1/2, JNK1/2 and p38 in PE‐treated AdshRNA and AdshUSP19 groups. n = 6, ** P < 0.01 vs AdshRNA/PE. B, Above, Western blots showing the phosphorylation and total protein levels of TAK1, ERK1/2, JNK1/2 and p38 in NRCMs infected with AdGFP or AdUSP19 followed by treatment with PBS or PE. Bottom, Statistical analysis of the phosphorylation levels of TAK1, ERK1/2, JNK1/2 and p38 in PE‐treated AdGFP and AdUSP19 groups. n = 6, ** P < 0.01 vs AdGFP/PE. C, Above, Western blots showing the phosphorylation and total protein levels of TAK1, ERK1/2, JNK1/2 and p38 in wild‐type (WT) or USP19‐knockout (KO) mice subjected to sham or transverse aortic constriction (TAC) surgery. Bottom, densitometry analyses of the phosphorylation levels of TAK1, ERK1/2, JNK1/2 and p38 in TAC‐treated WT and KO mice. n = 6, ** P < 0.01 vs WT/TAC. n.s., no significance

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway. Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway

    doi: 10.1111/jcmm.15724

    Figure Lengend Snippet: Ubiquitin‐specific protease 19 (USP19) reduces TAK1‐p38/JNK1/2 phosphorylation levels in hypertrophic hearts and cardiomyocytes. A, Above, Western blots showing the phosphorylation and total protein levels of TAK1, ERK1/2, JNK1/2 and p38 in primary neonatal rat cardiomyocytes (NRCMs) infected with AdshRNA or short hairpin RNA (AdshUSP19) followed by treatment with phosphate buffered saline (PBS) or phenylephrine (PE). Bottom, Statistical analysis of the phosphorylation levels of TAK1, ERK1/2, JNK1/2 and p38 in PE‐treated AdshRNA and AdshUSP19 groups. n = 6, ** P < 0.01 vs AdshRNA/PE. B, Above, Western blots showing the phosphorylation and total protein levels of TAK1, ERK1/2, JNK1/2 and p38 in NRCMs infected with AdGFP or AdUSP19 followed by treatment with PBS or PE. Bottom, Statistical analysis of the phosphorylation levels of TAK1, ERK1/2, JNK1/2 and p38 in PE‐treated AdGFP and AdUSP19 groups. n = 6, ** P < 0.01 vs AdGFP/PE. C, Above, Western blots showing the phosphorylation and total protein levels of TAK1, ERK1/2, JNK1/2 and p38 in wild‐type (WT) or USP19‐knockout (KO) mice subjected to sham or transverse aortic constriction (TAC) surgery. Bottom, densitometry analyses of the phosphorylation levels of TAK1, ERK1/2, JNK1/2 and p38 in TAC‐treated WT and KO mice. n = 6, ** P < 0.01 vs WT/TAC. n.s., no significance

    Article Snippet: 3.2 USP19 suppressed cardiac hypertrophy via blocking TAK1‐dependent pathwaySince the anti‐hypertrophy function of USP19 was confirmed, the investigators determined the underlying mechanism that mediated this effect.

    Techniques: Western Blot, Infection, shRNA, Knock-Out, Mouse Assay

    Ubiquitin‐specific protease 19 (USP19) deficiency exacerbates heart hypertrophy and dysfunction in response to transverse aortic constriction (TAC) in vivo. A, Western blot analysis revealing successful construction of USP19‐knockout (KO) mice. B‐D, Four weeks after TAC, the ratios of heart weight/bodyweight (HW/BW; left), lung weight/bodyweight (LW/BW; middle) and heart weight/tibia length (HW/TL, right) were assessed in wild‐type (WT) or KO groups subjected to sham or TAC operation (n = 10 mice per experimental group). E‐H, Echocardiographic assessment (n = 10 mice per experimental group) of left ventricular end‐diastolic diameter (LVEDd, left), left ventricular end‐systolic diameter (LVESd, middle left), fractional shortening (FS, middle right) and ejection fraction (EF, right). Data are expressed as mean ± SD. ** P < 0.01 or * P < 0.05 vs WT/sham; ## P < 0.01 or # P < 0.05 vs WT/TAC

