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  • 91
    Sino Biological il 33
    Serum IL-21 and <t>IL-33</t> are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p
    Il 33, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    il 33 - by Bioz Stars, 2021-01
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    93
    OriGene romo1
    Spearman’s correlation analyses evaluating correlation among <t>Romo1,</t> VEGF-C, VEGF-D, and ROS; Romo1 expression levels showed significant positive correlation with VEGF-C ( P =0.008, R =0.478) ( A ) and ROS ( P =0.016, R =0.436) ( C ) in tumor samples. ( B ) There was no significant correlation between Romo1 expression and VEGF-D levels. Abbreviations: Romo1, reactive oxygen species modulator-1; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.
    Romo1, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    OriGene lrrc32
    <t>LRRC32</t> contains an N-terminal signal peptide and a transmembrane region . a) Analysis of the amino acid sequence of LRRC32 by SignalP 3.0 software predicted a putative N-terminal cleavage site between alanines 17 and 18. Alternative potential cleavage sites are predicted and indicated as vertical solid lines on amino acids 19, 20, and 22. b) To address the actual cleavage site, N-terminal and C-terminal GFP-tagged constructs were designed as depicted and generated to facilitate further analysis of surface expression and cleavage. SP = signal peptide. TM = transmembrane region. GFP = green fluorescent protein. Arrow = putative cleavage site. c) Anti-GFP immunoblot analysis of total lysates from C-and N- terminal expressing clones revealed 99 kD (29 kD GFP + 70 kD LRRC32) and 31 kD (29 kD GFP + 2 kD LRRC32 signal peptide) fusion proteins respectively. Expected sizes of uncleaved fusion proteins, based upon predicted protein sequences, are shown in parentheses. The difference in protein size between the C- and N-terminal fusion proteins confirms a cleavage site in the N-terminus of the protein between alanines 17 and 18.
    Lrrc32, supplied by OriGene, used in various techniques. Bioz Stars score: 87/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Biotechnology Information orf finder
    Schematic diagram for the evaluation of promoter activity. ( a ) Outline of the construction of transformation vectors and transformations. After predicting putative <t>ORF</t> positions 32 , upstream regions of the <t>ORFs</t> were determined as potential promoter regions. Potential promoter regions amplified by PCR were used to construct the transformation vectors. The double-cassette vector containing the reporter gene egfp driven by each tested promoter and the antibiotic-resistant gene Sh ble driven by the promoter region of the fucoxanthin chlorophyll a / c -binding protein (FCP) A-1A gene derived from Cyl. fusiformis (termed CffcpA pro.) were constructed. ( b ) Assessment of promoter activity. Promoter activity was determined by averaging the ratios of egfp mRNA transcript levels to those of Sh ble mRNA transcripts in ten transformants to minimize the effects of copy numbers on the expression of transgenes. These transformants were also used to investigate eGFP protein expression patterns. CffcpA ter.: terminator region of the FCP A-1A gene derived from Cyl. fusiformis . The structure of the ClorDNA genome was modified from Tomaru et al. 32 . *For the transformation vector of the nitrate reductase gene promoter, we used pNICgfp 18 ( Supplementary Fig. 5a ).
    Orf Finder, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 90/100, based on 306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    OriGene cyr61
    Schematic diagram for the evaluation of promoter activity. ( a ) Outline of the construction of transformation vectors and transformations. After predicting putative <t>ORF</t> positions 32 , upstream regions of the <t>ORFs</t> were determined as potential promoter regions. Potential promoter regions amplified by PCR were used to construct the transformation vectors. The double-cassette vector containing the reporter gene egfp driven by each tested promoter and the antibiotic-resistant gene Sh ble driven by the promoter region of the fucoxanthin chlorophyll a / c -binding protein (FCP) A-1A gene derived from Cyl. fusiformis (termed CffcpA pro.) were constructed. ( b ) Assessment of promoter activity. Promoter activity was determined by averaging the ratios of egfp mRNA transcript levels to those of Sh ble mRNA transcripts in ten transformants to minimize the effects of copy numbers on the expression of transgenes. These transformants were also used to investigate eGFP protein expression patterns. CffcpA ter.: terminator region of the FCP A-1A gene derived from Cyl. fusiformis . The structure of the ClorDNA genome was modified from Tomaru et al. 32 . *For the transformation vector of the nitrate reductase gene promoter, we used pNICgfp 18 ( Supplementary Fig. 5a ).
    Cyr61, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    OriGene zdhhc17
    Genistein is a Lead Candidate for Inhibiting the <t>ZDHHC17-MAP2K4</t> Interaction in GBM. (A) GBM0378 and GBM1492, from patients with GBM, cell viability after 24h-treatment with indicated inhibitors at indicated concentration from the MAPK Compound Library. Data represent the means ± SD from three separate experiments (** p
    Zdhhc17, supplied by OriGene, used in various techniques. Bioz Stars score: 98/100, based on 116 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    OriGene fam60a
    <t>FAM60A</t> directly represses expression of genes in the TGF-beta pathway. A , Genes increased by microarray analysis in FAM60A and SDS3 knockdown cells that encode components of the TGF-beta signaling pathway. Fold-increase of the top probe for that gene
    Fam60a, supplied by OriGene, used in various techniques. Bioz Stars score: 86/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene samhd1
    <t>SAMHD1</t> restricts HIV-1 infection of monocytes and AM. AM and PBMC were obtained from the same healthy donor. CD14 + cells were selected from the PBMC, cultured in the presence of M-CSF overnight (monocytes) and infected (A, top ) or cultured an additional 6 days (MDM) before infection (B, top ) . AM were cultured without cytokines overnight, washed vigorously to remove contaminants, and infected 1 day (A, bottom ) or 1 week (B, bottom ) after isolation. Cells were infected with HIV NLAD8 in the absence or presence of SIV-VLP (+Vpx) as indicated. (A, B) Cells were stained and analyzed by flow cytometry 4 dpi. Circled populations are HIV p24 positive, CD4 downregulated, productively infected cells. Numbers in the plots indicate percent of total cells infected. (C, D) Productively infected cells from (A and B) were quantified and displayed as bar graphs in C (infected 1 day after isolation) and D (infected 1 week after isolation). Data are representative of three experiments. AM, alveolar macrophages; M-CSF, macrophage-colony stimulating factor; PBMC, peripheral blood mononuclear cells.
    Samhd1, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene aldh1a1
    <t>ALDH1A1</t> affects tumor growth and vascular flow in MCF-7 tumor xenograft in athymic nude mice. MCF-7 cells (1 × 10 7 with 50% v /v of Matrigel) were injected s.c. in flank of athymic female nude mice. β-estradiol were injected (3 mg/kg), every 7 days i.m.. All mice were sacrificed at day 23. Tumor volumes were detected twice a week using a caliper and calculated by the formula: shortest diameter × longest diameter × thickness of the tumor in mm ( n = 6 animals per group). a Tumor volume at day 23. ** p
    Aldh1a1, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Sino Biological igfbp4
    <t>ALDH1A1</t> affects tumor growth and vascular flow in MCF-7 tumor xenograft in athymic nude mice. MCF-7 cells (1 × 10 7 with 50% v /v of Matrigel) were injected s.c. in flank of athymic female nude mice. β-estradiol were injected (3 mg/kg), every 7 days i.m.. All mice were sacrificed at day 23. Tumor volumes were detected twice a week using a caliper and calculated by the formula: shortest diameter × longest diameter × thickness of the tumor in mm ( n = 6 animals per group). a Tumor volume at day 23. ** p
    Igfbp4, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    OriGene map2k4
    Genistein is a Lead Candidate for Inhibiting the <t>ZDHHC17-MAP2K4</t> Interaction in GBM. (A) GBM0378 and GBM1492, from patients with GBM, cell viability after 24h-treatment with indicated inhibitors at indicated concentration from the MAPK Compound Library. Data represent the means ± SD from three separate experiments (** p
    Map2k4, supplied by OriGene, used in various techniques. Bioz Stars score: 97/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Sino Biological il 28b
    Genistein is a Lead Candidate for Inhibiting the <t>ZDHHC17-MAP2K4</t> Interaction in GBM. (A) GBM0378 and GBM1492, from patients with GBM, cell viability after 24h-treatment with indicated inhibitors at indicated concentration from the MAPK Compound Library. Data represent the means ± SD from three separate experiments (** p
    Il 28b, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    OriGene trim21
    <t>Trim21</t> and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.
    Trim21, supplied by OriGene, used in various techniques. Bioz Stars score: 89/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    OriGene clec18b
    High level of <t>CLEC18B</t> predicted shorter overall survival (OS) in GBM patients. (a) Kaplan–Meier curves for OS according to CLEC18B expression in patients with GBM collected from TCGA. (b) Kaplan–Meier curves for OS according to CLEC18B expression in GBM patients collected at our institution. CLEC18B = C-type lectin domain family 18 member B; GBM = glioblastoma multiforme; TCGA = The Cancer Genome Atlas.
    Clec18b, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher protein coding regions
    High level of <t>CLEC18B</t> predicted shorter overall survival (OS) in GBM patients. (a) Kaplan–Meier curves for OS according to CLEC18B expression in patients with GBM collected from TCGA. (b) Kaplan–Meier curves for OS according to CLEC18B expression in GBM patients collected at our institution. CLEC18B = C-type lectin domain family 18 member B; GBM = glioblastoma multiforme; TCGA = The Cancer Genome Atlas.
    Protein Coding Regions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    OriGene pla2g3
    Overexpression of <t>Pla2g3</t> reduces IDE expression. A , B , Quantitative RT-PCR result of IDE in TR-AST cells and rat primary astrocytes. TR-AST cells and primary astrocytes were treated with hydrogen peroxide in indicated conditions. Fold changes to the non-treated cells as controls are indicated. C , Quantitative PCR results of IDE in Pla2g3 transfected HEK293 cells. Human Pla2g3 was transiently expressed and cells were harvested after 48 hours of transfection. Fold changes to the mock control are indicated. *p
    Pla2g3, supplied by OriGene, used in various techniques. Bioz Stars score: 89/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    OriGene adam10
    <t>ADAM10</t> expression in human renal tissues. ADAM10 expression (green) in renal biopsy samples from CKD patients (stages 2–3) was analyzed by immunofluorescence, with controls derived from living donor biopsies. Cell nuclei were stained with DAPI. Scale bar represents 50 µm. a – e Immunohistochemical staining of ADAM10 in the renal tissues of renal biopsy samples from CKD patients (stages 2–3). Original magnification, × 400
    Adam10, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Sino Biological covid 19
    <t>ADAM10</t> expression in human renal tissues. ADAM10 expression (green) in renal biopsy samples from CKD patients (stages 2–3) was analyzed by immunofluorescence, with controls derived from living donor biopsies. Cell nuclei were stained with DAPI. Scale bar represents 50 µm. a – e Immunohistochemical staining of ADAM10 in the renal tissues of renal biopsy samples from CKD patients (stages 2–3). Original magnification, × 400
    Covid 19, supplied by Sino Biological, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    OriGene zscan4
    <t>ZSCAN4</t> is Lysine 48 (K48Ub) polyubiquitinated A . Endogenous ZSCAN4 immunoprecipitation (IP) in treated (+MG132) or untreated (−MG132) WT cells followed by ZSCAN4 immunoblot. B . Denaturing conditions where used followed by ZSCAN4-IP and immunoblot with anti-Lysine 48 (K48) ubiquitin (anti-K48Ub). MG132 treated cells indicate that ZSCAN4 is polyubiquitinated. Untreated cells were used as controls. C. Co-immunostaining and confocal microscopy analyses in WT Tu167 cells treated with MG132 (+MG132) or untreated (−MG132) using anti-ZSCAN4 (green) and anti-K48 ubiquitin (red) indicates the ZSCAN4 overlaps with K48Ub. D . Colocalization analyses with ImageJ show an increase in the number of ZSCAN4 foci colocalized with K48Ub and E . the percent (%) of K48Ub colocalized ZSCAN4 foci (n = 6 per group and average of > 300 foci per group). Asterisks indicate * p
    Zscan4, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    OriGene s100a4
    Effects on <t>S100A4</t> expression and MTX sensitivity upon siS100A4 transfection of HT29 cells . A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p
    S100a4, supplied by OriGene, used in various techniques. Bioz Stars score: 94/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Sino Biological bmp 2
    Effects on <t>S100A4</t> expression and MTX sensitivity upon siS100A4 transfection of HT29 cells . A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p
    Bmp 2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene tfap2a
    Differential Binding of <t>TFAP2a</t> at SNP rs2595104
    Tfap2a, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    OriGene cyb5r2
    Analysis of the methylation status of the <t>CYB5R2</t> promoter region in NPC cell lines, NPC primary tumors and NNE samples. a MSP analysis in NPC cell lines and NNEs. M methylated alleles, U unmethylated alleles. In vitro methylated DNA was used as a methylation-positive control and DNA from normal lymphocytes was the positive control for unmethylated alleles. The blank control was water. b MSP analysis in NPC biopsies and NNE, representative data is shown. c Methylation status of the 39 CpG sites of the CYB5R2 promoter in two NPC cell lines (TW03 and CNE2), two NPC biopsies (NPC15 and 27) and one NNE biopsy (NNE7). Five clones were randomly selected and sequenced for each sample. Different size filled sectors of the circles represent the fraction of methylated CpG. MSP primer locations are indicated by frames. d Restoration of CYB5R2 expression by treatment with 5-aza-dC in NPC cell lines. One NNE sample and CYB5R2 expression plasmid DNA were used as positive controls
    Cyb5r2, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    OriGene eftud2
    eftu-2 <t>/Eftud2</t> regulates host defense in C. elegans . Depicted are survival plots for eri-1 mutant nematodes treated with either control RNAi, eftu-2 RNAi, or vha-11 RNAi (as indicated) that were subsequently exposed to either P. aeruginosa strain PA14
    Eftud2, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sino Biological trem2
    Top DEGs of R47H <t>TREM2</t> and TREM2 KO pMac overlap; furthermore, the R47H DEGs that are shared with TREM2 KO represent numerous biological processes, distributed between five protein-protein interaction (PPI) modules. a Heatmap of top upregulated and top downregulated DEGs for R47H TREM2 and TREM2 KO, with the relative expression for each gene represented by colours on the corresponding row. A selection of “relevant genes” was also included. Unbiased clustering (dendrogram on the left) shows that genes separate into two major clusters based on whether they are upregulated or downregulated in TREM2 KO or R47H TREM2, and there is no genotype-specific segregation. b Five modules of functionally related DEGs identified by PPI network analysis of TREM2 KO DEGs, with enriched gene ontology (GO) terms shown. R47H DEGs were only included where they were also differentially expressed in TREM2 KO and were overlaid onto clusters identified using TREM2 KO data. Clusters identified numerically on the x -axis, with TREM2 KO on the left and R47H TREM2 on the right, showing a similar pattern of dysregulated cell functions. Number of DEGs represented by circle size, and p value represented by colour
    Trem2, supplied by Sino Biological, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Highly characterized substrate for herpes simplex virus type 1 protease HSV 1 which is essential for viral nucleocapsid formation and for viral replication Enzyme activity was measured using HPLC for
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    Image Search Results


    Serum IL-21 and IL-33 are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p

    Journal: Nature Communications

    Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

    doi: 10.1038/s41467-017-02304-7

    Figure Lengend Snippet: Serum IL-21 and IL-33 are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p

    Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

    Techniques: Mouse Assay, Multiplex Assay, Two Tailed Test

    IL-21 or IL-33 treatments induce clearance of established BPS persistence. Plasmids expressing murine IL-21 or IL-33 and empty vector control were injected into BPS HDI mice at 4 and 7 weeks post injection (w.p.i.) (arrows). Sera were collected at indicated time points and levels of IL-21 and IL-33 ( a ), HBsAg ( b ), HBsAb ( c ) and HBeAg ( d ) were analysed using ELISA. Group means and s.e.m. within group are presented with group sizes ( n ) indicated. Group positivity percentage data are also presented for HBsAg ( b , right panel), HBsAb ( c , right panel) and HBeAg ( d , right panel). HBV DNA for each group were analysed using pooled serum in commercial quantitative assay ( e ). Dotted lines represent cut-off thresholds ( a , b – d , right panels) and lower limit of quantification ( e ) respectively. geq genome equivalents

    Journal: Nature Communications

    Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

    doi: 10.1038/s41467-017-02304-7

    Figure Lengend Snippet: IL-21 or IL-33 treatments induce clearance of established BPS persistence. Plasmids expressing murine IL-21 or IL-33 and empty vector control were injected into BPS HDI mice at 4 and 7 weeks post injection (w.p.i.) (arrows). Sera were collected at indicated time points and levels of IL-21 and IL-33 ( a ), HBsAg ( b ), HBsAb ( c ) and HBeAg ( d ) were analysed using ELISA. Group means and s.e.m. within group are presented with group sizes ( n ) indicated. Group positivity percentage data are also presented for HBsAg ( b , right panel), HBsAb ( c , right panel) and HBeAg ( d , right panel). HBV DNA for each group were analysed using pooled serum in commercial quantitative assay ( e ). Dotted lines represent cut-off thresholds ( a , b – d , right panels) and lower limit of quantification ( e ) respectively. geq genome equivalents

    Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

    Techniques: Expressing, Plasmid Preparation, Injection, Mouse Assay, Enzyme-linked Immunosorbent Assay

    IL-21-induced or IL-33-induced clearance of BPS persistence engenders protection from re-challenge. HDI mice harbouring BPS persistence were treated with injections of plasmids expressing IL-21 ( a ) or IL-33 ( b ) at 4 and 7 w.p.i. and those displaying at least 4 weeks of sustained HBsAg negativity (see Fig. 6 ) were re-challenged in the same way as initial BPS HDI. BPS HDI mice treated with vector control were used as control and re-challenged similarly ( c ). Sera were collected at indicated time points and levels of HBsAg (left), HBsAb (middle) and HBeAg (right) were analysed using ELISA. Dotted lines represent cut-off thresholds. w.p.i. weeks post injection of BPS re-challenge

    Journal: Nature Communications

    Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

    doi: 10.1038/s41467-017-02304-7

    Figure Lengend Snippet: IL-21-induced or IL-33-induced clearance of BPS persistence engenders protection from re-challenge. HDI mice harbouring BPS persistence were treated with injections of plasmids expressing IL-21 ( a ) or IL-33 ( b ) at 4 and 7 w.p.i. and those displaying at least 4 weeks of sustained HBsAg negativity (see Fig. 6 ) were re-challenged in the same way as initial BPS HDI. BPS HDI mice treated with vector control were used as control and re-challenged similarly ( c ). Sera were collected at indicated time points and levels of HBsAg (left), HBsAb (middle) and HBeAg (right) were analysed using ELISA. Dotted lines represent cut-off thresholds. w.p.i. weeks post injection of BPS re-challenge

    Article Snippet: Mouse IL-21 (MG50137-M-N) and IL-33 (MG50118-M-N) expression plasmids (Sino Biological, China) and rat monoclonal antibodies (anti-mIL-21: 16-9333; anti-mIL-33, 16-7211) (eBioscience, China) were purchased.

    Techniques: Mouse Assay, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Injection

    Spearman’s correlation analyses evaluating correlation among Romo1, VEGF-C, VEGF-D, and ROS; Romo1 expression levels showed significant positive correlation with VEGF-C ( P =0.008, R =0.478) ( A ) and ROS ( P =0.016, R =0.436) ( C ) in tumor samples. ( B ) There was no significant correlation between Romo1 expression and VEGF-D levels. Abbreviations: Romo1, reactive oxygen species modulator-1; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

    Journal: OncoTargets and therapy

    Article Title: Overexpression of Romo1 is an unfavorable prognostic biomarker and a predictor of lymphatic metastasis in non-small cell lung cancer patients

    doi: 10.2147/OTT.S161587

    Figure Lengend Snippet: Spearman’s correlation analyses evaluating correlation among Romo1, VEGF-C, VEGF-D, and ROS; Romo1 expression levels showed significant positive correlation with VEGF-C ( P =0.008, R =0.478) ( A ) and ROS ( P =0.016, R =0.436) ( C ) in tumor samples. ( B ) There was no significant correlation between Romo1 expression and VEGF-D levels. Abbreviations: Romo1, reactive oxygen species modulator-1; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

    Article Snippet: Then, we blocked the TMA slides with blocking buffer, and incubated them with antibodies for Romo1 (OriGene, Rockville, MD, USA) at a 1:150 dilution, VEGF-D (Abcam, Cambridge, UK) at a 1:50 dilution, and VEGF-C (Abnova, Taipei City, Taiwan) at a 1:25 dilution.

    Techniques: Expressing

    Kaplan–Meier curves of overall survival according to Romo1 ( A ), NLR ( B ), PLR ( C ), and CRP–albumin ratio ( D ). Abbreviations: CRP, C-reactive protein; NLR, neutrophil to lymphocyte ratio; PLR, platelet to lymphocyte ratio; Romo1, reactive oxygen species modulator-1.

    Journal: OncoTargets and therapy

    Article Title: Overexpression of Romo1 is an unfavorable prognostic biomarker and a predictor of lymphatic metastasis in non-small cell lung cancer patients

    doi: 10.2147/OTT.S161587

    Figure Lengend Snippet: Kaplan–Meier curves of overall survival according to Romo1 ( A ), NLR ( B ), PLR ( C ), and CRP–albumin ratio ( D ). Abbreviations: CRP, C-reactive protein; NLR, neutrophil to lymphocyte ratio; PLR, platelet to lymphocyte ratio; Romo1, reactive oxygen species modulator-1.

    Article Snippet: Then, we blocked the TMA slides with blocking buffer, and incubated them with antibodies for Romo1 (OriGene, Rockville, MD, USA) at a 1:150 dilution, VEGF-D (Abcam, Cambridge, UK) at a 1:50 dilution, and VEGF-C (Abnova, Taipei City, Taiwan) at a 1:25 dilution.

    Techniques:

    ROC analysis to set best cutoff for Romo1 and serologic inflammatory biomarkers (NLR, PLR, and CRP–albumin ratio). Abbreviations: AUC, area under the curve; CRP, C-reactive protein; NLR, neutrophil to lymphocyte ratio; PLR, platelet to lymphocyte ratio; ROC, receiver operating curve; Romo1, reactive oxygen species modulator-1.

    Journal: OncoTargets and therapy

    Article Title: Overexpression of Romo1 is an unfavorable prognostic biomarker and a predictor of lymphatic metastasis in non-small cell lung cancer patients

    doi: 10.2147/OTT.S161587

    Figure Lengend Snippet: ROC analysis to set best cutoff for Romo1 and serologic inflammatory biomarkers (NLR, PLR, and CRP–albumin ratio). Abbreviations: AUC, area under the curve; CRP, C-reactive protein; NLR, neutrophil to lymphocyte ratio; PLR, platelet to lymphocyte ratio; ROC, receiver operating curve; Romo1, reactive oxygen species modulator-1.

    Article Snippet: Then, we blocked the TMA slides with blocking buffer, and incubated them with antibodies for Romo1 (OriGene, Rockville, MD, USA) at a 1:150 dilution, VEGF-D (Abcam, Cambridge, UK) at a 1:50 dilution, and VEGF-C (Abnova, Taipei City, Taiwan) at a 1:25 dilution.

    Techniques:

    Representative IHC-stained tissue samples. Magnification, ×100; ( A ) Romo1, negative control, IHC score of 2, 10, and 15; ( B ) VEGF-C, negative control, IHC score of 2, 9, and 15; ( C ) VEGF-D, negative control, IHC score of 2, 8, and 15; ( D ) ROS, negative control, IHC score of 1, 7, and 15. Abbreviations: IHC, immunohistochemical; Romo1, reactive oxygen species modulator-1; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

    Journal: OncoTargets and therapy

    Article Title: Overexpression of Romo1 is an unfavorable prognostic biomarker and a predictor of lymphatic metastasis in non-small cell lung cancer patients

    doi: 10.2147/OTT.S161587

    Figure Lengend Snippet: Representative IHC-stained tissue samples. Magnification, ×100; ( A ) Romo1, negative control, IHC score of 2, 10, and 15; ( B ) VEGF-C, negative control, IHC score of 2, 9, and 15; ( C ) VEGF-D, negative control, IHC score of 2, 8, and 15; ( D ) ROS, negative control, IHC score of 1, 7, and 15. Abbreviations: IHC, immunohistochemical; Romo1, reactive oxygen species modulator-1; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

    Article Snippet: Then, we blocked the TMA slides with blocking buffer, and incubated them with antibodies for Romo1 (OriGene, Rockville, MD, USA) at a 1:150 dilution, VEGF-D (Abcam, Cambridge, UK) at a 1:50 dilution, and VEGF-C (Abnova, Taipei City, Taiwan) at a 1:25 dilution.

    Techniques: Immunohistochemistry, Staining, Negative Control

    Kaplan–Meier curves of disease-free survival according to Romo1 ( A ), NLR ( B ), PLR ( C ), and CRP–albumin ratio ( D ). Abbreviations: CRP, C-reactive protein; NLR, neutrophil to lymphocyte ratio; PLR, platelet to lymphocyte ratio; Romo1, reactive oxygen species modulator-1.

    Journal: OncoTargets and therapy

    Article Title: Overexpression of Romo1 is an unfavorable prognostic biomarker and a predictor of lymphatic metastasis in non-small cell lung cancer patients

    doi: 10.2147/OTT.S161587

    Figure Lengend Snippet: Kaplan–Meier curves of disease-free survival according to Romo1 ( A ), NLR ( B ), PLR ( C ), and CRP–albumin ratio ( D ). Abbreviations: CRP, C-reactive protein; NLR, neutrophil to lymphocyte ratio; PLR, platelet to lymphocyte ratio; Romo1, reactive oxygen species modulator-1.

    Article Snippet: Then, we blocked the TMA slides with blocking buffer, and incubated them with antibodies for Romo1 (OriGene, Rockville, MD, USA) at a 1:150 dilution, VEGF-D (Abcam, Cambridge, UK) at a 1:50 dilution, and VEGF-C (Abnova, Taipei City, Taiwan) at a 1:25 dilution.

    Techniques:

    LRRC32 contains an N-terminal signal peptide and a transmembrane region . a) Analysis of the amino acid sequence of LRRC32 by SignalP 3.0 software predicted a putative N-terminal cleavage site between alanines 17 and 18. Alternative potential cleavage sites are predicted and indicated as vertical solid lines on amino acids 19, 20, and 22. b) To address the actual cleavage site, N-terminal and C-terminal GFP-tagged constructs were designed as depicted and generated to facilitate further analysis of surface expression and cleavage. SP = signal peptide. TM = transmembrane region. GFP = green fluorescent protein. Arrow = putative cleavage site. c) Anti-GFP immunoblot analysis of total lysates from C-and N- terminal expressing clones revealed 99 kD (29 kD GFP + 70 kD LRRC32) and 31 kD (29 kD GFP + 2 kD LRRC32 signal peptide) fusion proteins respectively. Expected sizes of uncleaved fusion proteins, based upon predicted protein sequences, are shown in parentheses. The difference in protein size between the C- and N-terminal fusion proteins confirms a cleavage site in the N-terminus of the protein between alanines 17 and 18.

    Journal: BMC Biochemistry

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

    doi: 10.1186/1471-2091-12-27

    Figure Lengend Snippet: LRRC32 contains an N-terminal signal peptide and a transmembrane region . a) Analysis of the amino acid sequence of LRRC32 by SignalP 3.0 software predicted a putative N-terminal cleavage site between alanines 17 and 18. Alternative potential cleavage sites are predicted and indicated as vertical solid lines on amino acids 19, 20, and 22. b) To address the actual cleavage site, N-terminal and C-terminal GFP-tagged constructs were designed as depicted and generated to facilitate further analysis of surface expression and cleavage. SP = signal peptide. TM = transmembrane region. GFP = green fluorescent protein. Arrow = putative cleavage site. c) Anti-GFP immunoblot analysis of total lysates from C-and N- terminal expressing clones revealed 99 kD (29 kD GFP + 70 kD LRRC32) and 31 kD (29 kD GFP + 2 kD LRRC32 signal peptide) fusion proteins respectively. Expected sizes of uncleaved fusion proteins, based upon predicted protein sequences, are shown in parentheses. The difference in protein size between the C- and N-terminal fusion proteins confirms a cleavage site in the N-terminus of the protein between alanines 17 and 18.

    Article Snippet: The reported sequence was aligned with the reported cDNA sequence of LRRC32 obtained from OriGene using the Vector NTI (Invitrogen) software system, and sequence alignments were then confirmed prior to further utilization of the LRRC32 sequence-verified plasmids.

    Techniques: Sequencing, Software, Construct, Generated, Expressing, Clone Assay

    LRRC32 + CD4 + CD25 hi FoxP3 T regs appear to be more potent suppressors than LRRC32 - CD4 + CD25 hi FoxP3 and exhibit decreased CD62L upon activation . a) Expression of LRRC32 and LAP in CD4+ T cells rested overnight (top panel) or stimulated with plate bound anti-CD3 and soluble anti-CD28 (bottom panel). T regs were selected from the top 5% CD25-expressing and FoxP3 + populations, as previously described. Confirmation of activation by expression of the surface markers CD40L and CD69 are also shown (top of each panel). b) The expression patterns of various T reg and activation surface markers (CD62L, CD69, GITR, CTLA4, CD45RO, and HLA-DR) in FoxP3 + and LRRC32 + -gated populations of CD25 hi cells were studied using flow cytometry. Stimulated CD4 + FoxP3 + CD25 hi T regs (top panel) unstimulated CD4 + FoxP3 + CD25 hi T regs (bottom panel). c) Composite summary of phenotypic analysis of unstimulated LRRC32 - CD4 + CD25 hi FoxP3 + T regs and stimulated LRRC32 + and LRRC32 - CD4 + CD25 hi FoxP3 + T regs . Black bars = unstimulated LRRC32 - T regs . Dark grey bars = stimulated LRRC32 - T regs . Light grey bars = stimulated LRRC32 + T regs . Data are expressed as the mean ± SEM from 3 individuals. Heteroscedastic variances and an independent t-test comparing stimulated LRRC32 + and LRRC32 - subsets were used for calculations of the p values which are reported along the x-axis, below each surface marker (*). d) CD25 hi cells were sorted and activated overnight using anti-CD3-coated plates and soluble anti-CD28 (1 microgram/ml). Cells were then resorted based upon LRRC32 expression. The suppressive capacities of these LRRC32 + and LRRC32 - T regs were subsequently tested in a mixed lymphocyte reaction utilizing syngeneic effectors (T eff , 20,000/well) and allogenic antigen presenting cells (50,000/well). T reg :T eff ratios are depicted above. Data summarize 3 independent experiments. Results are expressed as the mean ± SEM. p = 0.0001 and R 2 = 0.7244. Absolute proliferation values for the 3 experiments were as follow: T effs alone: average of 31094 cpm to average of 47483 cpm (at least 6 replicates per assay), background: average of 24 cpm to 35 cpm (at least 6 replicates per assay); T reg :T eff ratio of 1:1: 89 cpm to 346 cpm. When titrating T regs vs. T effs , 3 replicates were performed at each titration for the LRRC32 + and LRRC32 - T regs except for in one assay set in which there was limited number of LRRC32 + T regs . In this case, only one replicate was performed at the 1:1 and 1:2 titrations, and two replicates were performed for the other titrations (0:1, 1:4, 1:8, and 1:16). We performed 3 replicates for each titration utilizing the LRRC32 - T regs .

    Journal: BMC Biochemistry

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

    doi: 10.1186/1471-2091-12-27

    Figure Lengend Snippet: LRRC32 + CD4 + CD25 hi FoxP3 T regs appear to be more potent suppressors than LRRC32 - CD4 + CD25 hi FoxP3 and exhibit decreased CD62L upon activation . a) Expression of LRRC32 and LAP in CD4+ T cells rested overnight (top panel) or stimulated with plate bound anti-CD3 and soluble anti-CD28 (bottom panel). T regs were selected from the top 5% CD25-expressing and FoxP3 + populations, as previously described. Confirmation of activation by expression of the surface markers CD40L and CD69 are also shown (top of each panel). b) The expression patterns of various T reg and activation surface markers (CD62L, CD69, GITR, CTLA4, CD45RO, and HLA-DR) in FoxP3 + and LRRC32 + -gated populations of CD25 hi cells were studied using flow cytometry. Stimulated CD4 + FoxP3 + CD25 hi T regs (top panel) unstimulated CD4 + FoxP3 + CD25 hi T regs (bottom panel). c) Composite summary of phenotypic analysis of unstimulated LRRC32 - CD4 + CD25 hi FoxP3 + T regs and stimulated LRRC32 + and LRRC32 - CD4 + CD25 hi FoxP3 + T regs . Black bars = unstimulated LRRC32 - T regs . Dark grey bars = stimulated LRRC32 - T regs . Light grey bars = stimulated LRRC32 + T regs . Data are expressed as the mean ± SEM from 3 individuals. Heteroscedastic variances and an independent t-test comparing stimulated LRRC32 + and LRRC32 - subsets were used for calculations of the p values which are reported along the x-axis, below each surface marker (*). d) CD25 hi cells were sorted and activated overnight using anti-CD3-coated plates and soluble anti-CD28 (1 microgram/ml). Cells were then resorted based upon LRRC32 expression. The suppressive capacities of these LRRC32 + and LRRC32 - T regs were subsequently tested in a mixed lymphocyte reaction utilizing syngeneic effectors (T eff , 20,000/well) and allogenic antigen presenting cells (50,000/well). T reg :T eff ratios are depicted above. Data summarize 3 independent experiments. Results are expressed as the mean ± SEM. p = 0.0001 and R 2 = 0.7244. Absolute proliferation values for the 3 experiments were as follow: T effs alone: average of 31094 cpm to average of 47483 cpm (at least 6 replicates per assay), background: average of 24 cpm to 35 cpm (at least 6 replicates per assay); T reg :T eff ratio of 1:1: 89 cpm to 346 cpm. When titrating T regs vs. T effs , 3 replicates were performed at each titration for the LRRC32 + and LRRC32 - T regs except for in one assay set in which there was limited number of LRRC32 + T regs . In this case, only one replicate was performed at the 1:1 and 1:2 titrations, and two replicates were performed for the other titrations (0:1, 1:4, 1:8, and 1:16). We performed 3 replicates for each titration utilizing the LRRC32 - T regs .

    Article Snippet: The reported sequence was aligned with the reported cDNA sequence of LRRC32 obtained from OriGene using the Vector NTI (Invitrogen) software system, and sequence alignments were then confirmed prior to further utilization of the LRRC32 sequence-verified plasmids.

    Techniques: Activation Assay, Expressing, Flow Cytometry, Cytometry, Marker, Titration

    Lrrc32 mRNA is preferentially expressed in naturally-occurring freshly-isolated non-expanded human T regs but is not observed on the surface of these cells . a) T cell sorting gates based upon CD25 surface expression. b) Lrrc32 mRNA expression comports with Foxp3 mRNA expression and CD25 surface expression, and Lrrc32 mRNA is preferentially expressed in T regs , compared to T effs . Relative expressions of 18SrRNA -normalized Foxp3 and Lrrc32 genes were determined using real-time PCR. Data summarize four independent experiments. Results are expressed as the mean ± SEM. * p = 0.01 compared to CD25 mid cells, ** p = 0.005 compared to CD25 mid cells. c) Flow cytometric analysis of freshly-isolated activated CD4 + human T cells shows that the CD25 hi subgroup (composed of the CD25hi+ and CD25hi++ subgroups denoted in figure 1b) demarcating T regs expresses LRRC32 on the cell surface (top right panel) but that the CD25 - subgroup demarcating T effs expresses negligible amounts of LRRC32 on the cell surface (bottom right panel). Analysis strategy is indicated via the arrows. d) Flow cytometric analysis of sorted resting CD4 + human T regs shows that they lack surface expression of LRRC32 protein (top left panel). However, after permeabilization of the cell membrane, a low expression of LRRC32 protein can be observed intracellularly (top right panel). After activation of the sorted T regs with a T reg expansion kit, LRRC32 can be seen on the surface of these activated T regs (bottom left panel), and evidence of intracellular expression of LRRC32 can also be observed after permeabilization of the cell membrane (bottom right panel).

    Journal: BMC Biochemistry

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

    doi: 10.1186/1471-2091-12-27

    Figure Lengend Snippet: Lrrc32 mRNA is preferentially expressed in naturally-occurring freshly-isolated non-expanded human T regs but is not observed on the surface of these cells . a) T cell sorting gates based upon CD25 surface expression. b) Lrrc32 mRNA expression comports with Foxp3 mRNA expression and CD25 surface expression, and Lrrc32 mRNA is preferentially expressed in T regs , compared to T effs . Relative expressions of 18SrRNA -normalized Foxp3 and Lrrc32 genes were determined using real-time PCR. Data summarize four independent experiments. Results are expressed as the mean ± SEM. * p = 0.01 compared to CD25 mid cells, ** p = 0.005 compared to CD25 mid cells. c) Flow cytometric analysis of freshly-isolated activated CD4 + human T cells shows that the CD25 hi subgroup (composed of the CD25hi+ and CD25hi++ subgroups denoted in figure 1b) demarcating T regs expresses LRRC32 on the cell surface (top right panel) but that the CD25 - subgroup demarcating T effs expresses negligible amounts of LRRC32 on the cell surface (bottom right panel). Analysis strategy is indicated via the arrows. d) Flow cytometric analysis of sorted resting CD4 + human T regs shows that they lack surface expression of LRRC32 protein (top left panel). However, after permeabilization of the cell membrane, a low expression of LRRC32 protein can be observed intracellularly (top right panel). After activation of the sorted T regs with a T reg expansion kit, LRRC32 can be seen on the surface of these activated T regs (bottom left panel), and evidence of intracellular expression of LRRC32 can also be observed after permeabilization of the cell membrane (bottom right panel).

    Article Snippet: The reported sequence was aligned with the reported cDNA sequence of LRRC32 obtained from OriGene using the Vector NTI (Invitrogen) software system, and sequence alignments were then confirmed prior to further utilization of the LRRC32 sequence-verified plasmids.

    Techniques: Isolation, FACS, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Activation Assay

    LRRC32 is a cell surface protein . a) C-and N- terminus GFP-tagged LRRC32 expressing HEK293 cell clones were surface biotinylated, and cell lysates were immunoprecipitated using antibody specific for GFP or using normal rabbit serum (NRS) as a control (left and right panels). Protein lysates were then electrophoresed, transferred to membrane PDVF, and probed for the presence of biotinylation using streptavidin-HRP (left panel only). Blots were also probed with anti-GFP (right panel only). b) Confocal analysis of untransfected (top row) HEK293 cells, C-GFP/CAT-transfected HEK293 cells (second row), N-GFP/LRRC32-transfected HEK293 cells (third row), C-GFP/LRRC32-transfected HEK293 cells (fourth row), and C-GFP/LRRC32ΔSP-transfected HEK293 cells (last row); Green = GFP, Red = anti-LRRC32 antibody, Blue = nuclear counterstain. The left column shows anti-LRRC32 only. The right column shows GFP only. The middle column shows the merged composite confocal picture (anti-LRRC32 + GFP) with the nuclear counterstain.

    Journal: BMC Biochemistry

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

    doi: 10.1186/1471-2091-12-27

    Figure Lengend Snippet: LRRC32 is a cell surface protein . a) C-and N- terminus GFP-tagged LRRC32 expressing HEK293 cell clones were surface biotinylated, and cell lysates were immunoprecipitated using antibody specific for GFP or using normal rabbit serum (NRS) as a control (left and right panels). Protein lysates were then electrophoresed, transferred to membrane PDVF, and probed for the presence of biotinylation using streptavidin-HRP (left panel only). Blots were also probed with anti-GFP (right panel only). b) Confocal analysis of untransfected (top row) HEK293 cells, C-GFP/CAT-transfected HEK293 cells (second row), N-GFP/LRRC32-transfected HEK293 cells (third row), C-GFP/LRRC32-transfected HEK293 cells (fourth row), and C-GFP/LRRC32ΔSP-transfected HEK293 cells (last row); Green = GFP, Red = anti-LRRC32 antibody, Blue = nuclear counterstain. The left column shows anti-LRRC32 only. The right column shows GFP only. The middle column shows the merged composite confocal picture (anti-LRRC32 + GFP) with the nuclear counterstain.

    Article Snippet: The reported sequence was aligned with the reported cDNA sequence of LRRC32 obtained from OriGene using the Vector NTI (Invitrogen) software system, and sequence alignments were then confirmed prior to further utilization of the LRRC32 sequence-verified plasmids.

    Techniques: Expressing, Clone Assay, Immunoprecipitation, Transfection

    A 17 AA signal peptide is required for the cell surface expression of LRRC32 . a) Untransfected HEK293 cells or cells transfected with either C-terminus GFP-tagged LRRC32 or C-terminus GFP-tagged LRRC32 with a deleted signal peptide region were analyzed by flow cytometry for surface expression of LRRC32 or GFP expression. b) Anti-LRRC32 immunoblot analysis of total lysates from C-and N- terminus GFP-tagged LRRC32 expressing clones revealed intact LRRC32 expression at 99 kD (fusion protein: 29 kD GFP + 70 kD LRRC32) and 70 kD, respectively. However, immunoblot analysis of total lysates from C- terminus GFP-tagged LRRC32 expressing clones lacking an intact signal peptide did not detect the presence of LRRC32 (rightmost lane). c) RT-PCR analysis of HEK293 cell lysates utilizing untransfected, C-CAT, C-terminus GFP-tagged LRRC32, or C-terminus GFP-tagged LRRC32 lacking an intact signal peptide was performed using primers for Lrrc32 (left panel) or GFP (right panel).

    Journal: BMC Biochemistry

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

    doi: 10.1186/1471-2091-12-27

    Figure Lengend Snippet: A 17 AA signal peptide is required for the cell surface expression of LRRC32 . a) Untransfected HEK293 cells or cells transfected with either C-terminus GFP-tagged LRRC32 or C-terminus GFP-tagged LRRC32 with a deleted signal peptide region were analyzed by flow cytometry for surface expression of LRRC32 or GFP expression. b) Anti-LRRC32 immunoblot analysis of total lysates from C-and N- terminus GFP-tagged LRRC32 expressing clones revealed intact LRRC32 expression at 99 kD (fusion protein: 29 kD GFP + 70 kD LRRC32) and 70 kD, respectively. However, immunoblot analysis of total lysates from C- terminus GFP-tagged LRRC32 expressing clones lacking an intact signal peptide did not detect the presence of LRRC32 (rightmost lane). c) RT-PCR analysis of HEK293 cell lysates utilizing untransfected, C-CAT, C-terminus GFP-tagged LRRC32, or C-terminus GFP-tagged LRRC32 lacking an intact signal peptide was performed using primers for Lrrc32 (left panel) or GFP (right panel).

    Article Snippet: The reported sequence was aligned with the reported cDNA sequence of LRRC32 obtained from OriGene using the Vector NTI (Invitrogen) software system, and sequence alignments were then confirmed prior to further utilization of the LRRC32 sequence-verified plasmids.

    Techniques: Expressing, Transfection, Flow Cytometry, Cytometry, Clone Assay, Reverse Transcription Polymerase Chain Reaction

    Schematic diagram for the evaluation of promoter activity. ( a ) Outline of the construction of transformation vectors and transformations. After predicting putative ORF positions 32 , upstream regions of the ORFs were determined as potential promoter regions. Potential promoter regions amplified by PCR were used to construct the transformation vectors. The double-cassette vector containing the reporter gene egfp driven by each tested promoter and the antibiotic-resistant gene Sh ble driven by the promoter region of the fucoxanthin chlorophyll a / c -binding protein (FCP) A-1A gene derived from Cyl. fusiformis (termed CffcpA pro.) were constructed. ( b ) Assessment of promoter activity. Promoter activity was determined by averaging the ratios of egfp mRNA transcript levels to those of Sh ble mRNA transcripts in ten transformants to minimize the effects of copy numbers on the expression of transgenes. These transformants were also used to investigate eGFP protein expression patterns. CffcpA ter.: terminator region of the FCP A-1A gene derived from Cyl. fusiformis . The structure of the ClorDNA genome was modified from Tomaru et al. 32 . *For the transformation vector of the nitrate reductase gene promoter, we used pNICgfp 18 ( Supplementary Fig. 5a ).

    Journal: Scientific Reports

    Article Title: Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum

    doi: 10.1038/srep18708

    Figure Lengend Snippet: Schematic diagram for the evaluation of promoter activity. ( a ) Outline of the construction of transformation vectors and transformations. After predicting putative ORF positions 32 , upstream regions of the ORFs were determined as potential promoter regions. Potential promoter regions amplified by PCR were used to construct the transformation vectors. The double-cassette vector containing the reporter gene egfp driven by each tested promoter and the antibiotic-resistant gene Sh ble driven by the promoter region of the fucoxanthin chlorophyll a / c -binding protein (FCP) A-1A gene derived from Cyl. fusiformis (termed CffcpA pro.) were constructed. ( b ) Assessment of promoter activity. Promoter activity was determined by averaging the ratios of egfp mRNA transcript levels to those of Sh ble mRNA transcripts in ten transformants to minimize the effects of copy numbers on the expression of transgenes. These transformants were also used to investigate eGFP protein expression patterns. CffcpA ter.: terminator region of the FCP A-1A gene derived from Cyl. fusiformis . The structure of the ClorDNA genome was modified from Tomaru et al. 32 . *For the transformation vector of the nitrate reductase gene promoter, we used pNICgfp 18 ( Supplementary Fig. 5a ).

    Article Snippet: The ORFs of the two DIVs were identified using an ORF finder (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/gorf/ ).

    Techniques: Activity Assay, Transformation Assay, Amplification, Polymerase Chain Reaction, Construct, Plasmid Preparation, Binding Assay, Derivative Assay, Expressing, Modification

    Genistein is a Lead Candidate for Inhibiting the ZDHHC17-MAP2K4 Interaction in GBM. (A) GBM0378 and GBM1492, from patients with GBM, cell viability after 24h-treatment with indicated inhibitors at indicated concentration from the MAPK Compound Library. Data represent the means ± SD from three separate experiments (** p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: Genistein is a Lead Candidate for Inhibiting the ZDHHC17-MAP2K4 Interaction in GBM. (A) GBM0378 and GBM1492, from patients with GBM, cell viability after 24h-treatment with indicated inhibitors at indicated concentration from the MAPK Compound Library. Data represent the means ± SD from three separate experiments (** p

    Article Snippet: The ZDHHC17 and MAP2K4 interaction mostly depended on the ZDHHC17 ankyrin-repeat (ANK) domain, as ZDHHC17 ΔANK lost the ability of binding MAP2K4 ( Figure F).