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway. Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway

    doi: 10.1111/jcmm.15724

    Figure Lengend Snippet: Ubiquitin‐specific protease 19 (USP19) deficiency exacerbates heart hypertrophy and dysfunction in response to transverse aortic constriction (TAC) in vivo. A, Western blot analysis revealing successful construction of USP19‐knockout (KO) mice. B‐D, Four weeks after TAC, the ratios of heart weight/bodyweight (HW/BW; left), lung weight/bodyweight (LW/BW; middle) and heart weight/tibia length (HW/TL, right) were assessed in wild‐type (WT) or KO groups subjected to sham or TAC operation (n = 10 mice per experimental group). E‐H, Echocardiographic assessment (n = 10 mice per experimental group) of left ventricular end‐diastolic diameter (LVEDd, left), left ventricular end‐systolic diameter (LVESd, middle left), fractional shortening (FS, middle right) and ejection fraction (EF, right). Data are expressed as mean ± SD. ** P < 0.01 or * P < 0.05 vs WT/sham; ## P < 0.01 or # P < 0.05 vs WT/TAC

    Article Snippet: 3.2 USP19 suppressed cardiac hypertrophy via blocking TAK1‐dependent pathwaySince the anti‐hypertrophy function of USP19 was confirmed, the investigators determined the underlying mechanism that mediated this effect.

    Techniques: In Vivo, Western Blot, Knock-Out, Mouse Assay

    Ubiquitin‐specific protease 19 (USP19) attenuates phenylephrine (PE)‐induced cardiomyocyte hypertrophy in vitro. A, Western blot bands of USP19 levels in primary cultured cardiomyocytes infected with AdshRNA or short hairpin RNA (AdshUSP19) (n = 3 independent experiments). B, Representative microscopic images of cardiomyocytes infected with AdshRNA or AdshUSP19 and treated with phosphate buffered saline (PBS) or phenylephrine (PE). Cells were double stained of α‐actin (green) and 4‐6‐diamidino‐2‐phenylindole (blue). Scale bar, 20 μm. C, Quantitative results of the relative mRNA levels of atriopeptin (ANP) and myosin heavy chain 7 (Myh7, Above), and cross‐sectional area (Bottom) of cardiomyocytes infected with AdshRNA and AdshUSP19 in response to PBS or PE (n = 3 independent experiments, ** P < 0.01 vs AdshRNA/PBS, ## P < 0.01 vs AdshRNA/PE). D, Western blot bands of USP19 levels in primary cultured cardiomyocytes infected with AdGFP or AdUSP19 (n = 3 independent experiments). E, Representative microscopic images of cardiomyocytes infected with AdGFP or AdUSP19 and treated with phosphate buffered saline (PBS) or phenylephrine (PE). F, Quantitative results of the relative mRNA levels of ANP and Myh7 (Above), and cross‐sectional area (Bottom) of cardiomyocytes infected with AdGFP and AdUSP19 in response to PBS or PE (n = 3 independent experiments, ** P < 0.01 vs AdGFP/PBS, ## P < 0.01 vs AdGFP/PE), n.s. (no significance)

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway. Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway

    doi: 10.1111/jcmm.15724

    Figure Lengend Snippet: Ubiquitin‐specific protease 19 (USP19) attenuates phenylephrine (PE)‐induced cardiomyocyte hypertrophy in vitro. A, Western blot bands of USP19 levels in primary cultured cardiomyocytes infected with AdshRNA or short hairpin RNA (AdshUSP19) (n = 3 independent experiments). B, Representative microscopic images of cardiomyocytes infected with AdshRNA or AdshUSP19 and treated with phosphate buffered saline (PBS) or phenylephrine (PE). Cells were double stained of α‐actin (green) and 4‐6‐diamidino‐2‐phenylindole (blue). Scale bar, 20 μm. C, Quantitative results of the relative mRNA levels of atriopeptin (ANP) and myosin heavy chain 7 (Myh7, Above), and cross‐sectional area (Bottom) of cardiomyocytes infected with AdshRNA and AdshUSP19 in response to PBS or PE (n = 3 independent experiments, ** P < 0.01 vs AdshRNA/PBS, ## P < 0.01 vs AdshRNA/PE). D, Western blot bands of USP19 levels in primary cultured cardiomyocytes infected with AdGFP or AdUSP19 (n = 3 independent experiments). E, Representative microscopic images of cardiomyocytes infected with AdGFP or AdUSP19 and treated with phosphate buffered saline (PBS) or phenylephrine (PE). F, Quantitative results of the relative mRNA levels of ANP and Myh7 (Above), and cross‐sectional area (Bottom) of cardiomyocytes infected with AdGFP and AdUSP19 in response to PBS or PE (n = 3 independent experiments, ** P < 0.01 vs AdGFP/PBS, ## P < 0.01 vs AdGFP/PE), n.s. (no significance)

    Article Snippet: 3.2 USP19 suppressed cardiac hypertrophy via blocking TAK1‐dependent pathwaySince the anti‐hypertrophy function of USP19 was confirmed, the investigators determined the underlying mechanism that mediated this effect.

    Techniques: In Vitro, Western Blot, Cell Culture, Infection, shRNA, Staining, Aqueous Normal-phase Chromatography

    PPD inhibited osteoclast differentiation by the NF-κB pathway. (A–C) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Nuclear protein was isolated for western blot analysis. Cell lysates were analyzed using western blotting with specific antibodies against NF-κB and p-NF-κB were quantified and were normalized to H3 using ImageJ software. (D) RAW264.7 cells that had been stably transfected with an NF-κB luciferase reporter construct were treated with the indicated concentrations of PPD for 1 h, followed by incubation in the absence or presence of RANKL for 8 h. Luciferase activity was measured using the Promega Luciferase Assay System. All experiments were performed at least 3 times; (E) BMDMs were cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL with or without 5 μM PPD for 0–5 days. Cell lysates were prepared and analyzed using western blotting. (F,G) The gray levels corresponding to the indicated proteins were quantified and normalized relative to β-actin using Image J for c-fos and NFATc1. Significant differences between the groups were determined by paired Student’s t -test. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: 20(S)-Protopanaxadiol Inhibits Titanium Particle-Induced Inflammatory Osteolysis and RANKL-Mediated Osteoclastogenesis via MAPK and NF-κB Signaling Pathways

    doi: 10.3389/fphar.2018.01538

    Figure Lengend Snippet: PPD inhibited osteoclast differentiation by the NF-κB pathway. (A–C) RAW264.7 cells were treated with or without 5 μM PPD for 2 h and then treated with 100 ng/mL RANKL for the indicated periods. Nuclear protein was isolated for western blot analysis. Cell lysates were analyzed using western blotting with specific antibodies against NF-κB and p-NF-κB were quantified and were normalized to H3 using ImageJ software. (D) RAW264.7 cells that had been stably transfected with an NF-κB luciferase reporter construct were treated with the indicated concentrations of PPD for 1 h, followed by incubation in the absence or presence of RANKL for 8 h. Luciferase activity was measured using the Promega Luciferase Assay System. All experiments were performed at least 3 times; (E) BMDMs were cultured with 30 ng/mL M-CSF and 100 ng/mL RANKL with or without 5 μM PPD for 0–5 days. Cell lysates were prepared and analyzed using western blotting. (F,G) The gray levels corresponding to the indicated proteins were quantified and normalized relative to β-actin using Image J for c-fos and NFATc1. Significant differences between the groups were determined by paired Student’s t -test. ∗ P

    Article Snippet: Antibodies against transforming growth factor activated kinase-1 (TAK1) (ab109526), phospho-TAK1(ab109404), NFATc1 (ab175134), were purchased from Abcam (Cambridge, United Kingdom).

    Techniques: Isolation, Western Blot, Software, Stable Transfection, Transfection, Luciferase, Construct, Incubation, Activity Assay, Cell Culture