    Techniques: Concentration Assay

    ZDHHC17-MAP2K4 Signaling Module Promotes Chemoradiotherapy Resistance in GBM Spheres. (A) mRNA expression analysis (Mao's dataset, GSE67089) of ZDHHC17 expression in mesenchymal (MES) GSCs compared to normal astrocytes, or proneural (PN) GSCs. (B) Genome-wide transcriptome microarray analysis (GSE67089) of MAPKKs showing MAP2K4 up-regulation in MES compared with PN GSCs. (C) RT-PCR for ZDHHC17 and MAP2K4 in post-radiation GSCs from U118MG (6 Gy) or temozolomide (TMZ)-treated (25 μM) versus naive GSCs. (D) Western blot for ZDHHC17, MAP2K4, and EZH2 in post-radiation GSCs from U118MG (6 Gy) or TMZ-treated GSCs (25 μM) versus naive GSCs. (E) Flow cytometric analysis for apoptosis in GSCs pre-transduced with control or ZDHHC17 dsRNA, then treated with or without genistein (2.5 μM), radiation (6 Gy), and TMZ (25 μM). (F) BALB/c mice were subcutaneously injected with GSCs from U118MG. After five days, the nude mice were treated with 20 Gy X-irradiation (4.5-4.6 Gy/min), TMZ (50 mg kg -1 two days -1 , gastric infusion), or genistein (100 mg/kg daily, tail vein injection). Tumor weight was quantified. Data represent the means ± SD from five separate experiments ( ns , not significant; ** p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module Promotes Chemoradiotherapy Resistance in GBM Spheres. (A) mRNA expression analysis (Mao's dataset, GSE67089) of ZDHHC17 expression in mesenchymal (MES) GSCs compared to normal astrocytes, or proneural (PN) GSCs. (B) Genome-wide transcriptome microarray analysis (GSE67089) of MAPKKs showing MAP2K4 up-regulation in MES compared with PN GSCs. (C) RT-PCR for ZDHHC17 and MAP2K4 in post-radiation GSCs from U118MG (6 Gy) or temozolomide (TMZ)-treated (25 μM) versus naive GSCs. (D) Western blot for ZDHHC17, MAP2K4, and EZH2 in post-radiation GSCs from U118MG (6 Gy) or TMZ-treated GSCs (25 μM) versus naive GSCs. (E) Flow cytometric analysis for apoptosis in GSCs pre-transduced with control or ZDHHC17 dsRNA, then treated with or without genistein (2.5 μM), radiation (6 Gy), and TMZ (25 μM). (F) BALB/c mice were subcutaneously injected with GSCs from U118MG. After five days, the nude mice were treated with 20 Gy X-irradiation (4.5-4.6 Gy/min), TMZ (50 mg kg -1 two days -1 , gastric infusion), or genistein (100 mg/kg daily, tail vein injection). Tumor weight was quantified. Data represent the means ± SD from five separate experiments ( ns , not significant; ** p

    Article Snippet: The ZDHHC17 and MAP2K4 interaction mostly depended on the ZDHHC17 ankyrin-repeat (ANK) domain, as ZDHHC17 ΔANK lost the ability of binding MAP2K4 ( Figure F).

    Techniques: Expressing, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Transduction, Mouse Assay, Injection, Irradiation

    ZDHHC17-MAP2K4 Signaling Module is Required for GBM Cell Neurosphere Formation. (A-C) Flow cytometry and quantitation of SOX2 (stem cell marker) (A, B) and western blot of SOX2, CD133, and Nestin using β-actin as a loading control (C) in SW1088 cells transfected with ZDHHC17 plasmid or control, further transfected with MAP2K4 dsRNA, or genistein (2.5 μM)-treated. Data represent the means ± SD from three separate experiments (*** p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module is Required for GBM Cell Neurosphere Formation. (A-C) Flow cytometry and quantitation of SOX2 (stem cell marker) (A, B) and western blot of SOX2, CD133, and Nestin using β-actin as a loading control (C) in SW1088 cells transfected with ZDHHC17 plasmid or control, further transfected with MAP2K4 dsRNA, or genistein (2.5 μM)-treated. Data represent the means ± SD from three separate experiments (*** p

    Article Snippet: The ZDHHC17 and MAP2K4 interaction mostly depended on the ZDHHC17 ankyrin-repeat (ANK) domain, as ZDHHC17 ΔANK lost the ability of binding MAP2K4 ( Figure F).

    Techniques: Flow Cytometry, Cytometry, Quantitation Assay, Marker, Western Blot, Transfection, Plasmid Preparation

    ZDHHC17 and MAP2K4 Interact Via Specific Binding Motifs. (A) ZDHHC17 ANK (2-4) domain is crucial for ZDHHC17-MAP2K4 interaction. Myc-tagged ZDHHC17 ANK (1-7) and various deletions (right). Interaction capability (positive or negative) is shown. (B) MAP2K4 PKc domain is crucial for the ZDHHC17-MAP2K4 interaction, independent of the DVD domain. FLAG-tagged MAP2K4 and various MAP2K4 deletion fragments (right). Interaction capability (positive or negative) is shown. (C) Surface representation of the complex. ZDHHC17 and MAP2K4 binding mode molecular docking was performed using the ZDOCK server. MAP2K4 (Magenta) binds to the concave ANK(1-7) (Green) region between ANK2 and ANK4. (D) Cartoon and stick representation of ZDHHC17 ANK (1-7) (Pale Green) and ANK (1-7) (Green), respectively. (E) IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 ANK (1-7) mutants, followed by IB with anti-Flag antibodies and anti-Myc. Data represent the means ± SD from three separate experiments ( ns , not significant; * p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17 and MAP2K4 Interact Via Specific Binding Motifs. (A) ZDHHC17 ANK (2-4) domain is crucial for ZDHHC17-MAP2K4 interaction. Myc-tagged ZDHHC17 ANK (1-7) and various deletions (right). Interaction capability (positive or negative) is shown. (B) MAP2K4 PKc domain is crucial for the ZDHHC17-MAP2K4 interaction, independent of the DVD domain. FLAG-tagged MAP2K4 and various MAP2K4 deletion fragments (right). Interaction capability (positive or negative) is shown. (C) Surface representation of the complex. ZDHHC17 and MAP2K4 binding mode molecular docking was performed using the ZDOCK server. MAP2K4 (Magenta) binds to the concave ANK(1-7) (Green) region between ANK2 and ANK4. (D) Cartoon and stick representation of ZDHHC17 ANK (1-7) (Pale Green) and ANK (1-7) (Green), respectively. (E) IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 ANK (1-7) mutants, followed by IB with anti-Flag antibodies and anti-Myc. Data represent the means ± SD from three separate experiments ( ns , not significant; * p

    Article Snippet: The ZDHHC17 and MAP2K4 interaction mostly depended on the ZDHHC17 ankyrin-repeat (ANK) domain, as ZDHHC17 ΔANK lost the ability of binding MAP2K4 ( Figure F).

    Techniques: Binding Assay, Expressing

    ZDHHC17 and MAP2K4 Expression Correlates with Glioma Tumor Grade and has Prognostic Significance . (A) Representative images of immunohistochemistry staining for ZDHHC17 and MAP2K4 in different grade gliomas and normal brain specimens. Scale bar, 100 µm. (B, C) Correlation between MAP2K4 and ZDHHC17 (B) or ZDHHC13 (C) in GBM using TCGA datasets. Correlation statistical significance was evaluated using a linear regression model (n = 171, r = 0.4394, p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17 and MAP2K4 Expression Correlates with Glioma Tumor Grade and has Prognostic Significance . (A) Representative images of immunohistochemistry staining for ZDHHC17 and MAP2K4 in different grade gliomas and normal brain specimens. Scale bar, 100 µm. (B, C) Correlation between MAP2K4 and ZDHHC17 (B) or ZDHHC13 (C) in GBM using TCGA datasets. Correlation statistical significance was evaluated using a linear regression model (n = 171, r = 0.4394, p

    Article Snippet: The ZDHHC17 and MAP2K4 interaction mostly depended on the ZDHHC17 ankyrin-repeat (ANK) domain, as ZDHHC17 ΔANK lost the ability of binding MAP2K4 ( Figure F).

    Techniques: Expressing, Immunohistochemistry, Staining

    ZDHHC17 is Important for Brain Glioma Development. (A, B) Migratory human neural stem cells (hNSCs) transfected with indicated lentiviruses in Transwell assays. Cells were stained with crystal violet for counting. Scale bar, 500 µm. Data represent the means ± SD from three separate experiments (* p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17 is Important for Brain Glioma Development. (A, B) Migratory human neural stem cells (hNSCs) transfected with indicated lentiviruses in Transwell assays. Cells were stained with crystal violet for counting. Scale bar, 500 µm. Data represent the means ± SD from three separate experiments (* p

    Article Snippet: The ZDHHC17 and MAP2K4 interaction mostly depended on the ZDHHC17 ankyrin-repeat (ANK) domain, as ZDHHC17 ΔANK lost the ability of binding MAP2K4 ( Figure F).

    Techniques: Transfection, Staining

    ZDHHC17-MAP2K4 Signaling Module is Necessary for GBM Cell Tumorigenic and Invasive Phenotypes. (A, B) Clonogenic survival of (A) U118MG cells transfected with control or ZDHHC17 dsRNA, further transfected with ZDHHC17 plasmid or MAP2K4 dsRNA, or treated with genistein (2.5 μM), and (B) SW1088 cells transfected with control or ZDHHC17 plasmid, further transfected with ZDHHC17 dsRNA or MAP2K4 plasmid, or genistein (2.5 μM) treated and ZDHHC17-expressing. Data represent the means ± SD from three separate experiments ( ns , non-significant; * p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module is Necessary for GBM Cell Tumorigenic and Invasive Phenotypes. (A, B) Clonogenic survival of (A) U118MG cells transfected with control or ZDHHC17 dsRNA, further transfected with ZDHHC17 plasmid or MAP2K4 dsRNA, or treated with genistein (2.5 μM), and (B) SW1088 cells transfected with control or ZDHHC17 plasmid, further transfected with ZDHHC17 dsRNA or MAP2K4 plasmid, or genistein (2.5 μM) treated and ZDHHC17-expressing. Data represent the means ± SD from three separate experiments ( ns , non-significant; * p

    Article Snippet: The ZDHHC17 and MAP2K4 interaction mostly depended on the ZDHHC17 ankyrin-repeat (ANK) domain, as ZDHHC17 ΔANK lost the ability of binding MAP2K4 ( Figure F).

    Techniques: Transfection, Plasmid Preparation, Expressing

    ZDHHC17-MAP2K4-JNK/p38 Signaling Module Formation in Glioblastoma Multiforme (GBM). (A) The expression of ERK1 (pT202, pY204), ERK2 (pT185, pY187), JNK1/2(pT183, pT185), JNK3 (pT221, pY223) and p38 (pT180, pY182) in GBM is summarized based on the immunohistochemistry results of the Human Protein Atlas. (B) The expression of different DHHCs in glioma is summarized based on the immunohistochemistry results of the Human Protein Atlas. (C) Venn diagram showing the relationship between expression patterns of different DHHCs and activation of ERK1, ERK2, JNK1/2, JNK3, and p38 in GBM. (D) Lysates from HEK293 cells expressing Myc-ZDHHC17 and Flag-MAPKKs were subjected to immunoprecipitation (IP), followed up by immunoblotting (IB) with anti-FLAG antibodies and anti-Myc. IP, immunoprecipitation; IB, immunoblotting. (E) GST pull-down utilizing purified GST-MAP2K4 (or GST-MAP2K7) and FLAG-ZDHHC17-expressing HEK293 cell lysates, followed by IB with an anti-FLAG antibody. (F) ZDHHC17 associates with MAP2K4 via ZDHHC17 ANK rather than PAT activity domain. IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 mutants, followed by IB with anti-Flag antibodies and anti-Myc. ZDHHC17 ΔDHHC: ZDHHC17 aa 440-487 deletion; ΔANK: ZDHHC17 aa 51-288 deletion. (G) ZDHHC17 protein recruits MAP2K4 in the Golgi and cytoplasmic vesicles in U118MG cells, in a ZDHHC17 ANK domain-dependent manner. Immunofluorescence of U118MG cells expressing Myc-ZDHHC17 (or -ZDHHC17 ΔDHHC or ΔANK) with anti-Myc (Pink), MAP2K4 (Red), and GM130 (Green) antibodies. Scale bar, 20 µm. (H) ZDHHC17 interacts with MAP2K4 wild-type (wt) and the kinase-inactive mutant (ki). IP of lysates from HEK293 cells expressing Myc-ZDHHC17 and FLAG-MAP2K4wt (or FLAG-MAP2K4ki), followed by IB with anti-FLAG antibodies and anti-Myc. (I) MAP2K4 contributes to the ZDHHC7-mediated JNK1 and p38 phosphorylation. FLAG-MAP2K4ki co-expression in HEK293 cells reduces Myc-ZDHHC17-mediated GFP-JNK1 (or GFP-p38) phosphorylation. (J) MAP2K4 and JNK1 (or p38) are recruited by ZDHHC17 in a signaling module. GFP-JNK1 (p38) and FLAG-MAP2K4 were introduced in HEK293 cells, with or without Myc-ZDHHC17. MAP2K4 presence upon JNK1 (or p38) IP is improved by ZDHHC17 co-expression.

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17-MAP2K4-JNK/p38 Signaling Module Formation in Glioblastoma Multiforme (GBM). (A) The expression of ERK1 (pT202, pY204), ERK2 (pT185, pY187), JNK1/2(pT183, pT185), JNK3 (pT221, pY223) and p38 (pT180, pY182) in GBM is summarized based on the immunohistochemistry results of the Human Protein Atlas. (B) The expression of different DHHCs in glioma is summarized based on the immunohistochemistry results of the Human Protein Atlas. (C) Venn diagram showing the relationship between expression patterns of different DHHCs and activation of ERK1, ERK2, JNK1/2, JNK3, and p38 in GBM. (D) Lysates from HEK293 cells expressing Myc-ZDHHC17 and Flag-MAPKKs were subjected to immunoprecipitation (IP), followed up by immunoblotting (IB) with anti-FLAG antibodies and anti-Myc. IP, immunoprecipitation; IB, immunoblotting. (E) GST pull-down utilizing purified GST-MAP2K4 (or GST-MAP2K7) and FLAG-ZDHHC17-expressing HEK293 cell lysates, followed by IB with an anti-FLAG antibody. (F) ZDHHC17 associates with MAP2K4 via ZDHHC17 ANK rather than PAT activity domain. IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 mutants, followed by IB with anti-Flag antibodies and anti-Myc. ZDHHC17 ΔDHHC: ZDHHC17 aa 440-487 deletion; ΔANK: ZDHHC17 aa 51-288 deletion. (G) ZDHHC17 protein recruits MAP2K4 in the Golgi and cytoplasmic vesicles in U118MG cells, in a ZDHHC17 ANK domain-dependent manner. Immunofluorescence of U118MG cells expressing Myc-ZDHHC17 (or -ZDHHC17 ΔDHHC or ΔANK) with anti-Myc (Pink), MAP2K4 (Red), and GM130 (Green) antibodies. Scale bar, 20 µm. (H) ZDHHC17 interacts with MAP2K4 wild-type (wt) and the kinase-inactive mutant (ki). IP of lysates from HEK293 cells expressing Myc-ZDHHC17 and FLAG-MAP2K4wt (or FLAG-MAP2K4ki), followed by IB with anti-FLAG antibodies and anti-Myc. (I) MAP2K4 contributes to the ZDHHC7-mediated JNK1 and p38 phosphorylation. FLAG-MAP2K4ki co-expression in HEK293 cells reduces Myc-ZDHHC17-mediated GFP-JNK1 (or GFP-p38) phosphorylation. (J) MAP2K4 and JNK1 (or p38) are recruited by ZDHHC17 in a signaling module. GFP-JNK1 (p38) and FLAG-MAP2K4 were introduced in HEK293 cells, with or without Myc-ZDHHC17. MAP2K4 presence upon JNK1 (or p38) IP is improved by ZDHHC17 co-expression.

    Article Snippet: The ZDHHC17 and MAP2K4 interaction mostly depended on the ZDHHC17 ankyrin-repeat (ANK) domain, as ZDHHC17 ΔANK lost the ability of binding MAP2K4 ( Figure F).

    Techniques: Expressing, Immunohistochemistry, Activation Assay, Immunoprecipitation, Purification, Activity Assay, Immunofluorescence, Mutagenesis

    FAM60A directly represses expression of genes in the TGF-beta pathway. A , Genes increased by microarray analysis in FAM60A and SDS3 knockdown cells that encode components of the TGF-beta signaling pathway. Fold-increase of the top probe for that gene

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex *

    doi: 10.1074/mcp.M112.020255

    Figure Lengend Snippet: FAM60A directly represses expression of genes in the TGF-beta pathway. A , Genes increased by microarray analysis in FAM60A and SDS3 knockdown cells that encode components of the TGF-beta signaling pathway. Fold-increase of the top probe for that gene

    Article Snippet: Sequences of primers and annealing temperatures are as follows: FAM60A (Origene): forward 5′-GACTCGTTCAGGAGACATCTGC-3′, reverse 5′-AGTCTTTAGACTGGGTCCAGCC-3′ (annealing at 58 °C); GAPDH: forward 5′-TCCTGCACCACCAACTGCTTAG-3′, reverse 5′-GTAGAGGCAGGGATGATGTTC-3′ (annealing at 58 °C or 60 °C); TGFβR1 (Origene): forward 5′-GACAACGTCAGGTTCTGGCTCA-3′, reverse 5′-CCGCCACTTTCCTCTCCAAACT-3′ (annealing at 60 °C); TGFβ1: forward 5′-TACCTGAACCCGTGTTGCTCTC-3′, reverse 5′-GTTGCTGAGGTATCGCCAGGAA-3′ (annealing at 60 °C); TGFβ2: forward 5′-AAGAAGCGTGCTTTGGATGCGG-3′, reverse 5′-ATGCTCCAGCACAGAAGTTGGC-3′ (annealing at 60 °C); SMAD2 (Origene): forward 5′-GGGTTTTGAAGCCGTCTATCAGC-3′, reverse 5′-CCAACCACTGTAGAGGTCCATTC-3′: ING2: forward 5′-ACATGCAGAGGAACGTGTCTGTG-3′, reverse 5′-ACCAATTCGAGCATTTGTGTAAC-3′.

    Techniques: Expressing, Microarray

    FAM60A regulates cell morphology and migration of HepG2 cells. A , Morphology changes in HepG2 cells transfected with control siRNAs or siRNAs against FAM60A. B , Western blots of whole cell extracts from HepG2 knockdown cells using the indicated antibodies.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex *

    doi: 10.1074/mcp.M112.020255

    Figure Lengend Snippet: FAM60A regulates cell morphology and migration of HepG2 cells. A , Morphology changes in HepG2 cells transfected with control siRNAs or siRNAs against FAM60A. B , Western blots of whole cell extracts from HepG2 knockdown cells using the indicated antibodies.

    Article Snippet: Sequences of primers and annealing temperatures are as follows: FAM60A (Origene): forward 5′-GACTCGTTCAGGAGACATCTGC-3′, reverse 5′-AGTCTTTAGACTGGGTCCAGCC-3′ (annealing at 58 °C); GAPDH: forward 5′-TCCTGCACCACCAACTGCTTAG-3′, reverse 5′-GTAGAGGCAGGGATGATGTTC-3′ (annealing at 58 °C or 60 °C); TGFβR1 (Origene): forward 5′-GACAACGTCAGGTTCTGGCTCA-3′, reverse 5′-CCGCCACTTTCCTCTCCAAACT-3′ (annealing at 60 °C); TGFβ1: forward 5′-TACCTGAACCCGTGTTGCTCTC-3′, reverse 5′-GTTGCTGAGGTATCGCCAGGAA-3′ (annealing at 60 °C); TGFβ2: forward 5′-AAGAAGCGTGCTTTGGATGCGG-3′, reverse 5′-ATGCTCCAGCACAGAAGTTGGC-3′ (annealing at 60 °C); SMAD2 (Origene): forward 5′-GGGTTTTGAAGCCGTCTATCAGC-3′, reverse 5′-CCAACCACTGTAGAGGTCCATTC-3′: ING2: forward 5′-ACATGCAGAGGAACGTGTCTGTG-3′, reverse 5′-ACCAATTCGAGCATTTGTGTAAC-3′.

    Techniques: Migration, Transfection, Western Blot

    Loss of FAM60A affects cell morphology and gene expression. A , A549 cells were transfected with control siRNAs, or siRNAs against FAM60A, SDS3, or ING2. B , Western blots of whole cell extracts from FAM60A, SDS3, and control knockdown using the indicated

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex *

    doi: 10.1074/mcp.M112.020255

    Figure Lengend Snippet: Loss of FAM60A affects cell morphology and gene expression. A , A549 cells were transfected with control siRNAs, or siRNAs against FAM60A, SDS3, or ING2. B , Western blots of whole cell extracts from FAM60A, SDS3, and control knockdown using the indicated

    Article Snippet: Sequences of primers and annealing temperatures are as follows: FAM60A (Origene): forward 5′-GACTCGTTCAGGAGACATCTGC-3′, reverse 5′-AGTCTTTAGACTGGGTCCAGCC-3′ (annealing at 58 °C); GAPDH: forward 5′-TCCTGCACCACCAACTGCTTAG-3′, reverse 5′-GTAGAGGCAGGGATGATGTTC-3′ (annealing at 58 °C or 60 °C); TGFβR1 (Origene): forward 5′-GACAACGTCAGGTTCTGGCTCA-3′, reverse 5′-CCGCCACTTTCCTCTCCAAACT-3′ (annealing at 60 °C); TGFβ1: forward 5′-TACCTGAACCCGTGTTGCTCTC-3′, reverse 5′-GTTGCTGAGGTATCGCCAGGAA-3′ (annealing at 60 °C); TGFβ2: forward 5′-AAGAAGCGTGCTTTGGATGCGG-3′, reverse 5′-ATGCTCCAGCACAGAAGTTGGC-3′ (annealing at 60 °C); SMAD2 (Origene): forward 5′-GGGTTTTGAAGCCGTCTATCAGC-3′, reverse 5′-CCAACCACTGTAGAGGTCCATTC-3′: ING2: forward 5′-ACATGCAGAGGAACGTGTCTGTG-3′, reverse 5′-ACCAATTCGAGCATTTGTGTAAC-3′.

    Techniques: Expressing, Transfection, Western Blot

    FAM60A is a metazoan-specific Sin3/HDAC interacting protein. A , Heat map for the relative protein abundances expressed as dNSAF in the core Sin3/HDAC complex. The data are represented in a bait-prey matrix, where each column corresponds to purification

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex *

    doi: 10.1074/mcp.M112.020255

    Figure Lengend Snippet: FAM60A is a metazoan-specific Sin3/HDAC interacting protein. A , Heat map for the relative protein abundances expressed as dNSAF in the core Sin3/HDAC complex. The data are represented in a bait-prey matrix, where each column corresponds to purification

    Article Snippet: Sequences of primers and annealing temperatures are as follows: FAM60A (Origene): forward 5′-GACTCGTTCAGGAGACATCTGC-3′, reverse 5′-AGTCTTTAGACTGGGTCCAGCC-3′ (annealing at 58 °C); GAPDH: forward 5′-TCCTGCACCACCAACTGCTTAG-3′, reverse 5′-GTAGAGGCAGGGATGATGTTC-3′ (annealing at 58 °C or 60 °C); TGFβR1 (Origene): forward 5′-GACAACGTCAGGTTCTGGCTCA-3′, reverse 5′-CCGCCACTTTCCTCTCCAAACT-3′ (annealing at 60 °C); TGFβ1: forward 5′-TACCTGAACCCGTGTTGCTCTC-3′, reverse 5′-GTTGCTGAGGTATCGCCAGGAA-3′ (annealing at 60 °C); TGFβ2: forward 5′-AAGAAGCGTGCTTTGGATGCGG-3′, reverse 5′-ATGCTCCAGCACAGAAGTTGGC-3′ (annealing at 60 °C); SMAD2 (Origene): forward 5′-GGGTTTTGAAGCCGTCTATCAGC-3′, reverse 5′-CCAACCACTGTAGAGGTCCATTC-3′: ING2: forward 5′-ACATGCAGAGGAACGTGTCTGTG-3′, reverse 5′-ACCAATTCGAGCATTTGTGTAAC-3′.

    Techniques: Purification

    FAM60A and SDS3 regulate common subsets of genes. A , Scatter plot showing the changes in gene expression in SDS3 knockdown cells relative to a nontargeting control siRNA. The y -axis indicates the ratio of expression as log2 (SDS3 knockdown/control), whereas

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex *

    doi: 10.1074/mcp.M112.020255

    Figure Lengend Snippet: FAM60A and SDS3 regulate common subsets of genes. A , Scatter plot showing the changes in gene expression in SDS3 knockdown cells relative to a nontargeting control siRNA. The y -axis indicates the ratio of expression as log2 (SDS3 knockdown/control), whereas

    Article Snippet: Sequences of primers and annealing temperatures are as follows: FAM60A (Origene): forward 5′-GACTCGTTCAGGAGACATCTGC-3′, reverse 5′-AGTCTTTAGACTGGGTCCAGCC-3′ (annealing at 58 °C); GAPDH: forward 5′-TCCTGCACCACCAACTGCTTAG-3′, reverse 5′-GTAGAGGCAGGGATGATGTTC-3′ (annealing at 58 °C or 60 °C); TGFβR1 (Origene): forward 5′-GACAACGTCAGGTTCTGGCTCA-3′, reverse 5′-CCGCCACTTTCCTCTCCAAACT-3′ (annealing at 60 °C); TGFβ1: forward 5′-TACCTGAACCCGTGTTGCTCTC-3′, reverse 5′-GTTGCTGAGGTATCGCCAGGAA-3′ (annealing at 60 °C); TGFβ2: forward 5′-AAGAAGCGTGCTTTGGATGCGG-3′, reverse 5′-ATGCTCCAGCACAGAAGTTGGC-3′ (annealing at 60 °C); SMAD2 (Origene): forward 5′-GGGTTTTGAAGCCGTCTATCAGC-3′, reverse 5′-CCAACCACTGTAGAGGTCCATTC-3′: ING2: forward 5′-ACATGCAGAGGAACGTGTCTGTG-3′, reverse 5′-ACCAATTCGAGCATTTGTGTAAC-3′.

    Techniques: Expressing

    Loss of FAM60A or SDS3 increases cell migration in A549 lung cancer cells. A , Migration assays in control, FAM60A, or SDS3 knockdown cells. A representative example is shown for each siRNA. B , Quantification of migration assays shown in ( A ). The average

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex *

    doi: 10.1074/mcp.M112.020255

    Figure Lengend Snippet: Loss of FAM60A or SDS3 increases cell migration in A549 lung cancer cells. A , Migration assays in control, FAM60A, or SDS3 knockdown cells. A representative example is shown for each siRNA. B , Quantification of migration assays shown in ( A ). The average

    Article Snippet: Sequences of primers and annealing temperatures are as follows: FAM60A (Origene): forward 5′-GACTCGTTCAGGAGACATCTGC-3′, reverse 5′-AGTCTTTAGACTGGGTCCAGCC-3′ (annealing at 58 °C); GAPDH: forward 5′-TCCTGCACCACCAACTGCTTAG-3′, reverse 5′-GTAGAGGCAGGGATGATGTTC-3′ (annealing at 58 °C or 60 °C); TGFβR1 (Origene): forward 5′-GACAACGTCAGGTTCTGGCTCA-3′, reverse 5′-CCGCCACTTTCCTCTCCAAACT-3′ (annealing at 60 °C); TGFβ1: forward 5′-TACCTGAACCCGTGTTGCTCTC-3′, reverse 5′-GTTGCTGAGGTATCGCCAGGAA-3′ (annealing at 60 °C); TGFβ2: forward 5′-AAGAAGCGTGCTTTGGATGCGG-3′, reverse 5′-ATGCTCCAGCACAGAAGTTGGC-3′ (annealing at 60 °C); SMAD2 (Origene): forward 5′-GGGTTTTGAAGCCGTCTATCAGC-3′, reverse 5′-CCAACCACTGTAGAGGTCCATTC-3′: ING2: forward 5′-ACATGCAGAGGAACGTGTCTGTG-3′, reverse 5′-ACCAATTCGAGCATTTGTGTAAC-3′.

    Techniques: Migration

    SAMHD1 restricts HIV-1 infection of monocytes and AM. AM and PBMC were obtained from the same healthy donor. CD14 + cells were selected from the PBMC, cultured in the presence of M-CSF overnight (monocytes) and infected (A, top ) or cultured an additional 6 days (MDM) before infection (B, top ) . AM were cultured without cytokines overnight, washed vigorously to remove contaminants, and infected 1 day (A, bottom ) or 1 week (B, bottom ) after isolation. Cells were infected with HIV NLAD8 in the absence or presence of SIV-VLP (+Vpx) as indicated. (A, B) Cells were stained and analyzed by flow cytometry 4 dpi. Circled populations are HIV p24 positive, CD4 downregulated, productively infected cells. Numbers in the plots indicate percent of total cells infected. (C, D) Productively infected cells from (A and B) were quantified and displayed as bar graphs in C (infected 1 day after isolation) and D (infected 1 week after isolation). Data are representative of three experiments. AM, alveolar macrophages; M-CSF, macrophage-colony stimulating factor; PBMC, peripheral blood mononuclear cells.

    Journal: AIDS Research and Human Retroviruses

    Article Title: Brain Microglial Cells Are Highly Susceptible to HIV-1 Infection and Spread

    doi: 10.1089/aid.2017.0004

    Figure Lengend Snippet: SAMHD1 restricts HIV-1 infection of monocytes and AM. AM and PBMC were obtained from the same healthy donor. CD14 + cells were selected from the PBMC, cultured in the presence of M-CSF overnight (monocytes) and infected (A, top ) or cultured an additional 6 days (MDM) before infection (B, top ) . AM were cultured without cytokines overnight, washed vigorously to remove contaminants, and infected 1 day (A, bottom ) or 1 week (B, bottom ) after isolation. Cells were infected with HIV NLAD8 in the absence or presence of SIV-VLP (+Vpx) as indicated. (A, B) Cells were stained and analyzed by flow cytometry 4 dpi. Circled populations are HIV p24 positive, CD4 downregulated, productively infected cells. Numbers in the plots indicate percent of total cells infected. (C, D) Productively infected cells from (A and B) were quantified and displayed as bar graphs in C (infected 1 day after isolation) and D (infected 1 week after isolation). Data are representative of three experiments. AM, alveolar macrophages; M-CSF, macrophage-colony stimulating factor; PBMC, peripheral blood mononuclear cells.

    Article Snippet: Cells were stained for SAMHD1 (Clone 1A1; OriGene, Rockville, MD) in staining buffer for 15 min at room temperature.

    Techniques: Infection, Cell Culture, Isolation, Staining, Flow Cytometry, Cytometry

    SAMHD1 restricts HIV-1 infection in MDM. MDM were infected with the HIV-1 strains HIV NLAD8 (NLAD8) (A) or HIV NLAD8-GFP-Nef (NLAD8-GFP) (B) alone or in the presence of SIV-VLP (+Vpx) as indicated. Cells were stained and analyzed by flow cytometry 4 and 8 dpi. Circled populations are HIV p24-positive (HIV NLAD8 ) or GPF-positive (HIV NLAD8-GFP-Nef ), CD4 downregulated productively infected cells. Numbers in the plots indicate percent of total cells infected. (C) MDM were cultured on glass coverslips and infected with HIV NLAD8-GFP-Nef without or with SIV-VLP (+Vpx), fixed 4 or 8 dpi and stained for SAMHD1 ( red ) and nuclear DNA ( blue ). Adjacent fields were imaged, stitched together, and rendered into single whole cell volume projections. GFP ( green ) marks productively infected cells. Scale bar: 15 μm. (D) MDM were infected with HIV NLAD8-GFP-Nef without or with SIV-VLP (+Vpx) for the indicated time and quantified by flow cytometry as above. MVC (2.5 μM) was added 5 dpi and every 3 days thereafter where indicated to block the potential spread of infection. Infection was quantified using flow cytometry as in (A) . Data shown are representative of at least three independent experiments. dpi, day postinfection; HIV-1, human immunodeficiency virus type 1; MDM, monocyte-derived macrophages; MVC, Maraviroc; SAMHD1, sterile alpha motif and histidine/aspartic acid domain-containing protein 1; SIV-VLP, simian immunodeficiency virus–virus-like particles.

    Journal: AIDS Research and Human Retroviruses

    Article Title: Brain Microglial Cells Are Highly Susceptible to HIV-1 Infection and Spread

    doi: 10.1089/aid.2017.0004

    Figure Lengend Snippet: SAMHD1 restricts HIV-1 infection in MDM. MDM were infected with the HIV-1 strains HIV NLAD8 (NLAD8) (A) or HIV NLAD8-GFP-Nef (NLAD8-GFP) (B) alone or in the presence of SIV-VLP (+Vpx) as indicated. Cells were stained and analyzed by flow cytometry 4 and 8 dpi. Circled populations are HIV p24-positive (HIV NLAD8 ) or GPF-positive (HIV NLAD8-GFP-Nef ), CD4 downregulated productively infected cells. Numbers in the plots indicate percent of total cells infected. (C) MDM were cultured on glass coverslips and infected with HIV NLAD8-GFP-Nef without or with SIV-VLP (+Vpx), fixed 4 or 8 dpi and stained for SAMHD1 ( red ) and nuclear DNA ( blue ). Adjacent fields were imaged, stitched together, and rendered into single whole cell volume projections. GFP ( green ) marks productively infected cells. Scale bar: 15 μm. (D) MDM were infected with HIV NLAD8-GFP-Nef without or with SIV-VLP (+Vpx) for the indicated time and quantified by flow cytometry as above. MVC (2.5 μM) was added 5 dpi and every 3 days thereafter where indicated to block the potential spread of infection. Infection was quantified using flow cytometry as in (A) . Data shown are representative of at least three independent experiments. dpi, day postinfection; HIV-1, human immunodeficiency virus type 1; MDM, monocyte-derived macrophages; MVC, Maraviroc; SAMHD1, sterile alpha motif and histidine/aspartic acid domain-containing protein 1; SIV-VLP, simian immunodeficiency virus–virus-like particles.

    Article Snippet: Cells were stained for SAMHD1 (Clone 1A1; OriGene, Rockville, MD) in staining buffer for 15 min at room temperature.

    Techniques: Infection, Staining, Flow Cytometry, Cytometry, Cell Culture, Blocking Assay, Derivative Assay

    Microglia are highly permissive to HIV-1 infection even in the presence of SAMHD1. Human microglia were cultured overnight on chamber slides and infected with HIV NLAD8-GFP-Nef (NLAD8) without or with SIV-VLP (+Vpx) as indicated. Cells were fixed and stained for SAMHD1 ( red ) and nuclear DNA ( blue ) and imaged. Adjacent fields were stitched together and rendered into single whole cell volume projections. GFP ( green ) marks productively infected cells. Representative images of multiple fields are shown. (A, B) Standard titer (150 ng/ml) of virus was used, and cells were analyzed 3 and 6 dpi as indicated. (C, D) Low titer (15 ng/ml = 10% of standard titer) of virus was used, and cells were analyzed 4 and 8 dpi as indicated. (B, D) GFP-positive cells in multiple fields were quantified, including those shown in (A, C) , and plotted as average GFP-positive nuclei per field. Bars indicate the standard error of the mean ( n = 3 fields, ∼1,500 cells). Data are representative of three independent experiments. Scale bars: 15 μm (A) , 50 μm (C) .

    Journal: AIDS Research and Human Retroviruses

    Article Title: Brain Microglial Cells Are Highly Susceptible to HIV-1 Infection and Spread

    doi: 10.1089/aid.2017.0004

    Figure Lengend Snippet: Microglia are highly permissive to HIV-1 infection even in the presence of SAMHD1. Human microglia were cultured overnight on chamber slides and infected with HIV NLAD8-GFP-Nef (NLAD8) without or with SIV-VLP (+Vpx) as indicated. Cells were fixed and stained for SAMHD1 ( red ) and nuclear DNA ( blue ) and imaged. Adjacent fields were stitched together and rendered into single whole cell volume projections. GFP ( green ) marks productively infected cells. Representative images of multiple fields are shown. (A, B) Standard titer (150 ng/ml) of virus was used, and cells were analyzed 3 and 6 dpi as indicated. (C, D) Low titer (15 ng/ml = 10% of standard titer) of virus was used, and cells were analyzed 4 and 8 dpi as indicated. (B, D) GFP-positive cells in multiple fields were quantified, including those shown in (A, C) , and plotted as average GFP-positive nuclei per field. Bars indicate the standard error of the mean ( n = 3 fields, ∼1,500 cells). Data are representative of three independent experiments. Scale bars: 15 μm (A) , 50 μm (C) .

    Article Snippet: Cells were stained for SAMHD1 (Clone 1A1; OriGene, Rockville, MD) in staining buffer for 15 min at room temperature.

    Techniques: Infection, Cell Culture, Staining

    SAMHD1 restricts HIV-1 infection of PM. Macrophages (PM) were isolated from peritoneal exudates by Ficoll centrifugation and CD14 + magnetic bead selection. PM were infected 1 day after isolation with HIV NLAD8-GFP-Nef (NLAD8) without or with SIV-VLP (+Vpx) as indicated. (A) PM were cultured on glass coverslips, fixed, and stained for SAMHD1 ( red ) and nuclear DNA ( blue ) 4 and 8 dpi. Adjacent fields were imaged, stitched together, and rendered into single whole cell volume projections. GFP ( green ) marks productively infected cells. Scale bar: 15 μm. (B) Cells were cultured overnight, infected as above, and analyzed by flow cytometry 4 and 8 dpi. The mean percent infected cells from three independent experiments are shown; error bars represent standard error of the mean ( n = 3). PM, peritoneal macrophages.

    Journal: AIDS Research and Human Retroviruses

    Article Title: Brain Microglial Cells Are Highly Susceptible to HIV-1 Infection and Spread

    doi: 10.1089/aid.2017.0004

    Figure Lengend Snippet: SAMHD1 restricts HIV-1 infection of PM. Macrophages (PM) were isolated from peritoneal exudates by Ficoll centrifugation and CD14 + magnetic bead selection. PM were infected 1 day after isolation with HIV NLAD8-GFP-Nef (NLAD8) without or with SIV-VLP (+Vpx) as indicated. (A) PM were cultured on glass coverslips, fixed, and stained for SAMHD1 ( red ) and nuclear DNA ( blue ) 4 and 8 dpi. Adjacent fields were imaged, stitched together, and rendered into single whole cell volume projections. GFP ( green ) marks productively infected cells. Scale bar: 15 μm. (B) Cells were cultured overnight, infected as above, and analyzed by flow cytometry 4 and 8 dpi. The mean percent infected cells from three independent experiments are shown; error bars represent standard error of the mean ( n = 3). PM, peritoneal macrophages.

    Article Snippet: Cells were stained for SAMHD1 (Clone 1A1; OriGene, Rockville, MD) in staining buffer for 15 min at room temperature.

    Techniques: Infection, Isolation, Centrifugation, Selection, Cell Culture, Staining, Flow Cytometry, Cytometry

    ALDH1A1 affects tumor growth and vascular flow in MCF-7 tumor xenograft in athymic nude mice. MCF-7 cells (1 × 10 7 with 50% v /v of Matrigel) were injected s.c. in flank of athymic female nude mice. β-estradiol were injected (3 mg/kg), every 7 days i.m.. All mice were sacrificed at day 23. Tumor volumes were detected twice a week using a caliper and calculated by the formula: shortest diameter × longest diameter × thickness of the tumor in mm ( n = 6 animals per group). a Tumor volume at day 23. ** p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells

    doi: 10.1186/s13046-018-0975-0

    Figure Lengend Snippet: ALDH1A1 affects tumor growth and vascular flow in MCF-7 tumor xenograft in athymic nude mice. MCF-7 cells (1 × 10 7 with 50% v /v of Matrigel) were injected s.c. in flank of athymic female nude mice. β-estradiol were injected (3 mg/kg), every 7 days i.m.. All mice were sacrificed at day 23. Tumor volumes were detected twice a week using a caliper and calculated by the formula: shortest diameter × longest diameter × thickness of the tumor in mm ( n = 6 animals per group). a Tumor volume at day 23. ** p

    Article Snippet: To generate a stable ALDH1A1 overexpression (ALDH1A1+ ), breast cancer cells were seeded on 6-multiplates and transfected with lentiviral particles containing nucleotide sequences encoding for ALDH1A1 (Origene RC200723 LentiORF particles, ALDH1A1 (Myc-DDK tagged) - Human).

    Techniques: Flow Cytometry, Mouse Assay, Injection

    ALDH1A1 influences tumor angiogenesis and VEGF production in vivo. a Evaluation of VEGF, HIF-1α and ALDH1A1 RNA in tumor samples. Frozen tumors were homogenized and RNA was extracted to perform RT-PCR analysis of VEGF, HIF-1α and ALDH1A1 mRNA. Data are reported as ΔCt (Ct gene of interest-Ct Housekeeping gene). Each bar is the mean of 6 different tumors. The experiment was repeated two times. * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells

    doi: 10.1186/s13046-018-0975-0

    Figure Lengend Snippet: ALDH1A1 influences tumor angiogenesis and VEGF production in vivo. a Evaluation of VEGF, HIF-1α and ALDH1A1 RNA in tumor samples. Frozen tumors were homogenized and RNA was extracted to perform RT-PCR analysis of VEGF, HIF-1α and ALDH1A1 mRNA. Data are reported as ΔCt (Ct gene of interest-Ct Housekeeping gene). Each bar is the mean of 6 different tumors. The experiment was repeated two times. * p

    Article Snippet: To generate a stable ALDH1A1 overexpression (ALDH1A1+ ), breast cancer cells were seeded on 6-multiplates and transfected with lentiviral particles containing nucleotide sequences encoding for ALDH1A1 (Origene RC200723 LentiORF particles, ALDH1A1 (Myc-DDK tagged) - Human).

    Techniques: In Vivo, Reverse Transcription Polymerase Chain Reaction

    Expression and activity of ALDH1A1 in breast cancer cells. a RT-PCR analysis of ALDH1A1 in breast cancer cells grown with 10% FBS. b Western blot analysis for ALDH1A1. β-Actin was used as loading control. Gel shown is representative of three experiments with similar results. c Variation of ALDH1A1 activity, measured by the formation of NADH in tumor cells. Breast tumor cell lysates were pretreated with CM037 (50 μM, 10 min) and absorbance at 340 nm (corresponding to NADH production) was measured. ** p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells

    doi: 10.1186/s13046-018-0975-0

    Figure Lengend Snippet: Expression and activity of ALDH1A1 in breast cancer cells. a RT-PCR analysis of ALDH1A1 in breast cancer cells grown with 10% FBS. b Western blot analysis for ALDH1A1. β-Actin was used as loading control. Gel shown is representative of three experiments with similar results. c Variation of ALDH1A1 activity, measured by the formation of NADH in tumor cells. Breast tumor cell lysates were pretreated with CM037 (50 μM, 10 min) and absorbance at 340 nm (corresponding to NADH production) was measured. ** p

    Article Snippet: To generate a stable ALDH1A1 overexpression (ALDH1A1+ ), breast cancer cells were seeded on 6-multiplates and transfected with lentiviral particles containing nucleotide sequences encoding for ALDH1A1 (Origene RC200723 LentiORF particles, ALDH1A1 (Myc-DDK tagged) - Human).

    Techniques: Expressing, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

    MCF-7 ALDH1A1 affects in vitro stemness. a Representative images of tumorspheres (4x magnification) showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. b Representative images of tumorspheres (4x magnification) of MCF-7 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1 + , showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. b1, b2, b3. Representative images of tumorspheres (10x magnification) of MCF-1 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1 + , showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. c Quantification of MCF-7 tumorspheres. Tumorspheres area were calculated using ImageJ Software. Ten pictures for each well were quantified. Tumorspheres > 10.000 pixel square were considered. ** p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells

    doi: 10.1186/s13046-018-0975-0

    Figure Lengend Snippet: MCF-7 ALDH1A1 affects in vitro stemness. a Representative images of tumorspheres (4x magnification) showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. b Representative images of tumorspheres (4x magnification) of MCF-7 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1 + , showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. b1, b2, b3. Representative images of tumorspheres (10x magnification) of MCF-1 Scr, MCF-7 ALDH1A1KD and MCF-7 ALDH1A1 + , showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 μm. c Quantification of MCF-7 tumorspheres. Tumorspheres area were calculated using ImageJ Software. Ten pictures for each well were quantified. Tumorspheres > 10.000 pixel square were considered. ** p

    Article Snippet: To generate a stable ALDH1A1 overexpression (ALDH1A1+ ), breast cancer cells were seeded on 6-multiplates and transfected with lentiviral particles containing nucleotide sequences encoding for ALDH1A1 (Origene RC200723 LentiORF particles, ALDH1A1 (Myc-DDK tagged) - Human).

    Techniques: In Vitro, Software

    MCF-7 ALDH1A1 regulates endothelial angiogenic features in VEGF dependent manner. a Viability of MCF-7 (Scr, ALDH1A1KD, ALDH1A1 + ) exposed to exogenous serum (10% FBS) or VEGF (2 and 20 ng/ml) at 72 h and evaluated by MTT assay. Data are reported as absorbance at 540 nm. *** p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells

    doi: 10.1186/s13046-018-0975-0

    Figure Lengend Snippet: MCF-7 ALDH1A1 regulates endothelial angiogenic features in VEGF dependent manner. a Viability of MCF-7 (Scr, ALDH1A1KD, ALDH1A1 + ) exposed to exogenous serum (10% FBS) or VEGF (2 and 20 ng/ml) at 72 h and evaluated by MTT assay. Data are reported as absorbance at 540 nm. *** p

    Article Snippet: To generate a stable ALDH1A1 overexpression (ALDH1A1+ ), breast cancer cells were seeded on 6-multiplates and transfected with lentiviral particles containing nucleotide sequences encoding for ALDH1A1 (Origene RC200723 LentiORF particles, ALDH1A1 (Myc-DDK tagged) - Human).

    Techniques: MTT Assay

    MCF-7 ALDH1A1 regulates angiogenic factor output via retinoic acid signalling. a Angiogenic factor release evaluated by ELISA plate array in supernatants of MCF-7 treated with CM037 (1 μM) for 48 h. The experiment was performed 2 times in duplicate. b MCF-7 cells were exposed to CM037 at different concentrations (1 and 10 μM) for 18 h and western blot was carried out. β-Actin was used to normalize loading. c Cells were treated with CM037 (1 μM, 18 h) and VEGF levels were measured by ELISA assay in MCF-7 conditioned media. After 18 h supernatants were harvested and cells fixed, stained and counted. The number of counted cells was not significantly different. Data are reported as pg/ml. ** p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells

    doi: 10.1186/s13046-018-0975-0

    Figure Lengend Snippet: MCF-7 ALDH1A1 regulates angiogenic factor output via retinoic acid signalling. a Angiogenic factor release evaluated by ELISA plate array in supernatants of MCF-7 treated with CM037 (1 μM) for 48 h. The experiment was performed 2 times in duplicate. b MCF-7 cells were exposed to CM037 at different concentrations (1 and 10 μM) for 18 h and western blot was carried out. β-Actin was used to normalize loading. c Cells were treated with CM037 (1 μM, 18 h) and VEGF levels were measured by ELISA assay in MCF-7 conditioned media. After 18 h supernatants were harvested and cells fixed, stained and counted. The number of counted cells was not significantly different. Data are reported as pg/ml. ** p

    Article Snippet: To generate a stable ALDH1A1 overexpression (ALDH1A1+ ), breast cancer cells were seeded on 6-multiplates and transfected with lentiviral particles containing nucleotide sequences encoding for ALDH1A1 (Origene RC200723 LentiORF particles, ALDH1A1 (Myc-DDK tagged) - Human).

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Staining

    Genistein is a Lead Candidate for Inhibiting the ZDHHC17-MAP2K4 Interaction in GBM. (A) GBM0378 and GBM1492, from patients with GBM, cell viability after 24h-treatment with indicated inhibitors at indicated concentration from the MAPK Compound Library. Data represent the means ± SD from three separate experiments (** p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: Genistein is a Lead Candidate for Inhibiting the ZDHHC17-MAP2K4 Interaction in GBM. (A) GBM0378 and GBM1492, from patients with GBM, cell viability after 24h-treatment with indicated inhibitors at indicated concentration from the MAPK Compound Library. Data represent the means ± SD from three separate experiments (** p

    Article Snippet: The association with MAP2K4 was independent of ZDHHC17 PAT activity, because both ZDHHC17 wild-type (wt) and ΔDHHC could bind MAP2K4 by IP ( Figure F).

    Techniques: Concentration Assay

    ZDHHC17-MAP2K4 Signaling Module Promotes Chemoradiotherapy Resistance in GBM Spheres. (A) mRNA expression analysis (Mao's dataset, GSE67089) of ZDHHC17 expression in mesenchymal (MES) GSCs compared to normal astrocytes, or proneural (PN) GSCs. (B) Genome-wide transcriptome microarray analysis (GSE67089) of MAPKKs showing MAP2K4 up-regulation in MES compared with PN GSCs. (C) RT-PCR for ZDHHC17 and MAP2K4 in post-radiation GSCs from U118MG (6 Gy) or temozolomide (TMZ)-treated (25 μM) versus naive GSCs. (D) Western blot for ZDHHC17, MAP2K4, and EZH2 in post-radiation GSCs from U118MG (6 Gy) or TMZ-treated GSCs (25 μM) versus naive GSCs. (E) Flow cytometric analysis for apoptosis in GSCs pre-transduced with control or ZDHHC17 dsRNA, then treated with or without genistein (2.5 μM), radiation (6 Gy), and TMZ (25 μM). (F) BALB/c mice were subcutaneously injected with GSCs from U118MG. After five days, the nude mice were treated with 20 Gy X-irradiation (4.5-4.6 Gy/min), TMZ (50 mg kg -1 two days -1 , gastric infusion), or genistein (100 mg/kg daily, tail vein injection). Tumor weight was quantified. Data represent the means ± SD from five separate experiments ( ns , not significant; ** p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module Promotes Chemoradiotherapy Resistance in GBM Spheres. (A) mRNA expression analysis (Mao's dataset, GSE67089) of ZDHHC17 expression in mesenchymal (MES) GSCs compared to normal astrocytes, or proneural (PN) GSCs. (B) Genome-wide transcriptome microarray analysis (GSE67089) of MAPKKs showing MAP2K4 up-regulation in MES compared with PN GSCs. (C) RT-PCR for ZDHHC17 and MAP2K4 in post-radiation GSCs from U118MG (6 Gy) or temozolomide (TMZ)-treated (25 μM) versus naive GSCs. (D) Western blot for ZDHHC17, MAP2K4, and EZH2 in post-radiation GSCs from U118MG (6 Gy) or TMZ-treated GSCs (25 μM) versus naive GSCs. (E) Flow cytometric analysis for apoptosis in GSCs pre-transduced with control or ZDHHC17 dsRNA, then treated with or without genistein (2.5 μM), radiation (6 Gy), and TMZ (25 μM). (F) BALB/c mice were subcutaneously injected with GSCs from U118MG. After five days, the nude mice were treated with 20 Gy X-irradiation (4.5-4.6 Gy/min), TMZ (50 mg kg -1 two days -1 , gastric infusion), or genistein (100 mg/kg daily, tail vein injection). Tumor weight was quantified. Data represent the means ± SD from five separate experiments ( ns , not significant; ** p

    Article Snippet: The association with MAP2K4 was independent of ZDHHC17 PAT activity, because both ZDHHC17 wild-type (wt) and ΔDHHC could bind MAP2K4 by IP ( Figure F).

    Techniques: Expressing, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Western Blot, Flow Cytometry, Transduction, Mouse Assay, Injection, Irradiation

    ZDHHC17-MAP2K4 Signaling Module is Required for GBM Cell Neurosphere Formation. (A-C) Flow cytometry and quantitation of SOX2 (stem cell marker) (A, B) and western blot of SOX2, CD133, and Nestin using β-actin as a loading control (C) in SW1088 cells transfected with ZDHHC17 plasmid or control, further transfected with MAP2K4 dsRNA, or genistein (2.5 μM)-treated. Data represent the means ± SD from three separate experiments (*** p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module is Required for GBM Cell Neurosphere Formation. (A-C) Flow cytometry and quantitation of SOX2 (stem cell marker) (A, B) and western blot of SOX2, CD133, and Nestin using β-actin as a loading control (C) in SW1088 cells transfected with ZDHHC17 plasmid or control, further transfected with MAP2K4 dsRNA, or genistein (2.5 μM)-treated. Data represent the means ± SD from three separate experiments (*** p

    Article Snippet: The association with MAP2K4 was independent of ZDHHC17 PAT activity, because both ZDHHC17 wild-type (wt) and ΔDHHC could bind MAP2K4 by IP ( Figure F).

    Techniques: Flow Cytometry, Cytometry, Quantitation Assay, Marker, Western Blot, Transfection, Plasmid Preparation

    ZDHHC17 and MAP2K4 Interact Via Specific Binding Motifs. (A) ZDHHC17 ANK (2-4) domain is crucial for ZDHHC17-MAP2K4 interaction. Myc-tagged ZDHHC17 ANK (1-7) and various deletions (right). Interaction capability (positive or negative) is shown. (B) MAP2K4 PKc domain is crucial for the ZDHHC17-MAP2K4 interaction, independent of the DVD domain. FLAG-tagged MAP2K4 and various MAP2K4 deletion fragments (right). Interaction capability (positive or negative) is shown. (C) Surface representation of the complex. ZDHHC17 and MAP2K4 binding mode molecular docking was performed using the ZDOCK server. MAP2K4 (Magenta) binds to the concave ANK(1-7) (Green) region between ANK2 and ANK4. (D) Cartoon and stick representation of ZDHHC17 ANK (1-7) (Pale Green) and ANK (1-7) (Green), respectively. (E) IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 ANK (1-7) mutants, followed by IB with anti-Flag antibodies and anti-Myc. Data represent the means ± SD from three separate experiments ( ns , not significant; * p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17 and MAP2K4 Interact Via Specific Binding Motifs. (A) ZDHHC17 ANK (2-4) domain is crucial for ZDHHC17-MAP2K4 interaction. Myc-tagged ZDHHC17 ANK (1-7) and various deletions (right). Interaction capability (positive or negative) is shown. (B) MAP2K4 PKc domain is crucial for the ZDHHC17-MAP2K4 interaction, independent of the DVD domain. FLAG-tagged MAP2K4 and various MAP2K4 deletion fragments (right). Interaction capability (positive or negative) is shown. (C) Surface representation of the complex. ZDHHC17 and MAP2K4 binding mode molecular docking was performed using the ZDOCK server. MAP2K4 (Magenta) binds to the concave ANK(1-7) (Green) region between ANK2 and ANK4. (D) Cartoon and stick representation of ZDHHC17 ANK (1-7) (Pale Green) and ANK (1-7) (Green), respectively. (E) IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 ANK (1-7) mutants, followed by IB with anti-Flag antibodies and anti-Myc. Data represent the means ± SD from three separate experiments ( ns , not significant; * p

    Article Snippet: The association with MAP2K4 was independent of ZDHHC17 PAT activity, because both ZDHHC17 wild-type (wt) and ΔDHHC could bind MAP2K4 by IP ( Figure F).

    Techniques: Binding Assay, Expressing

    ZDHHC17 and MAP2K4 Expression Correlates with Glioma Tumor Grade and has Prognostic Significance . (A) Representative images of immunohistochemistry staining for ZDHHC17 and MAP2K4 in different grade gliomas and normal brain specimens. Scale bar, 100 µm. (B, C) Correlation between MAP2K4 and ZDHHC17 (B) or ZDHHC13 (C) in GBM using TCGA datasets. Correlation statistical significance was evaluated using a linear regression model (n = 171, r = 0.4394, p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17 and MAP2K4 Expression Correlates with Glioma Tumor Grade and has Prognostic Significance . (A) Representative images of immunohistochemistry staining for ZDHHC17 and MAP2K4 in different grade gliomas and normal brain specimens. Scale bar, 100 µm. (B, C) Correlation between MAP2K4 and ZDHHC17 (B) or ZDHHC13 (C) in GBM using TCGA datasets. Correlation statistical significance was evaluated using a linear regression model (n = 171, r = 0.4394, p

    Article Snippet: The association with MAP2K4 was independent of ZDHHC17 PAT activity, because both ZDHHC17 wild-type (wt) and ΔDHHC could bind MAP2K4 by IP ( Figure F).

    Techniques: Expressing, Immunohistochemistry, Staining

    ZDHHC17-MAP2K4 Signaling Module is Necessary for GBM Cell Tumorigenic and Invasive Phenotypes. (A, B) Clonogenic survival of (A) U118MG cells transfected with control or ZDHHC17 dsRNA, further transfected with ZDHHC17 plasmid or MAP2K4 dsRNA, or treated with genistein (2.5 μM), and (B) SW1088 cells transfected with control or ZDHHC17 plasmid, further transfected with ZDHHC17 dsRNA or MAP2K4 plasmid, or genistein (2.5 μM) treated and ZDHHC17-expressing. Data represent the means ± SD from three separate experiments ( ns , non-significant; * p

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17-MAP2K4 Signaling Module is Necessary for GBM Cell Tumorigenic and Invasive Phenotypes. (A, B) Clonogenic survival of (A) U118MG cells transfected with control or ZDHHC17 dsRNA, further transfected with ZDHHC17 plasmid or MAP2K4 dsRNA, or treated with genistein (2.5 μM), and (B) SW1088 cells transfected with control or ZDHHC17 plasmid, further transfected with ZDHHC17 dsRNA or MAP2K4 plasmid, or genistein (2.5 μM) treated and ZDHHC17-expressing. Data represent the means ± SD from three separate experiments ( ns , non-significant; * p

    Article Snippet: The association with MAP2K4 was independent of ZDHHC17 PAT activity, because both ZDHHC17 wild-type (wt) and ΔDHHC could bind MAP2K4 by IP ( Figure F).

    Techniques: Transfection, Plasmid Preparation, Expressing

    ZDHHC17-MAP2K4-JNK/p38 Signaling Module Formation in Glioblastoma Multiforme (GBM). (A) The expression of ERK1 (pT202, pY204), ERK2 (pT185, pY187), JNK1/2(pT183, pT185), JNK3 (pT221, pY223) and p38 (pT180, pY182) in GBM is summarized based on the immunohistochemistry results of the Human Protein Atlas. (B) The expression of different DHHCs in glioma is summarized based on the immunohistochemistry results of the Human Protein Atlas. (C) Venn diagram showing the relationship between expression patterns of different DHHCs and activation of ERK1, ERK2, JNK1/2, JNK3, and p38 in GBM. (D) Lysates from HEK293 cells expressing Myc-ZDHHC17 and Flag-MAPKKs were subjected to immunoprecipitation (IP), followed up by immunoblotting (IB) with anti-FLAG antibodies and anti-Myc. IP, immunoprecipitation; IB, immunoblotting. (E) GST pull-down utilizing purified GST-MAP2K4 (or GST-MAP2K7) and FLAG-ZDHHC17-expressing HEK293 cell lysates, followed by IB with an anti-FLAG antibody. (F) ZDHHC17 associates with MAP2K4 via ZDHHC17 ANK rather than PAT activity domain. IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 mutants, followed by IB with anti-Flag antibodies and anti-Myc. ZDHHC17 ΔDHHC: ZDHHC17 aa 440-487 deletion; ΔANK: ZDHHC17 aa 51-288 deletion. (G) ZDHHC17 protein recruits MAP2K4 in the Golgi and cytoplasmic vesicles in U118MG cells, in a ZDHHC17 ANK domain-dependent manner. Immunofluorescence of U118MG cells expressing Myc-ZDHHC17 (or -ZDHHC17 ΔDHHC or ΔANK) with anti-Myc (Pink), MAP2K4 (Red), and GM130 (Green) antibodies. Scale bar, 20 µm. (H) ZDHHC17 interacts with MAP2K4 wild-type (wt) and the kinase-inactive mutant (ki). IP of lysates from HEK293 cells expressing Myc-ZDHHC17 and FLAG-MAP2K4wt (or FLAG-MAP2K4ki), followed by IB with anti-FLAG antibodies and anti-Myc. (I) MAP2K4 contributes to the ZDHHC7-mediated JNK1 and p38 phosphorylation. FLAG-MAP2K4ki co-expression in HEK293 cells reduces Myc-ZDHHC17-mediated GFP-JNK1 (or GFP-p38) phosphorylation. (J) MAP2K4 and JNK1 (or p38) are recruited by ZDHHC17 in a signaling module. GFP-JNK1 (p38) and FLAG-MAP2K4 were introduced in HEK293 cells, with or without Myc-ZDHHC17. MAP2K4 presence upon JNK1 (or p38) IP is improved by ZDHHC17 co-expression.

    Journal: Theranostics

    Article Title: Activation of JNK and p38 MAPK Mediated by ZDHHC17 Drives Glioblastoma Multiforme Development and Malignant Progression

    doi: 10.7150/thno.40076

    Figure Lengend Snippet: ZDHHC17-MAP2K4-JNK/p38 Signaling Module Formation in Glioblastoma Multiforme (GBM). (A) The expression of ERK1 (pT202, pY204), ERK2 (pT185, pY187), JNK1/2(pT183, pT185), JNK3 (pT221, pY223) and p38 (pT180, pY182) in GBM is summarized based on the immunohistochemistry results of the Human Protein Atlas. (B) The expression of different DHHCs in glioma is summarized based on the immunohistochemistry results of the Human Protein Atlas. (C) Venn diagram showing the relationship between expression patterns of different DHHCs and activation of ERK1, ERK2, JNK1/2, JNK3, and p38 in GBM. (D) Lysates from HEK293 cells expressing Myc-ZDHHC17 and Flag-MAPKKs were subjected to immunoprecipitation (IP), followed up by immunoblotting (IB) with anti-FLAG antibodies and anti-Myc. IP, immunoprecipitation; IB, immunoblotting. (E) GST pull-down utilizing purified GST-MAP2K4 (or GST-MAP2K7) and FLAG-ZDHHC17-expressing HEK293 cell lysates, followed by IB with an anti-FLAG antibody. (F) ZDHHC17 associates with MAP2K4 via ZDHHC17 ANK rather than PAT activity domain. IP of lysates from HEK293 cells expressing Flag-MAP2K4 and Myc-ZDHHC17 mutants, followed by IB with anti-Flag antibodies and anti-Myc. ZDHHC17 ΔDHHC: ZDHHC17 aa 440-487 deletion; ΔANK: ZDHHC17 aa 51-288 deletion. (G) ZDHHC17 protein recruits MAP2K4 in the Golgi and cytoplasmic vesicles in U118MG cells, in a ZDHHC17 ANK domain-dependent manner. Immunofluorescence of U118MG cells expressing Myc-ZDHHC17 (or -ZDHHC17 ΔDHHC or ΔANK) with anti-Myc (Pink), MAP2K4 (Red), and GM130 (Green) antibodies. Scale bar, 20 µm. (H) ZDHHC17 interacts with MAP2K4 wild-type (wt) and the kinase-inactive mutant (ki). IP of lysates from HEK293 cells expressing Myc-ZDHHC17 and FLAG-MAP2K4wt (or FLAG-MAP2K4ki), followed by IB with anti-FLAG antibodies and anti-Myc. (I) MAP2K4 contributes to the ZDHHC7-mediated JNK1 and p38 phosphorylation. FLAG-MAP2K4ki co-expression in HEK293 cells reduces Myc-ZDHHC17-mediated GFP-JNK1 (or GFP-p38) phosphorylation. (J) MAP2K4 and JNK1 (or p38) are recruited by ZDHHC17 in a signaling module. GFP-JNK1 (p38) and FLAG-MAP2K4 were introduced in HEK293 cells, with or without Myc-ZDHHC17. MAP2K4 presence upon JNK1 (or p38) IP is improved by ZDHHC17 co-expression.

    Article Snippet: The association with MAP2K4 was independent of ZDHHC17 PAT activity, because both ZDHHC17 wild-type (wt) and ΔDHHC could bind MAP2K4 by IP ( Figure F).

    Techniques: Expressing, Immunohistochemistry, Activation Assay, Immunoprecipitation, Purification, Activity Assay, Immunofluorescence, Mutagenesis

    Trim21 and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Trim21 and LFG expression. (A) Results of real-time PCR-analysis of Trim21-expression after 24 h under different culture conditions. Mock, Untreated MDA-MB-231 as negative control, Trim21, 2.0 μl recombinant human Trim21 protein added to culture medium; siControl, adenoviral transfection of MDA-MB-231 with empty vector; siLFG, adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG. (B) Expression analysis of LFG protein under different culture conditions by western blot analysis using 25 μg of total protein. a, Adenoviral transfection of MDA-MB-231 with vector coding for siRNA against LFG (24 h). b, Adenoviral transfection of MDA-MB-231 with empty vector. c, Untreated MDA-MB-231 as negative control. d, MDA-MB231 cultivated with 2.0 μg recombinant Trim21-protein in culture medium.

    Article Snippet: The order of increasing signal intensity of TRIM21 is MOCK, TRIM21, siControl and siLFG.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Negative Control, Recombinant, Transfection, Plasmid Preparation, Western Blot

    TRIM21 domain. (A) (Left) Vector map of the used pMK-RQ vector (Life Technologies), (right) amino acid sequence of TRIM21 and the sequences of an individual domains. (B) Western blot analysis after native 12.5% polyacrylamide gel. Detection of TRIM21 with Li-Cor 800 anti-goat, detection of LFG with Li-Cor-680 anti-rabbit. Sample 1a (PRY domain), sample 1b (SPRY domain), sample 2 (coiled-coil domain), sample 3 (B-box domain), sample 4 (RING domain), marker (M). The samples 2–4 show clearly visible bands of ~80 and 60 kDa. The bands at 80 kDa are larger but slightly less visible as the bands at the height of 60 kDa. In samples 1a and 1b no bands are visible.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: TRIM21 domain. (A) (Left) Vector map of the used pMK-RQ vector (Life Technologies), (right) amino acid sequence of TRIM21 and the sequences of an individual domains. (B) Western blot analysis after native 12.5% polyacrylamide gel. Detection of TRIM21 with Li-Cor 800 anti-goat, detection of LFG with Li-Cor-680 anti-rabbit. Sample 1a (PRY domain), sample 1b (SPRY domain), sample 2 (coiled-coil domain), sample 3 (B-box domain), sample 4 (RING domain), marker (M). The samples 2–4 show clearly visible bands of ~80 and 60 kDa. The bands at 80 kDa are larger but slightly less visible as the bands at the height of 60 kDa. In samples 1a and 1b no bands are visible.

    Article Snippet: The order of increasing signal intensity of TRIM21 is MOCK, TRIM21, siControl and siLFG.

    Techniques: Plasmid Preparation, Sequencing, Western Blot, Marker

    Arrays. (A) Tissue samples of breast tumors having different degrees of malignity (US Biomax, Inc.) (a and b). Specific antibody against Trim21 and fluorescence labeled second antibody (Li-Cor-800), and staining of nucleic acid with Syto-60. Detection was carried out using Odyssey (Li-Cor Biosciences). (B) Bar chart of the fluorescence intensity of the tissue samples, degrees of malignity (IIa, IIb, IIIa, IIIb and IV); * P

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Arrays. (A) Tissue samples of breast tumors having different degrees of malignity (US Biomax, Inc.) (a and b). Specific antibody against Trim21 and fluorescence labeled second antibody (Li-Cor-800), and staining of nucleic acid with Syto-60. Detection was carried out using Odyssey (Li-Cor Biosciences). (B) Bar chart of the fluorescence intensity of the tissue samples, degrees of malignity (IIa, IIb, IIIa, IIIb and IV); * P

    Article Snippet: The order of increasing signal intensity of TRIM21 is MOCK, TRIM21, siControl and siLFG.

    Techniques: Fluorescence, Labeling, Staining

    NF-κB array. Bar chart of gene expression of common NF-κB regulated genes after real-time PCR-analysis. Trim21, MDA-MB-231 cultivated with 2.0 μg/ml recombinant human Trim21-protein for 24 h. Control, MDA-MB-231 cells cultivated for 24 h under standard conditions.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: NF-κB array. Bar chart of gene expression of common NF-κB regulated genes after real-time PCR-analysis. Trim21, MDA-MB-231 cultivated with 2.0 μg/ml recombinant human Trim21-protein for 24 h. Control, MDA-MB-231 cells cultivated for 24 h under standard conditions.

    Article Snippet: The order of increasing signal intensity of TRIM21 is MOCK, TRIM21, siControl and siLFG.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Recombinant

    Co-immunoprecipitation. (A) Native western blot analysis after co-immunoprecipitation of LFG and TRIM21. LFG was specifically isolated from the cell lysate of MDA-MB-231 after transfection with vectors coding for LFG and TRIM21 (24 h) by use of antibody coated magnetic beads (Dynabeads; Invitrogen). Detection of LFG (FAIM2) and TRIM21 (Ssa1/2) (both from Santa Cruz Biotechnology, Inc.) by specific first antibodies and fluorescence labeled second antibodies. LFG is visible as a green signal (Alexa Fluor 546), TRIM21 is visible as a red signal (Alexa Fluor 488), and combined signals are visible as a yellow signal. (B) Analysis of LFG and TRIM21 protein in the MDA-MB-231 breast cancer cells 24 h after transfection with vectors coding for LFG and TRIM21, by specific first and fluorescence labeled second antibodies. a, Green signal for LFG; b, red signal for TRIM21; c, blue signal for DAPI-stained core; d, overlay of the single signals with yellow signals in places where green and red signals appear in the same spot.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Co-immunoprecipitation. (A) Native western blot analysis after co-immunoprecipitation of LFG and TRIM21. LFG was specifically isolated from the cell lysate of MDA-MB-231 after transfection with vectors coding for LFG and TRIM21 (24 h) by use of antibody coated magnetic beads (Dynabeads; Invitrogen). Detection of LFG (FAIM2) and TRIM21 (Ssa1/2) (both from Santa Cruz Biotechnology, Inc.) by specific first antibodies and fluorescence labeled second antibodies. LFG is visible as a green signal (Alexa Fluor 546), TRIM21 is visible as a red signal (Alexa Fluor 488), and combined signals are visible as a yellow signal. (B) Analysis of LFG and TRIM21 protein in the MDA-MB-231 breast cancer cells 24 h after transfection with vectors coding for LFG and TRIM21, by specific first and fluorescence labeled second antibodies. a, Green signal for LFG; b, red signal for TRIM21; c, blue signal for DAPI-stained core; d, overlay of the single signals with yellow signals in places where green and red signals appear in the same spot.

    Article Snippet: The order of increasing signal intensity of TRIM21 is MOCK, TRIM21, siControl and siLFG.

    Techniques: Immunoprecipitation, Western Blot, Isolation, Multiple Displacement Amplification, Transfection, Magnetic Beads, Fluorescence, Labeling, Staining

    Caspase-3-activity and cell cycle-analysis. (A) Analysis of caspase-3-activity in MDA-MB-231 cells. Cultivation for 48 h under different conditions and addition of 100 ng agonistic anti-Fas (clone CH11; Abcam). Measurement of caspase-3-activity after 24 h. TRIM21, addition of 1.0 μg/ml recombinant human TRIM21-protein every 24 h. Control, untreated MDA-MB-231 cultivated for 48 h. Detection of LFG, TRIM21 and actin by specific first antibodies and fluorescence labeled second antibodies. (B) Plots of cell cycle-analysis of MDA-MB-231 after 24 h cultivation in presence of different concentration of recombinant human TRIM21-protein after staining with propidium iodide. a-1, Cultivation of MDA-MB-231 without addition of TRIM21-recombinant protein. b-1, Addition of 0.25 μg/ml TRIM21-recombinant protein to the culture medium. c-1, Addition of 0.5 μg/ml TRIM21-recombinant protein to the culture medium. d-1, Addition of 0.75 μg/ml TRIM21-recombinant protein to the culture medium. e-1, Addition of 1.0 μg/ml TRIM21-recombinant protein to the culture medium.

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Caspase-3-activity and cell cycle-analysis. (A) Analysis of caspase-3-activity in MDA-MB-231 cells. Cultivation for 48 h under different conditions and addition of 100 ng agonistic anti-Fas (clone CH11; Abcam). Measurement of caspase-3-activity after 24 h. TRIM21, addition of 1.0 μg/ml recombinant human TRIM21-protein every 24 h. Control, untreated MDA-MB-231 cultivated for 48 h. Detection of LFG, TRIM21 and actin by specific first antibodies and fluorescence labeled second antibodies. (B) Plots of cell cycle-analysis of MDA-MB-231 after 24 h cultivation in presence of different concentration of recombinant human TRIM21-protein after staining with propidium iodide. a-1, Cultivation of MDA-MB-231 without addition of TRIM21-recombinant protein. b-1, Addition of 0.25 μg/ml TRIM21-recombinant protein to the culture medium. c-1, Addition of 0.5 μg/ml TRIM21-recombinant protein to the culture medium. d-1, Addition of 0.75 μg/ml TRIM21-recombinant protein to the culture medium. e-1, Addition of 1.0 μg/ml TRIM21-recombinant protein to the culture medium.

    Article Snippet: The order of increasing signal intensity of TRIM21 is MOCK, TRIM21, siControl and siLFG.

    Techniques: Activity Assay, Cell Cycle Assay, Multiple Displacement Amplification, Recombinant, Fluorescence, Labeling, Concentration Assay, Staining

    Analysis of the interaction. Array analysis of the interaction of over 9,000 different proteins with LFG. Recombinant LFG protein (10 μM) was used and detected by specific first antibody (FAIM2; Santa Cruz Biotechnology, Inc.) and fluorescence labeled second antibody (Alexa Fluor 546). The display window shows the signal stimulated by the interaction of LFG with TRIM21 (white signal).

    Journal: International Journal of Oncology

    Article Title: TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro

    doi: 10.3892/ijo.2015.3169

    Figure Lengend Snippet: Analysis of the interaction. Array analysis of the interaction of over 9,000 different proteins with LFG. Recombinant LFG protein (10 μM) was used and detected by specific first antibody (FAIM2; Santa Cruz Biotechnology, Inc.) and fluorescence labeled second antibody (Alexa Fluor 546). The display window shows the signal stimulated by the interaction of LFG with TRIM21 (white signal).

    Article Snippet: The order of increasing signal intensity of TRIM21 is MOCK, TRIM21, siControl and siLFG.

    Techniques: Recombinant, Fluorescence, Labeling

    High level of CLEC18B predicted shorter overall survival (OS) in GBM patients. (a) Kaplan–Meier curves for OS according to CLEC18B expression in patients with GBM collected from TCGA. (b) Kaplan–Meier curves for OS according to CLEC18B expression in GBM patients collected at our institution. CLEC18B = C-type lectin domain family 18 member B; GBM = glioblastoma multiforme; TCGA = The Cancer Genome Atlas.

    Journal: ASN NEURO

    Article Title: Overexpression of CLEC18B Associates With the Proliferation, Migration, and Prognosis of Glioblastoma

    doi: 10.1177/1759091418781949

    Figure Lengend Snippet: High level of CLEC18B predicted shorter overall survival (OS) in GBM patients. (a) Kaplan–Meier curves for OS according to CLEC18B expression in patients with GBM collected from TCGA. (b) Kaplan–Meier curves for OS according to CLEC18B expression in GBM patients collected at our institution. CLEC18B = C-type lectin domain family 18 member B; GBM = glioblastoma multiforme; TCGA = The Cancer Genome Atlas.

    Article Snippet: The membranes were incubated overnight with the primary antibodies including CLEC18B (1:1000; OriGene Technologies Inc., Rockville, MD, USA), nucleus β-catenin (1:1000), Histone (1:1000), C-Myc (1:1000), and Cyclin D1 (1:1000; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Expressing

    Growth and colony-formation abilities of GBM cells were inhibited by CLEC18B knockdown. (a to c) Transfection efficacy of CLEC18B si-RNA in U87 and U251 cells was analyzed by qPCR and Western blot assay, respectively. (d) Assessing the effects of CLEC18B downregulation on the proliferation ability of U87 and U251 cells using CCK-8 assay. (e) Evaluating the effects of CLEC18B knockdown on the colony-formation ability of U87 and U251 cells. All values are shown as mean ± SD, ** p

    Journal: ASN NEURO

    Article Title: Overexpression of CLEC18B Associates With the Proliferation, Migration, and Prognosis of Glioblastoma

    doi: 10.1177/1759091418781949

    Figure Lengend Snippet: Growth and colony-formation abilities of GBM cells were inhibited by CLEC18B knockdown. (a to c) Transfection efficacy of CLEC18B si-RNA in U87 and U251 cells was analyzed by qPCR and Western blot assay, respectively. (d) Assessing the effects of CLEC18B downregulation on the proliferation ability of U87 and U251 cells using CCK-8 assay. (e) Evaluating the effects of CLEC18B knockdown on the colony-formation ability of U87 and U251 cells. All values are shown as mean ± SD, ** p

    Article Snippet: The membranes were incubated overnight with the primary antibodies including CLEC18B (1:1000; OriGene Technologies Inc., Rockville, MD, USA), nucleus β-catenin (1:1000), Histone (1:1000), C-Myc (1:1000), and Cyclin D1 (1:1000; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay

    Migration and invasion capabilities of U87 and U251 cells were prohibited by CLEC18B knockdown. (a) The mobility of U87 cells was measured by wound-healing assay. (b) The migration and invasion of U87 cells were tested by transwell assay. (c) The mobility of U251 cells was measured by wound-healing assay. (d) The migration and invasion of U251 cells were tested by transwell assay. All values are shown as mean ± SD, ** p

    Journal: ASN NEURO

    Article Title: Overexpression of CLEC18B Associates With the Proliferation, Migration, and Prognosis of Glioblastoma

    doi: 10.1177/1759091418781949

    Figure Lengend Snippet: Migration and invasion capabilities of U87 and U251 cells were prohibited by CLEC18B knockdown. (a) The mobility of U87 cells was measured by wound-healing assay. (b) The migration and invasion of U87 cells were tested by transwell assay. (c) The mobility of U251 cells was measured by wound-healing assay. (d) The migration and invasion of U251 cells were tested by transwell assay. All values are shown as mean ± SD, ** p

    Article Snippet: The membranes were incubated overnight with the primary antibodies including CLEC18B (1:1000; OriGene Technologies Inc., Rockville, MD, USA), nucleus β-catenin (1:1000), Histone (1:1000), C-Myc (1:1000), and Cyclin D1 (1:1000; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Migration, Wound Healing Assay, Transwell Assay

    Elevated expression level of CLEC18B was observed in GBM tissues and cells. (a) Box plots of The Cancer Genome Atlas (TCGA) RNA expression analysis for CLEC18B. (b) Box plots of Oncomine datasets RNA expression analysis for CLEC18B. (c) RNA expression level of CLEC18B in clinical samples. (d) RNA expression level of CLEC18B in GBM cells and normal brain glial cell lines, HEB. All values are shown as mean ± SD, ** p

    Journal: ASN NEURO

    Article Title: Overexpression of CLEC18B Associates With the Proliferation, Migration, and Prognosis of Glioblastoma

    doi: 10.1177/1759091418781949

    Figure Lengend Snippet: Elevated expression level of CLEC18B was observed in GBM tissues and cells. (a) Box plots of The Cancer Genome Atlas (TCGA) RNA expression analysis for CLEC18B. (b) Box plots of Oncomine datasets RNA expression analysis for CLEC18B. (c) RNA expression level of CLEC18B in clinical samples. (d) RNA expression level of CLEC18B in GBM cells and normal brain glial cell lines, HEB. All values are shown as mean ± SD, ** p

    Article Snippet: The membranes were incubated overnight with the primary antibodies including CLEC18B (1:1000; OriGene Technologies Inc., Rockville, MD, USA), nucleus β-catenin (1:1000), Histone (1:1000), C-Myc (1:1000), and Cyclin D1 (1:1000; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Expressing, RNA Expression

    The Wnt/β-catenin signaling was suppressed by knockdown of CLEC18B. (a) The key markers of Wnt/β-catenin signaling were measured by Western blot assay. (b) Box–whisker plot presenting the difference in protein levels in si-CLEC18B and si-NC groups. All values are shown as mean ± SD, ** p

    Journal: ASN NEURO

    Article Title: Overexpression of CLEC18B Associates With the Proliferation, Migration, and Prognosis of Glioblastoma

    doi: 10.1177/1759091418781949

    Figure Lengend Snippet: The Wnt/β-catenin signaling was suppressed by knockdown of CLEC18B. (a) The key markers of Wnt/β-catenin signaling were measured by Western blot assay. (b) Box–whisker plot presenting the difference in protein levels in si-CLEC18B and si-NC groups. All values are shown as mean ± SD, ** p

    Article Snippet: The membranes were incubated overnight with the primary antibodies including CLEC18B (1:1000; OriGene Technologies Inc., Rockville, MD, USA), nucleus β-catenin (1:1000), Histone (1:1000), C-Myc (1:1000), and Cyclin D1 (1:1000; Cell Signaling Technology, Danvers, MA, USA).

    Techniques: Western Blot, Whisker Assay

    Overexpression of Pla2g3 reduces IDE expression. A , B , Quantitative RT-PCR result of IDE in TR-AST cells and rat primary astrocytes. TR-AST cells and primary astrocytes were treated with hydrogen peroxide in indicated conditions. Fold changes to the non-treated cells as controls are indicated. C , Quantitative PCR results of IDE in Pla2g3 transfected HEK293 cells. Human Pla2g3 was transiently expressed and cells were harvested after 48 hours of transfection. Fold changes to the mock control are indicated. *p

    Journal: PLoS ONE

    Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

    doi: 10.1371/journal.pone.0143518

    Figure Lengend Snippet: Overexpression of Pla2g3 reduces IDE expression. A , B , Quantitative RT-PCR result of IDE in TR-AST cells and rat primary astrocytes. TR-AST cells and primary astrocytes were treated with hydrogen peroxide in indicated conditions. Fold changes to the non-treated cells as controls are indicated. C , Quantitative PCR results of IDE in Pla2g3 transfected HEK293 cells. Human Pla2g3 was transiently expressed and cells were harvested after 48 hours of transfection. Fold changes to the mock control are indicated. *p

    Article Snippet: Membranes were blocked with Tris-buffer saline containing 0.1% Tween 20 and 5% nonfat milk for 1 hour, then incubated with primary antibody to Pla2g3 (1:1000; Origene), alpha-tubulin (1:2000; MBL), and were then visualized with appropriate HRP-conjugated secondary antibodies.

    Techniques: Over Expression, Expressing, Quantitative RT-PCR, AST Assay, Real-time Polymerase Chain Reaction, Transfection

    Induction of Pla2g3 expression in cerebrum by oxidative stress. A , B , Quantitative RT-PCR results of Pla2g3 in cerebrum and cerebellum are shown. Fold changes to the aged wild-type mice on normal diet are indicated. n = 4 in each group. C , Western blots for Pla2g3 and alpha-tubulin are shown. Abbreviation used; WT normal; wild type 29 months old mice fed on normal diet, WT deficient; 29 months old wild type mice fed on vitamin E deficient diet, ttpKO deficient; 29 months old Ttpa -/- mice fed on vitamin E deficient diet. *p

    Journal: PLoS ONE

    Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

    doi: 10.1371/journal.pone.0143518

    Figure Lengend Snippet: Induction of Pla2g3 expression in cerebrum by oxidative stress. A , B , Quantitative RT-PCR results of Pla2g3 in cerebrum and cerebellum are shown. Fold changes to the aged wild-type mice on normal diet are indicated. n = 4 in each group. C , Western blots for Pla2g3 and alpha-tubulin are shown. Abbreviation used; WT normal; wild type 29 months old mice fed on normal diet, WT deficient; 29 months old wild type mice fed on vitamin E deficient diet, ttpKO deficient; 29 months old Ttpa -/- mice fed on vitamin E deficient diet. *p

    Article Snippet: Membranes were blocked with Tris-buffer saline containing 0.1% Tween 20 and 5% nonfat milk for 1 hour, then incubated with primary antibody to Pla2g3 (1:1000; Origene), alpha-tubulin (1:2000; MBL), and were then visualized with appropriate HRP-conjugated secondary antibodies.

    Techniques: Expressing, Quantitative RT-PCR, Mouse Assay, Western Blot

    Astrocytic expression of Pla2g3 by chronic oxidative stress. Double immunostaining of Pla2g3 (green) and GFAP (red) in 29 months old wild type mouse (A, B), 29 months old Ttpa -/- mouse (C, D), Ischemic site (E, F) and traumatic injury site (G, H) of cerebral cortex of wild type mice. Arrow heads indicate the strong Pla2g3 expression in astrocytes of Ttpa -/- cortex. Scale bar: 50 μm.

    Journal: PLoS ONE

    Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

    doi: 10.1371/journal.pone.0143518

    Figure Lengend Snippet: Astrocytic expression of Pla2g3 by chronic oxidative stress. Double immunostaining of Pla2g3 (green) and GFAP (red) in 29 months old wild type mouse (A, B), 29 months old Ttpa -/- mouse (C, D), Ischemic site (E, F) and traumatic injury site (G, H) of cerebral cortex of wild type mice. Arrow heads indicate the strong Pla2g3 expression in astrocytes of Ttpa -/- cortex. Scale bar: 50 μm.

    Article Snippet: Membranes were blocked with Tris-buffer saline containing 0.1% Tween 20 and 5% nonfat milk for 1 hour, then incubated with primary antibody to Pla2g3 (1:1000; Origene), alpha-tubulin (1:2000; MBL), and were then visualized with appropriate HRP-conjugated secondary antibodies.

    Techniques: Expressing, Double Immunostaining, Mouse Assay

    Increased expression of Pla2g3 in frontal cortex of AD patients. A , Pla2g3 expression in normal control brain and AD brain. The images are representative of 6 cases in each group. Frontal cortex was stained with anti-Pla2g3 antibody. B , Analysis of Pla2g3-positive cell numbers in frontal cortex. *p

    Journal: PLoS ONE

    Article Title: Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

    doi: 10.1371/journal.pone.0143518

    Figure Lengend Snippet: Increased expression of Pla2g3 in frontal cortex of AD patients. A , Pla2g3 expression in normal control brain and AD brain. The images are representative of 6 cases in each group. Frontal cortex was stained with anti-Pla2g3 antibody. B , Analysis of Pla2g3-positive cell numbers in frontal cortex. *p

    Article Snippet: Membranes were blocked with Tris-buffer saline containing 0.1% Tween 20 and 5% nonfat milk for 1 hour, then incubated with primary antibody to Pla2g3 (1:1000; Origene), alpha-tubulin (1:2000; MBL), and were then visualized with appropriate HRP-conjugated secondary antibodies.

    Techniques: Expressing, Staining

    ADAM10 expression in human renal tissues. ADAM10 expression (green) in renal biopsy samples from CKD patients (stages 2–3) was analyzed by immunofluorescence, with controls derived from living donor biopsies. Cell nuclei were stained with DAPI. Scale bar represents 50 µm. a – e Immunohistochemical staining of ADAM10 in the renal tissues of renal biopsy samples from CKD patients (stages 2–3). Original magnification, × 400

    Journal: International Urology and Nephrology

    Article Title: PAX2 may induce ADAM10 expression in renal tubular epithelial cells and contribute to epithelial-to-mesenchymal transition

    doi: 10.1007/s11255-018-1956-0

    Figure Lengend Snippet: ADAM10 expression in human renal tissues. ADAM10 expression (green) in renal biopsy samples from CKD patients (stages 2–3) was analyzed by immunofluorescence, with controls derived from living donor biopsies. Cell nuclei were stained with DAPI. Scale bar represents 50 µm. a – e Immunohistochemical staining of ADAM10 in the renal tissues of renal biopsy samples from CKD patients (stages 2–3). Original magnification, × 400

    Article Snippet: We then incubated slides at 4 °C overnight with primary antibodies, against ADAM10 (1:50; OriGene), E-cadherin (1:50; Abcam; ab76055), α-SMA (1:100; Abcam; ab5694), or PAX2 (1:50; Santa Cruz Biotechnology; sc-130387) for immunohistochemical examination, or ADAM10 (1:100; Diaclone, Besancon, France) for immunofluorescence staining.

    Techniques: Expressing, Immunofluorescence, Derivative Assay, Staining, Immunohistochemistry

    ADAM10 participates in UUO-induced renal fibrosis. a Immunohistochemical staining of E-cadherin, α-SMA, PAX2, and ADAM10 in the renal tissues of sham surgery and UUO rats. Original magnification, × 400. b Total kidney lysates from rats that underwent UUO or sham surgery rats were prepared. Protein expression was assessed by western blot relative to β-tubulin. Data represent mean ± SD ( n = 3). * P

    Journal: International Urology and Nephrology

    Article Title: PAX2 may induce ADAM10 expression in renal tubular epithelial cells and contribute to epithelial-to-mesenchymal transition

    doi: 10.1007/s11255-018-1956-0

    Figure Lengend Snippet: ADAM10 participates in UUO-induced renal fibrosis. a Immunohistochemical staining of E-cadherin, α-SMA, PAX2, and ADAM10 in the renal tissues of sham surgery and UUO rats. Original magnification, × 400. b Total kidney lysates from rats that underwent UUO or sham surgery rats were prepared. Protein expression was assessed by western blot relative to β-tubulin. Data represent mean ± SD ( n = 3). * P

    Article Snippet: We then incubated slides at 4 °C overnight with primary antibodies, against ADAM10 (1:50; OriGene), E-cadherin (1:50; Abcam; ab76055), α-SMA (1:100; Abcam; ab5694), or PAX2 (1:50; Santa Cruz Biotechnology; sc-130387) for immunohistochemical examination, or ADAM10 (1:100; Diaclone, Besancon, France) for immunofluorescence staining.

    Techniques: Immunohistochemistry, Staining, Expressing, Western Blot

    ADAM10 induces EMT. a E-cadherin (green) and α-SMA expression (red) were analyzed by immunofluorescence in HK-2 transfected with pRK5M–ADAM10 or empty vectors. HK-2 nuclei were stained with DAPI. Scale bars represent 50 µm. b Total cell lysates of HK-2 transfected with pRK5M–ADAM10 or empty vectors were prepared. Protein expression was normalized to β-tubulin. Data represent mean ± SD ( n = 3). c Messenger RNA was isolated from HK-2 transfected with pRK5M–ADAM10 or empty vectors. Data represent mean ± SD ( n = 3). * P

    Journal: International Urology and Nephrology

    Article Title: PAX2 may induce ADAM10 expression in renal tubular epithelial cells and contribute to epithelial-to-mesenchymal transition

    doi: 10.1007/s11255-018-1956-0

    Figure Lengend Snippet: ADAM10 induces EMT. a E-cadherin (green) and α-SMA expression (red) were analyzed by immunofluorescence in HK-2 transfected with pRK5M–ADAM10 or empty vectors. HK-2 nuclei were stained with DAPI. Scale bars represent 50 µm. b Total cell lysates of HK-2 transfected with pRK5M–ADAM10 or empty vectors were prepared. Protein expression was normalized to β-tubulin. Data represent mean ± SD ( n = 3). c Messenger RNA was isolated from HK-2 transfected with pRK5M–ADAM10 or empty vectors. Data represent mean ± SD ( n = 3). * P

    Article Snippet: We then incubated slides at 4 °C overnight with primary antibodies, against ADAM10 (1:50; OriGene), E-cadherin (1:50; Abcam; ab76055), α-SMA (1:100; Abcam; ab5694), or PAX2 (1:50; Santa Cruz Biotechnology; sc-130387) for immunohistochemical examination, or ADAM10 (1:100; Diaclone, Besancon, France) for immunofluorescence staining.

    Techniques: Expressing, Immunofluorescence, Transfection, Staining, Isolation

    Western blot indicating E-cadherin and α-SMA levels in PAX2-overexpressing NRK52E treated with the ADAM10 inhibitor, GI254023X (* P

    Journal: International Urology and Nephrology

    Article Title: PAX2 may induce ADAM10 expression in renal tubular epithelial cells and contribute to epithelial-to-mesenchymal transition

    doi: 10.1007/s11255-018-1956-0

    Figure Lengend Snippet: Western blot indicating E-cadherin and α-SMA levels in PAX2-overexpressing NRK52E treated with the ADAM10 inhibitor, GI254023X (* P

    Article Snippet: We then incubated slides at 4 °C overnight with primary antibodies, against ADAM10 (1:50; OriGene), E-cadherin (1:50; Abcam; ab76055), α-SMA (1:100; Abcam; ab5694), or PAX2 (1:50; Santa Cruz Biotechnology; sc-130387) for immunohistochemical examination, or ADAM10 (1:100; Diaclone, Besancon, France) for immunofluorescence staining.

    Techniques: Western Blot

    PAX2 directly binds the ADAM10 promoter. a A PAX2 consensus binding motif from the JASPAR CORE database ( http://jaspar.genereg.net/ ). b The putative PAX2 binding site (− 281 bp) within the ADAM10 promoter of rat renal tubular epithelial cells. c DNA sonication revealed that the majority of DNA had been sheared to fragments between 200 and 500 bp in length. d Results of a qChIP assay of the ADAM10 promoter region in NRK52E cells transfected with pGC–LV–PAX2 compared to those transfected with empty vectors. Data represent mean ± SD ( n = 3). IgG was used as a control

    Journal: International Urology and Nephrology

    Article Title: PAX2 may induce ADAM10 expression in renal tubular epithelial cells and contribute to epithelial-to-mesenchymal transition

    doi: 10.1007/s11255-018-1956-0

    Figure Lengend Snippet: PAX2 directly binds the ADAM10 promoter. a A PAX2 consensus binding motif from the JASPAR CORE database ( http://jaspar.genereg.net/ ). b The putative PAX2 binding site (− 281 bp) within the ADAM10 promoter of rat renal tubular epithelial cells. c DNA sonication revealed that the majority of DNA had been sheared to fragments between 200 and 500 bp in length. d Results of a qChIP assay of the ADAM10 promoter region in NRK52E cells transfected with pGC–LV–PAX2 compared to those transfected with empty vectors. Data represent mean ± SD ( n = 3). IgG was used as a control

    Article Snippet: We then incubated slides at 4 °C overnight with primary antibodies, against ADAM10 (1:50; OriGene), E-cadherin (1:50; Abcam; ab76055), α-SMA (1:100; Abcam; ab5694), or PAX2 (1:50; Santa Cruz Biotechnology; sc-130387) for immunohistochemical examination, or ADAM10 (1:100; Diaclone, Besancon, France) for immunofluorescence staining.

    Techniques: Binding Assay, Sonication, Transfection, Pyrolysis Gas Chromatography

    PAX2 influences ADAM10 expression. a Total cell lysates of NRK52E transfected with pGC–LV–PAX2, assessed by western blot. Protein levels were normalized to β-tubulin. Data represent mean ± SD ( n = 3). b ADAM10 expression (red) analyzed by immunofluorescence in NRK52E transfected with pGC–LV–PAX2 or empty vectors. The nuclei of NRK52E cells are stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars represent 50 µm. c Messenger RNA isolated from NRK52E transfected with pGC–LV–PAX2 or empty vectors. Data represent mean ± SD ( n = 3). * P

    Journal: International Urology and Nephrology

    Article Title: PAX2 may induce ADAM10 expression in renal tubular epithelial cells and contribute to epithelial-to-mesenchymal transition

    doi: 10.1007/s11255-018-1956-0

    Figure Lengend Snippet: PAX2 influences ADAM10 expression. a Total cell lysates of NRK52E transfected with pGC–LV–PAX2, assessed by western blot. Protein levels were normalized to β-tubulin. Data represent mean ± SD ( n = 3). b ADAM10 expression (red) analyzed by immunofluorescence in NRK52E transfected with pGC–LV–PAX2 or empty vectors. The nuclei of NRK52E cells are stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars represent 50 µm. c Messenger RNA isolated from NRK52E transfected with pGC–LV–PAX2 or empty vectors. Data represent mean ± SD ( n = 3). * P

    Article Snippet: We then incubated slides at 4 °C overnight with primary antibodies, against ADAM10 (1:50; OriGene), E-cadherin (1:50; Abcam; ab76055), α-SMA (1:100; Abcam; ab5694), or PAX2 (1:50; Santa Cruz Biotechnology; sc-130387) for immunohistochemical examination, or ADAM10 (1:100; Diaclone, Besancon, France) for immunofluorescence staining.

    Techniques: Expressing, Transfection, Pyrolysis Gas Chromatography, Western Blot, Immunofluorescence, Staining, Isolation

    ZSCAN4 is Lysine 48 (K48Ub) polyubiquitinated A . Endogenous ZSCAN4 immunoprecipitation (IP) in treated (+MG132) or untreated (−MG132) WT cells followed by ZSCAN4 immunoblot. B . Denaturing conditions where used followed by ZSCAN4-IP and immunoblot with anti-Lysine 48 (K48) ubiquitin (anti-K48Ub). MG132 treated cells indicate that ZSCAN4 is polyubiquitinated. Untreated cells were used as controls. C. Co-immunostaining and confocal microscopy analyses in WT Tu167 cells treated with MG132 (+MG132) or untreated (−MG132) using anti-ZSCAN4 (green) and anti-K48 ubiquitin (red) indicates the ZSCAN4 overlaps with K48Ub. D . Colocalization analyses with ImageJ show an increase in the number of ZSCAN4 foci colocalized with K48Ub and E . the percent (%) of K48Ub colocalized ZSCAN4 foci (n = 6 per group and average of > 300 foci per group). Asterisks indicate * p

    Journal: Biochemical and biophysical research communications

    Article Title: ZSCAN4 is negatively regulated by the ubiquitin-proteasome system and the E3 ubiquitin ligase RNF20

    doi: 10.1016/j.bbrc.2018.02.155

    Figure Lengend Snippet: ZSCAN4 is Lysine 48 (K48Ub) polyubiquitinated A . Endogenous ZSCAN4 immunoprecipitation (IP) in treated (+MG132) or untreated (−MG132) WT cells followed by ZSCAN4 immunoblot. B . Denaturing conditions where used followed by ZSCAN4-IP and immunoblot with anti-Lysine 48 (K48) ubiquitin (anti-K48Ub). MG132 treated cells indicate that ZSCAN4 is polyubiquitinated. Untreated cells were used as controls. C. Co-immunostaining and confocal microscopy analyses in WT Tu167 cells treated with MG132 (+MG132) or untreated (−MG132) using anti-ZSCAN4 (green) and anti-K48 ubiquitin (red) indicates the ZSCAN4 overlaps with K48Ub. D . Colocalization analyses with ImageJ show an increase in the number of ZSCAN4 foci colocalized with K48Ub and E . the percent (%) of K48Ub colocalized ZSCAN4 foci (n = 6 per group and average of > 300 foci per group). Asterisks indicate * p

    Article Snippet: Primary antibody was incubated overnight at 4 °C for the following antigens: ZSCAN4 (Origene, 1:1000), RNF20 (Cell Signaling, 1:2000), and Ubiquitin Lysine 48 (Millipore 1: 2000).

    Techniques: Immunoprecipitation, Immunostaining, Confocal Microscopy

    ZSCAN4 degradation is proteasome dependent A . Immunoblot analyses indicate that inhibition of autophagy with Bafilomycin A1 and Chloroquine results in accumulation of autophagy targets p62 and LC-3. However, ZSCAN4 was not affected. B. Induction of ZSCAN4 by Dox followed by treatment with the proteasomal inhibitor MG132 demonstrates ZSCAN4 accumulates in the cells. C–D. Kinetics experiments show that accumulation of ZSCAN4 following by MG132 treatment is time and dose dependent. E. Similar studies performed in WT cells show the accumulation of ZSCAN4 following MG132 treatment.

    Journal: Biochemical and biophysical research communications

    Article Title: ZSCAN4 is negatively regulated by the ubiquitin-proteasome system and the E3 ubiquitin ligase RNF20

    doi: 10.1016/j.bbrc.2018.02.155

    Figure Lengend Snippet: ZSCAN4 degradation is proteasome dependent A . Immunoblot analyses indicate that inhibition of autophagy with Bafilomycin A1 and Chloroquine results in accumulation of autophagy targets p62 and LC-3. However, ZSCAN4 was not affected. B. Induction of ZSCAN4 by Dox followed by treatment with the proteasomal inhibitor MG132 demonstrates ZSCAN4 accumulates in the cells. C–D. Kinetics experiments show that accumulation of ZSCAN4 following by MG132 treatment is time and dose dependent. E. Similar studies performed in WT cells show the accumulation of ZSCAN4 following MG132 treatment.

    Article Snippet: Primary antibody was incubated overnight at 4 °C for the following antigens: ZSCAN4 (Origene, 1:1000), RNF20 (Cell Signaling, 1:2000), and Ubiquitin Lysine 48 (Millipore 1: 2000).

    Techniques: Inhibition

    Human ZSCAN4 protein half-life A . Immunoblot analysis of ZSCAN4 in wild type (WT) Tu167 cells after treatment with cycloheximide (CHX) results in decreased ZSCAN4 protein. B. Protein half-life analysis indicates that endogenous ZSCAN4 protein half-life is 8.3 h. C. Immunoblot in tet-ZSCAN4 cell lines show addition of doxycycline (Dox+) to medium for 0–48 h results in ZSCAN4-FLAG induction in as early as 12 h. D. Cells fractionation to cytoplasmic (C) and nuclear (N) proteins, show the endogenous ZSCAN4 (anti-ZSCAN4) and FLAG-tagged ZSCAN4 (anti-FLAG), are localized to the nucleus. E. Immunoblot in isogenic (Dox+) tet-ZSCAN4 cells after treatment with CHX and F . Protein half-life analyses indicate that the ectopic ZSCAN4 half-life is 8.0 h. Images and results represent data of at least three independent experiments.

    Journal: Biochemical and biophysical research communications

    Article Title: ZSCAN4 is negatively regulated by the ubiquitin-proteasome system and the E3 ubiquitin ligase RNF20

    doi: 10.1016/j.bbrc.2018.02.155

    Figure Lengend Snippet: Human ZSCAN4 protein half-life A . Immunoblot analysis of ZSCAN4 in wild type (WT) Tu167 cells after treatment with cycloheximide (CHX) results in decreased ZSCAN4 protein. B. Protein half-life analysis indicates that endogenous ZSCAN4 protein half-life is 8.3 h. C. Immunoblot in tet-ZSCAN4 cell lines show addition of doxycycline (Dox+) to medium for 0–48 h results in ZSCAN4-FLAG induction in as early as 12 h. D. Cells fractionation to cytoplasmic (C) and nuclear (N) proteins, show the endogenous ZSCAN4 (anti-ZSCAN4) and FLAG-tagged ZSCAN4 (anti-FLAG), are localized to the nucleus. E. Immunoblot in isogenic (Dox+) tet-ZSCAN4 cells after treatment with CHX and F . Protein half-life analyses indicate that the ectopic ZSCAN4 half-life is 8.0 h. Images and results represent data of at least three independent experiments.

    Article Snippet: Primary antibody was incubated overnight at 4 °C for the following antigens: ZSCAN4 (Origene, 1:1000), RNF20 (Cell Signaling, 1:2000), and Ubiquitin Lysine 48 (Millipore 1: 2000).

    Techniques: Fractionation

    The E3 ubiquitin ligase RNF20 negatively regulates and interacts with ZSCAN4 A . Co-IP of ZSCAN4 and immunoblot with anti-RNF20 displays interaction. B. Confocal microscope images in untreated tet-ZSCAN4 cells (Dox−) show co-localization of RNF20 (red) and ZSCAN4 (green). DNA is stained with DAPI (blue). Furthermore, co-localization increases following ZSCAN4 induction (Dox+). C. Immunoblot analyses show successful RNF20 knockdown by SMARTpool siRNA and two different siRNA (RNAi 1 and RNAi 2) compared to non-template controls (NTC). Conversely, RNF20 depletion results in a significant increase in ZSCAN4. D. qRT-PCR analysis in RNF20 depleted by the indicated siRNAs show no significant change in ZSCAN4 expression compared to NTC-siRNA controls. E–F. Protein half-life analyses after treatment with CHX and immunoblot with corresponding antibodies indicate that NTC-siRNA do not alter the ZSCAN4 protein half-life which remains about 8 h, yet, as shown in G–H. Knockdown of RNF20 (pool siRNA) leads to stabilization of ZSCAN4 protein. All data shown represent three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Biochemical and biophysical research communications

    Article Title: ZSCAN4 is negatively regulated by the ubiquitin-proteasome system and the E3 ubiquitin ligase RNF20

    doi: 10.1016/j.bbrc.2018.02.155

    Figure Lengend Snippet: The E3 ubiquitin ligase RNF20 negatively regulates and interacts with ZSCAN4 A . Co-IP of ZSCAN4 and immunoblot with anti-RNF20 displays interaction. B. Confocal microscope images in untreated tet-ZSCAN4 cells (Dox−) show co-localization of RNF20 (red) and ZSCAN4 (green). DNA is stained with DAPI (blue). Furthermore, co-localization increases following ZSCAN4 induction (Dox+). C. Immunoblot analyses show successful RNF20 knockdown by SMARTpool siRNA and two different siRNA (RNAi 1 and RNAi 2) compared to non-template controls (NTC). Conversely, RNF20 depletion results in a significant increase in ZSCAN4. D. qRT-PCR analysis in RNF20 depleted by the indicated siRNAs show no significant change in ZSCAN4 expression compared to NTC-siRNA controls. E–F. Protein half-life analyses after treatment with CHX and immunoblot with corresponding antibodies indicate that NTC-siRNA do not alter the ZSCAN4 protein half-life which remains about 8 h, yet, as shown in G–H. Knockdown of RNF20 (pool siRNA) leads to stabilization of ZSCAN4 protein. All data shown represent three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: Primary antibody was incubated overnight at 4 °C for the following antigens: ZSCAN4 (Origene, 1:1000), RNF20 (Cell Signaling, 1:2000), and Ubiquitin Lysine 48 (Millipore 1: 2000).

    Techniques: Co-Immunoprecipitation Assay, Microscopy, Staining, Quantitative RT-PCR, Expressing

    Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 cells . A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p

    Journal: BMC Cancer

    Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

    doi: 10.1186/1471-2407-10-250

    Figure Lengend Snippet: Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 cells . A) HT29 cells (30,000) were transfected with siS100A4 as described in Methods. Total RNA was extracted after 48 h and S100A4 mRNA levels were determined by RT-Real-Time PCR. B) S100A4 protein levels were determined by Western Blotting 72 h after transfection, using specific antibodies against S100A4 and Actin to normalize the results. C) Chemosensitization assays toward methotrexate: cells were treated with siS100A4 for 48 h and then incubated with MTX. Cell viability was determined 3 days after MTX treatment. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. A representative image of Western Blots is presented. *p

    Article Snippet: S100A4 is secreted by HT29 cells transfected with pCMV-S100A4 Given that it had been described that S100A4 has extracellular functions [ , ], we explored S100A4 protein levels in the culture media of pCMV-S100A4 transfected cells.

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Incubation

    Effects on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells . A) mRNA levels of S100A4 determined by RT-Real-Time PCR 48 h after treatment of HT29 cells (30,000) with 250 ng of the expression vector for S100A4 (pCMV-S100A4). B) A representative image of the intracellular protein levels of S100A4 determined by Western Blotting 72 h after ectopic transfection with its expression vector is shown in the upper panel, and the quantification of the blots is shown in the lower panel. Purified S100A4 protein was used as a reference marker (Abnova; first lane). An additional panel showing endogenous S100A4 protein levels in HT29 sensitive (S) and resistant (R) cells is also provided. C) Effects of S100A4 overexpression on cell viability. HT29 cells (100,000) were treated with 1 μg of pCMV-S100A4 and 5 × 10 -8 M MTX was added 48 h later. Cell viability was assessed by the MTT assay six days after MTX treatment. D) Extracellular S100A4 protein levels quantified by ELISA 72 h after S100A4 overexpression upon pCMV-S100A4 transfection. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p

    Journal: BMC Cancer

    Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

    doi: 10.1186/1471-2407-10-250

    Figure Lengend Snippet: Effects on S100A4 expression and MTX sensitivity upon pCMV-S100A4 transfection of HT29 cells . A) mRNA levels of S100A4 determined by RT-Real-Time PCR 48 h after treatment of HT29 cells (30,000) with 250 ng of the expression vector for S100A4 (pCMV-S100A4). B) A representative image of the intracellular protein levels of S100A4 determined by Western Blotting 72 h after ectopic transfection with its expression vector is shown in the upper panel, and the quantification of the blots is shown in the lower panel. Purified S100A4 protein was used as a reference marker (Abnova; first lane). An additional panel showing endogenous S100A4 protein levels in HT29 sensitive (S) and resistant (R) cells is also provided. C) Effects of S100A4 overexpression on cell viability. HT29 cells (100,000) were treated with 1 μg of pCMV-S100A4 and 5 × 10 -8 M MTX was added 48 h later. Cell viability was assessed by the MTT assay six days after MTX treatment. D) Extracellular S100A4 protein levels quantified by ELISA 72 h after S100A4 overexpression upon pCMV-S100A4 transfection. The expression and viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p

    Article Snippet: S100A4 is secreted by HT29 cells transfected with pCMV-S100A4 Given that it had been described that S100A4 has extracellular functions [ , ], we explored S100A4 protein levels in the culture media of pCMV-S100A4 transfected cells.

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot, Purification, Marker, Over Expression, MTT Assay, Enzyme-linked Immunosorbent Assay

    Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 MTX-resistant cells . A) S100A4 mRNA levels were determined by RT-Real-time PCR as described in Methods 48 h after siS100A4 treatment. B) Cells were treated with MTX after a 48 h-pre-incubation with siS100A4, and cell viability was determined 3 days later by the MTT assay. The levels of expression and the viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p

    Journal: BMC Cancer

    Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

    doi: 10.1186/1471-2407-10-250

    Figure Lengend Snippet: Effects on S100A4 expression and MTX sensitivity upon siS100A4 transfection of HT29 MTX-resistant cells . A) S100A4 mRNA levels were determined by RT-Real-time PCR as described in Methods 48 h after siS100A4 treatment. B) Cells were treated with MTX after a 48 h-pre-incubation with siS100A4, and cell viability was determined 3 days later by the MTT assay. The levels of expression and the viability results are expressed as percentages referred to the untreated cells. Values are the mean of three independent experiments ± SE. *p

    Article Snippet: S100A4 is secreted by HT29 cells transfected with pCMV-S100A4 Given that it had been described that S100A4 has extracellular functions [ , ], we explored S100A4 protein levels in the culture media of pCMV-S100A4 transfected cells.

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Incubation, MTT Assay

    Effects of transfecting an expression vector encoding for β-Catenin on S100A4 mRNA levels . Transfection with β-Catenin expression vector (pcDNA3-β-Catenin) was performed in HT29 cells, both sensitive (Figure 4A) and resistant (Figure 4B) as described in Methods. S100A4 mRNA levels were determined by RT-Real-Time PCR 48 h after transfection. All results are expressed as percentages referred to untreated cells. Values are the mean of three independent experiments ± SE. * p

    Journal: BMC Cancer

    Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate

    doi: 10.1186/1471-2407-10-250

    Figure Lengend Snippet: Effects of transfecting an expression vector encoding for β-Catenin on S100A4 mRNA levels . Transfection with β-Catenin expression vector (pcDNA3-β-Catenin) was performed in HT29 cells, both sensitive (Figure 4A) and resistant (Figure 4B) as described in Methods. S100A4 mRNA levels were determined by RT-Real-Time PCR 48 h after transfection. All results are expressed as percentages referred to untreated cells. Values are the mean of three independent experiments ± SE. * p

    Article Snippet: S100A4 is secreted by HT29 cells transfected with pCMV-S100A4 Given that it had been described that S100A4 has extracellular functions [ , ], we explored S100A4 protein levels in the culture media of pCMV-S100A4 transfected cells.

    Techniques: Expressing, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction

    Differential Binding of TFAP2a at SNP rs2595104

    Journal: American Journal of Human Genetics

    Article Title: A Functional Variant Associated with Atrial Fibrillation Regulates PITX2c Expression through TFAP2a

    doi: 10.1016/j.ajhg.2016.10.001

    Figure Lengend Snippet: Differential Binding of TFAP2a at SNP rs2595104

    Article Snippet: In brief, the double-stranded probes (20 fmol) were incubated with purified TFAP2a (Origene) at room temperature for 20 min in the presence of 100 mM Tris (pH 7.5), 500 mM KCl, and 10 mM DTT in a 20 μL reaction.

    Techniques: Binding Assay

    Analysis of the methylation status of the CYB5R2 promoter region in NPC cell lines, NPC primary tumors and NNE samples. a MSP analysis in NPC cell lines and NNEs. M methylated alleles, U unmethylated alleles. In vitro methylated DNA was used as a methylation-positive control and DNA from normal lymphocytes was the positive control for unmethylated alleles. The blank control was water. b MSP analysis in NPC biopsies and NNE, representative data is shown. c Methylation status of the 39 CpG sites of the CYB5R2 promoter in two NPC cell lines (TW03 and CNE2), two NPC biopsies (NPC15 and 27) and one NNE biopsy (NNE7). Five clones were randomly selected and sequenced for each sample. Different size filled sectors of the circles represent the fraction of methylated CpG. MSP primer locations are indicated by frames. d Restoration of CYB5R2 expression by treatment with 5-aza-dC in NPC cell lines. One NNE sample and CYB5R2 expression plasmid DNA were used as positive controls

    Journal: Tumour Biology

    Article Title: Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma

    doi: 10.1007/s13277-013-1497-1

    Figure Lengend Snippet: Analysis of the methylation status of the CYB5R2 promoter region in NPC cell lines, NPC primary tumors and NNE samples. a MSP analysis in NPC cell lines and NNEs. M methylated alleles, U unmethylated alleles. In vitro methylated DNA was used as a methylation-positive control and DNA from normal lymphocytes was the positive control for unmethylated alleles. The blank control was water. b MSP analysis in NPC biopsies and NNE, representative data is shown. c Methylation status of the 39 CpG sites of the CYB5R2 promoter in two NPC cell lines (TW03 and CNE2), two NPC biopsies (NPC15 and 27) and one NNE biopsy (NNE7). Five clones were randomly selected and sequenced for each sample. Different size filled sectors of the circles represent the fraction of methylated CpG. MSP primer locations are indicated by frames. d Restoration of CYB5R2 expression by treatment with 5-aza-dC in NPC cell lines. One NNE sample and CYB5R2 expression plasmid DNA were used as positive controls

    Article Snippet: Vector construction and transfection The full-length coding sequence of CYB5R2 from Origene (USA) was subcloned into the pCMV-Tag3A vector (Stratagene, USA).

    Techniques: Methylation, In Vitro, Positive Control, Clone Assay, Expressing, Plasmid Preparation

    CYB5R2 reexpression reduces tumorigenicity of NPC cells in nude mice. a Growth curves of tumors in nude mice. The mean volume of tumors from CYB5R2 -HONE1 and empty vector-HONE1 cells was evaluated every 2 days after inoculation. Data are mean ± SD ( n = 8). b Representative tumors were removed from the nude mice at day 14 after transplantation. c Mean ± SD weight of tumors ( n = 8). * P

    Journal: Tumour Biology

    Article Title: Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma

    doi: 10.1007/s13277-013-1497-1

    Figure Lengend Snippet: CYB5R2 reexpression reduces tumorigenicity of NPC cells in nude mice. a Growth curves of tumors in nude mice. The mean volume of tumors from CYB5R2 -HONE1 and empty vector-HONE1 cells was evaluated every 2 days after inoculation. Data are mean ± SD ( n = 8). b Representative tumors were removed from the nude mice at day 14 after transplantation. c Mean ± SD weight of tumors ( n = 8). * P

    Article Snippet: Vector construction and transfection The full-length coding sequence of CYB5R2 from Origene (USA) was subcloned into the pCMV-Tag3A vector (Stratagene, USA).

    Techniques: Mouse Assay, Plasmid Preparation, Transplantation Assay

    Ectopic expression of CYB5R2 inhibits colony formation, migration and proliferation of NPC cells. a Colony formation ability of CYB5R2 -transfected and empty vector-transfected HONE1cells. Colony numbers in the bar graph represent the mean ± SD of three independent experiments. b Migration of CYB5R2 -transfected and empty vector-transfected HONE1cells examined by wound healing assay. c Growth curves of CYB5R2 -transfected, empty vector-transfected HONE1cells and parental cells. Data are mean ± SD ( n = 5). * P

    Journal: Tumour Biology

    Article Title: Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma

    doi: 10.1007/s13277-013-1497-1

    Figure Lengend Snippet: Ectopic expression of CYB5R2 inhibits colony formation, migration and proliferation of NPC cells. a Colony formation ability of CYB5R2 -transfected and empty vector-transfected HONE1cells. Colony numbers in the bar graph represent the mean ± SD of three independent experiments. b Migration of CYB5R2 -transfected and empty vector-transfected HONE1cells examined by wound healing assay. c Growth curves of CYB5R2 -transfected, empty vector-transfected HONE1cells and parental cells. Data are mean ± SD ( n = 5). * P

    Article Snippet: Vector construction and transfection The full-length coding sequence of CYB5R2 from Origene (USA) was subcloned into the pCMV-Tag3A vector (Stratagene, USA).

    Techniques: Expressing, Migration, Transfection, Plasmid Preparation, Wound Healing Assay

    Expression of cytochrome b5 reductase 2 ( CYB5R2 ) in NPC cell lines, NPC biopsies and NNE samples. a RT-PCR analysis of expression of CYB5R2 in NPC cell lines and NNEs. PC: positive control. GAPDH was the internal control. Water was the blank control. b Semi-quantitative RT-PCR of CYB5R2 expression in NNEs and primary NPC tumors. CYB5R2 expression was normalized to that of GAPDH

    Journal: Tumour Biology

    Article Title: Cytochrome b5 reductase 2 is a novel candidate tumor suppressor gene frequently inactivated by promoter hypermethylation in human nasopharyngeal carcinoma

    doi: 10.1007/s13277-013-1497-1

    Figure Lengend Snippet: Expression of cytochrome b5 reductase 2 ( CYB5R2 ) in NPC cell lines, NPC biopsies and NNE samples. a RT-PCR analysis of expression of CYB5R2 in NPC cell lines and NNEs. PC: positive control. GAPDH was the internal control. Water was the blank control. b Semi-quantitative RT-PCR of CYB5R2 expression in NNEs and primary NPC tumors. CYB5R2 expression was normalized to that of GAPDH

    Article Snippet: Vector construction and transfection The full-length coding sequence of CYB5R2 from Origene (USA) was subcloned into the pCMV-Tag3A vector (Stratagene, USA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR

    eftu-2 /Eftud2 regulates host defense in C. elegans . Depicted are survival plots for eri-1 mutant nematodes treated with either control RNAi, eftu-2 RNAi, or vha-11 RNAi (as indicated) that were subsequently exposed to either P. aeruginosa strain PA14

    Journal: Genetics

    Article Title: Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

    doi: 10.1534/genetics.113.160499

    Figure Lengend Snippet: eftu-2 /Eftud2 regulates host defense in C. elegans . Depicted are survival plots for eri-1 mutant nematodes treated with either control RNAi, eftu-2 RNAi, or vha-11 RNAi (as indicated) that were subsequently exposed to either P. aeruginosa strain PA14

    Article Snippet: The effect of Eftud2 was not limited to IL-6 as production of the proinflammatory cytokine TNFα also was diminished when Eftud2 was inhibited by siRNA ( ).

    Techniques: Mutagenesis

    Overexpression studies demonstrate that Eftud2 and Atp6v1c1 regulate IL-6 expression. Plasmids overexpressing the indicated genes were cotransfected along with plasmids expressing IL-6-luciferase and SV40-rluc into RAW264.7 cells, the cells were stimulated

    Journal: Genetics

    Article Title: Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

    doi: 10.1534/genetics.113.160499

    Figure Lengend Snippet: Overexpression studies demonstrate that Eftud2 and Atp6v1c1 regulate IL-6 expression. Plasmids overexpressing the indicated genes were cotransfected along with plasmids expressing IL-6-luciferase and SV40-rluc into RAW264.7 cells, the cells were stimulated

    Article Snippet: The effect of Eftud2 was not limited to IL-6 as production of the proinflammatory cytokine TNFα also was diminished when Eftud2 was inhibited by siRNA ( ).

    Techniques: Over Expression, Expressing, Luciferase

    Eftud2 regulates innate immunity by controlling the alternative mRNA splicing of MyD88. (A and B) Cells were treated with either Eftud2 siRNA or control (CT) nontargeting siRNA, were stimulated with 20 ng/ml LPS for 6 hr, and MyD88 L and MyD88 S mRNA levels

    Journal: Genetics

    Article Title: Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

    doi: 10.1534/genetics.113.160499

    Figure Lengend Snippet: Eftud2 regulates innate immunity by controlling the alternative mRNA splicing of MyD88. (A and B) Cells were treated with either Eftud2 siRNA or control (CT) nontargeting siRNA, were stimulated with 20 ng/ml LPS for 6 hr, and MyD88 L and MyD88 S mRNA levels

    Article Snippet: The effect of Eftud2 was not limited to IL-6 as production of the proinflammatory cytokine TNFα also was diminished when Eftud2 was inhibited by siRNA ( ).

    Techniques:

    Eftud2 regulates the innate immune response in mouse macrophages. (A–C) Cells were treated with either Eftud2 siRNA or control (CT) siRNA; were exposed to 20 ng/ml LPS for 6 hr; and IL-6 protein (A), IL-6 mRNA (B), or TNFα protein (C)

    Journal: Genetics

    Article Title: Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

    doi: 10.1534/genetics.113.160499

    Figure Lengend Snippet: Eftud2 regulates the innate immune response in mouse macrophages. (A–C) Cells were treated with either Eftud2 siRNA or control (CT) siRNA; were exposed to 20 ng/ml LPS for 6 hr; and IL-6 protein (A), IL-6 mRNA (B), or TNFα protein (C)

    Article Snippet: The effect of Eftud2 was not limited to IL-6 as production of the proinflammatory cytokine TNFα also was diminished when Eftud2 was inhibited by siRNA ( ).

    Techniques:

    Top DEGs of R47H TREM2 and TREM2 KO pMac overlap; furthermore, the R47H DEGs that are shared with TREM2 KO represent numerous biological processes, distributed between five protein-protein interaction (PPI) modules. a Heatmap of top upregulated and top downregulated DEGs for R47H TREM2 and TREM2 KO, with the relative expression for each gene represented by colours on the corresponding row. A selection of “relevant genes” was also included. Unbiased clustering (dendrogram on the left) shows that genes separate into two major clusters based on whether they are upregulated or downregulated in TREM2 KO or R47H TREM2, and there is no genotype-specific segregation. b Five modules of functionally related DEGs identified by PPI network analysis of TREM2 KO DEGs, with enriched gene ontology (GO) terms shown. R47H DEGs were only included where they were also differentially expressed in TREM2 KO and were overlaid onto clusters identified using TREM2 KO data. Clusters identified numerically on the x -axis, with TREM2 KO on the left and R47H TREM2 on the right, showing a similar pattern of dysregulated cell functions. Number of DEGs represented by circle size, and p value represented by colour

    Journal: Alzheimer's Research & Therapy

    Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

    doi: 10.1186/s13195-020-00709-z

    Figure Lengend Snippet: Top DEGs of R47H TREM2 and TREM2 KO pMac overlap; furthermore, the R47H DEGs that are shared with TREM2 KO represent numerous biological processes, distributed between five protein-protein interaction (PPI) modules. a Heatmap of top upregulated and top downregulated DEGs for R47H TREM2 and TREM2 KO, with the relative expression for each gene represented by colours on the corresponding row. A selection of “relevant genes” was also included. Unbiased clustering (dendrogram on the left) shows that genes separate into two major clusters based on whether they are upregulated or downregulated in TREM2 KO or R47H TREM2, and there is no genotype-specific segregation. b Five modules of functionally related DEGs identified by PPI network analysis of TREM2 KO DEGs, with enriched gene ontology (GO) terms shown. R47H DEGs were only included where they were also differentially expressed in TREM2 KO and were overlaid onto clusters identified using TREM2 KO data. Clusters identified numerically on the x -axis, with TREM2 KO on the left and R47H TREM2 on the right, showing a similar pattern of dysregulated cell functions. Number of DEGs represented by circle size, and p value represented by colour

    Article Snippet: TREM2 functions at the cell surface; therefore, proteins were purified by cell surface biotinylation and TREM2 detected by Western blotting, with comparison to the input cell homogenate (Fig. b).

    Techniques: Expressing, Selection

    Extracellular matrix-adhesion modifiers and adhesion to vitronectin are dysregulated in R47H TREM2 and TREM2 KO pMac. TGFβ treatment does not rescue adhesion deficits. a Validation of selected RNA-seq hits by qRT-PCR of unstimulated pMac. Means ± SEM, for N = 7 harvests, including the 3 samples originally used for RNA-seq (open symbols) plus 4 samples harvested independently from a separate differentiation (filled symbols). Repeated-measures 1-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to the WT. b Effect of TGFβ-stimulation (50 ng/mL, 24 h) on mRNA levels of selected RNA-seq hits, measured by qRT-PCR. Means ± SEM, for N = 3 samples harvested independently to Fig. 5 a. Two-way ANOVA, with Sidak’s post hoc test. c TREM2 KO pMac secrete reduced levels of TGFβ1 compared with WT, and TGFβ1 secretion is partly SYK-dependent. Total (inactive and active) TGFβ1 measured from supernatants by ELISA, cells treated ± OXSI-2 (2 μM, 24 h) to inhibit SYK. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. d Fibronectin protein expression is reduced in both R47H TREM2 and TREM2 KO versus WT. Fibronectin measured by Western blotting of pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Means ± SEM, quantified for N = 3 harvests on separate blots. Two-way ANOVA, with Sidak’s post hoc test. TGFβ1 vs unstimulated was not significant. e αVβ3 complex formation is reduced in both R47H TREM2 and TREM2 KO versus WT. Intact surface integrins αVβ3 and αVβ5 measured by flow cytometry in pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Data is expressed as the difference in median fluorescence intensity of the specific antibody versus isotype control, normalised (by subtraction) to the average for the harvest. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. f Adhesion to vitronectin is reduced for both R47H TREM2 and TREM2 KO versus WT, and treatment with TGFβ (50 ng/mL, 24 h prior to assay) increases αVβ3/5-dependent adhesion. Adhesion measured after 3 h by crystal violet colorimetric assay, and normalised to BSA-blocked wells (by division, and the result subtracted from 1). αVβ3/5 inhibitor (10 μM cilengitide) was added at the start of the assay to determine αVβ3/5-specific adhesion to vitronectin (striped bars). Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Dunnett’s post hoc test. Black annotations compare stimulations to unstimulated control. Grey annotations compare R47H or KO versus WT for each condition. WT = grey circles, R47H = orange squares, TREM2 KO = burgundy triangles. * p

    Journal: Alzheimer's Research & Therapy

    Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

    doi: 10.1186/s13195-020-00709-z

    Figure Lengend Snippet: Extracellular matrix-adhesion modifiers and adhesion to vitronectin are dysregulated in R47H TREM2 and TREM2 KO pMac. TGFβ treatment does not rescue adhesion deficits. a Validation of selected RNA-seq hits by qRT-PCR of unstimulated pMac. Means ± SEM, for N = 7 harvests, including the 3 samples originally used for RNA-seq (open symbols) plus 4 samples harvested independently from a separate differentiation (filled symbols). Repeated-measures 1-way ANOVA, with Dunnett’s post hoc test, pairwise comparisons to the WT. b Effect of TGFβ-stimulation (50 ng/mL, 24 h) on mRNA levels of selected RNA-seq hits, measured by qRT-PCR. Means ± SEM, for N = 3 samples harvested independently to Fig. 5 a. Two-way ANOVA, with Sidak’s post hoc test. c TREM2 KO pMac secrete reduced levels of TGFβ1 compared with WT, and TGFβ1 secretion is partly SYK-dependent. Total (inactive and active) TGFβ1 measured from supernatants by ELISA, cells treated ± OXSI-2 (2 μM, 24 h) to inhibit SYK. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. d Fibronectin protein expression is reduced in both R47H TREM2 and TREM2 KO versus WT. Fibronectin measured by Western blotting of pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Means ± SEM, quantified for N = 3 harvests on separate blots. Two-way ANOVA, with Sidak’s post hoc test. TGFβ1 vs unstimulated was not significant. e αVβ3 complex formation is reduced in both R47H TREM2 and TREM2 KO versus WT. Intact surface integrins αVβ3 and αVβ5 measured by flow cytometry in pMac ± TGFβ1 stimulation (50 ng/mL, 24 h). Data is expressed as the difference in median fluorescence intensity of the specific antibody versus isotype control, normalised (by subtraction) to the average for the harvest. Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Sidak’s post hoc test. f Adhesion to vitronectin is reduced for both R47H TREM2 and TREM2 KO versus WT, and treatment with TGFβ (50 ng/mL, 24 h prior to assay) increases αVβ3/5-dependent adhesion. Adhesion measured after 3 h by crystal violet colorimetric assay, and normalised to BSA-blocked wells (by division, and the result subtracted from 1). αVβ3/5 inhibitor (10 μM cilengitide) was added at the start of the assay to determine αVβ3/5-specific adhesion to vitronectin (striped bars). Means ± SEM, for N = 3 harvests. Two-way ANOVA, with Dunnett’s post hoc test. Black annotations compare stimulations to unstimulated control. Grey annotations compare R47H or KO versus WT for each condition. WT = grey circles, R47H = orange squares, TREM2 KO = burgundy triangles. * p

    Article Snippet: TREM2 functions at the cell surface; therefore, proteins were purified by cell surface biotinylation and TREM2 detected by Western blotting, with comparison to the input cell homogenate (Fig. b).

    Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Flow Cytometry, Fluorescence, Colorimetric Assay

    Reduced cell surface localization of R47H TREM2 does not impair antibody-mediated activation of TREM2 in pMac. a Schematic of microglia phenotypes investigated in this study. b , c Reduced cell surface expression of R47H TREM2. Cell surface pro teins on pMac were biotinylated and pulled down, the level of TREM2 protein enrichment was measured by Western blotting vs whole cell lysate, probed on separate blots. c Means ± SEM, for N = 5 harvests measured on separate Western blots. 2-tailed paired t-test: ** p = 0.0011 for R47H versus WT. d Increased sTREM2 production from R47H TREM2 pMac. sTREM2 were measured from unstimulated pMac supernatants by ELISA. Means ± SEM, for N = 3 harvests measured on same ELISA plate, data for each harvest was normalised to the average cell count. 1-way ANOVA, with Dunnett’s post hoc test: p = 0.0005 R47H versus WT (***), p = 0.0066 KO versus WT (**). e , f Co-localization of TREM2 with subcellular compartment markers in fixed and permeabilized pMac, images are a confocal slice at 4 μm, taken with Opera Phenix microscope. Inset panels are × 3 magnification of a selected cell. Calnexin used as a marker for ER, TGN46 for TGN, and LAMP1 for lysosomes. f Co-localization expressed as a ratio of TREM2 intensity to compartment marker intensity, in regions automatically segmented by high marker staining. Means ± SEM, for N = 3 harvests, each in triplicate wells. 2-way ANOVA, with Bonferroni’s post hoc test, no significant differences. g , h TREM2-activating antibody stimulation (used at concentration of 2.4 μg/1 × 10 6 cells and 3.84 μg/mL, for 10 min) of both WT and R47H TREM2 pMac caused SYK phosphorylation, measured by Western blotting. No response seen in TREM2 antibody-stimulated TREM2 KO cells, or cells treated with a goat IgG isotype control. h Means ± SEM, for N = 3 harvests measured on separate Western blots. 2-way ANOVA, with Sidak’s post hoc test, pairwise comparisons to WT for each treatment: p

    Journal: Alzheimer's Research & Therapy

    Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

    doi: 10.1186/s13195-020-00709-z

    Figure Lengend Snippet: Reduced cell surface localization of R47H TREM2 does not impair antibody-mediated activation of TREM2 in pMac. a Schematic of microglia phenotypes investigated in this study. b , c Reduced cell surface expression of R47H TREM2. Cell surface pro teins on pMac were biotinylated and pulled down, the level of TREM2 protein enrichment was measured by Western blotting vs whole cell lysate, probed on separate blots. c Means ± SEM, for N = 5 harvests measured on separate Western blots. 2-tailed paired t-test: ** p = 0.0011 for R47H versus WT. d Increased sTREM2 production from R47H TREM2 pMac. sTREM2 were measured from unstimulated pMac supernatants by ELISA. Means ± SEM, for N = 3 harvests measured on same ELISA plate, data for each harvest was normalised to the average cell count. 1-way ANOVA, with Dunnett’s post hoc test: p = 0.0005 R47H versus WT (***), p = 0.0066 KO versus WT (**). e , f Co-localization of TREM2 with subcellular compartment markers in fixed and permeabilized pMac, images are a confocal slice at 4 μm, taken with Opera Phenix microscope. Inset panels are × 3 magnification of a selected cell. Calnexin used as a marker for ER, TGN46 for TGN, and LAMP1 for lysosomes. f Co-localization expressed as a ratio of TREM2 intensity to compartment marker intensity, in regions automatically segmented by high marker staining. Means ± SEM, for N = 3 harvests, each in triplicate wells. 2-way ANOVA, with Bonferroni’s post hoc test, no significant differences. g , h TREM2-activating antibody stimulation (used at concentration of 2.4 μg/1 × 10 6 cells and 3.84 μg/mL, for 10 min) of both WT and R47H TREM2 pMac caused SYK phosphorylation, measured by Western blotting. No response seen in TREM2 antibody-stimulated TREM2 KO cells, or cells treated with a goat IgG isotype control. h Means ± SEM, for N = 3 harvests measured on separate Western blots. 2-way ANOVA, with Sidak’s post hoc test, pairwise comparisons to WT for each treatment: p

    Article Snippet: TREM2 functions at the cell surface; therefore, proteins were purified by cell surface biotinylation and TREM2 detected by Western blotting, with comparison to the input cell homogenate (Fig. b).

    Techniques: Activation Assay, Expressing, Protein Enrichment, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Counting, Microscopy, Marker, Staining, Concentration Assay

    Phagocytosis of dead SH-SY5Ys and synaptosomes is reduced in TREM2 KO, but not R47H TREM2, relative to WT pMac. a After 3 h of phagocytosis of pHrodo-labelled dead SH-SY5Ys, immunofluorescence staining shows that TREM2 is highly recruited to the phagocytic cup (marked by white arrow) during engulfment of cells expressing the neuronal marker TUJ1, whereas in b TREM2 is lost before maturation to RAB9+ endosomes (marked by white arrow). c – f Phagocytosis is impaired in TREM2 KO pMac only. Representative images of phagocytosis of SH-SY5Ys ( c ) or synaptosomes ( e ) shown in yellow, by pMac (red cytoplasm and blue nucleus), taken at 3 h with INCell 6000. Inset is a section of the image magnified 3-fold. d , f Means were quantified for the parameters: number of spots per cell, sum of spot areas (μm 2 ) per cell, percentage of cells containing phagocytosed particles per field. Data was normalised to mean for each genotype per experiment. Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, Dunnett’s post hoc test, pairwise comparisons to the WT for each time: * p

    Journal: Alzheimer's Research & Therapy

    Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

    doi: 10.1186/s13195-020-00709-z

    Figure Lengend Snippet: Phagocytosis of dead SH-SY5Ys and synaptosomes is reduced in TREM2 KO, but not R47H TREM2, relative to WT pMac. a After 3 h of phagocytosis of pHrodo-labelled dead SH-SY5Ys, immunofluorescence staining shows that TREM2 is highly recruited to the phagocytic cup (marked by white arrow) during engulfment of cells expressing the neuronal marker TUJ1, whereas in b TREM2 is lost before maturation to RAB9+ endosomes (marked by white arrow). c – f Phagocytosis is impaired in TREM2 KO pMac only. Representative images of phagocytosis of SH-SY5Ys ( c ) or synaptosomes ( e ) shown in yellow, by pMac (red cytoplasm and blue nucleus), taken at 3 h with INCell 6000. Inset is a section of the image magnified 3-fold. d , f Means were quantified for the parameters: number of spots per cell, sum of spot areas (μm 2 ) per cell, percentage of cells containing phagocytosed particles per field. Data was normalised to mean for each genotype per experiment. Means ± SEM, for N = 3 harvests. Repeated-measures 2-way ANOVA, Dunnett’s post hoc test, pairwise comparisons to the WT for each time: * p

    Article Snippet: TREM2 functions at the cell surface; therefore, proteins were purified by cell surface biotinylation and TREM2 detected by Western blotting, with comparison to the input cell homogenate (Fig. b).

    Techniques: Immunofluorescence, Staining, Expressing, Marker

    Transcriptomics reveal high overlap in dysregulated genes of R47H TREM2 and TREM2 KO pMac, relative to WT. a Validation of pMac “microglial identity” by dendrogram comparison to Abud et al. transcriptomes of iPS cells, iPSC-haematopoietic progenitor cells (iHPC), iPSC-microglia-like cells (iMGL), iMGL without TGFβ1 or without CD200 supplementation, iMGL co-cultured with rat cortical neurons, blood-derived monocytes, blood-derived dendritic cells, and primary foetal and adult human microglia [ 42 ]. b Plot of principal component analysis (PCA) with the first two principal components separating the RNA-seq samples by differentiation age and genotype. Ages represented by shapes, and genotypes represented by colour. c Venn diagram of differentially expressed genes (DEGs) identified relative to the WT line, showing the overlap between R47H TREM2 and TREM2 KO DEGs. DEGs are separately categorised as “upregulated” or “downregulated” relative to the WT

    Journal: Alzheimer's Research & Therapy

    Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

    doi: 10.1186/s13195-020-00709-z

    Figure Lengend Snippet: Transcriptomics reveal high overlap in dysregulated genes of R47H TREM2 and TREM2 KO pMac, relative to WT. a Validation of pMac “microglial identity” by dendrogram comparison to Abud et al. transcriptomes of iPS cells, iPSC-haematopoietic progenitor cells (iHPC), iPSC-microglia-like cells (iMGL), iMGL without TGFβ1 or without CD200 supplementation, iMGL co-cultured with rat cortical neurons, blood-derived monocytes, blood-derived dendritic cells, and primary foetal and adult human microglia [ 42 ]. b Plot of principal component analysis (PCA) with the first two principal components separating the RNA-seq samples by differentiation age and genotype. Ages represented by shapes, and genotypes represented by colour. c Venn diagram of differentially expressed genes (DEGs) identified relative to the WT line, showing the overlap between R47H TREM2 and TREM2 KO DEGs. DEGs are separately categorised as “upregulated” or “downregulated” relative to the WT

    Article Snippet: TREM2 functions at the cell surface; therefore, proteins were purified by cell surface biotinylation and TREM2 detected by Western blotting, with comparison to the input cell homogenate (Fig. b).

    Techniques: Cell Culture, Derivative Assay, RNA Sequencing Assay

    Divergent phenotypes in TREM2 KO and R47H TREM2 pMac, regarding cell morphology, migration, survival, and inflammatory responses. a , b TREM2 KO pMac are smaller and rounder than WT. Cell morphology measured by microscopy of pMac stained with CellTracker Deep Red, cells were fixed and imaged on INCell 6000 microscope. Representative images shown ( a ), and mean cell area (μm 2 ) and roundness were automatically quantified from 9 fields per well in triplicate wells using Columbus software ( b ). Means ± SEM, for N = 3 harvests. 1-way ANOVA with Dunnett’s post hoc test: p = 0.019 for cell area in KO versus WT (*), p = 0.007 for roundness in KO versus WT (**). c , d TREM2 KO pMac migrate slower towards C5a, but not ADP, compared with WT. Cell migration measured by transwell assay. Migration of pMac towards 30 μM ADP or 3 nM C5a is compared to unstimulated migration over 6 h ( c ), and inhibitors used to unmask the contribution of purinergic receptors P2RY1 (3 μM MRS2179), P2RY12 (30 μM PSB0739), and P2RY13 (10 μM MRS2211) to ADP-induced migration ( d ). Data is expressed as the percentage of migrated cells and was normalised to average migration for the harvest. Means ± SEM, for N = 4 harvests. 2-way ANOVA with Dunnett’s post hoc test. Black annotations compare stimulation to unstimulated control, or ADP + purinergic inhibitors to the ADP-only control. Grey annotations compare R47H or KO versus WT for each stimulation. * p

    Journal: Alzheimer's Research & Therapy

    Article Title: TREM2 Alzheimer’s variant R47H causes similar transcriptional dysregulation to knockout, yet only subtle functional phenotypes in human iPSC-derived macrophages

    doi: 10.1186/s13195-020-00709-z

    Figure Lengend Snippet: Divergent phenotypes in TREM2 KO and R47H TREM2 pMac, regarding cell morphology, migration, survival, and inflammatory responses. a , b TREM2 KO pMac are smaller and rounder than WT. Cell morphology measured by microscopy of pMac stained with CellTracker Deep Red, cells were fixed and imaged on INCell 6000 microscope. Representative images shown ( a ), and mean cell area (μm 2 ) and roundness were automatically quantified from 9 fields per well in triplicate wells using Columbus software ( b ). Means ± SEM, for N = 3 harvests. 1-way ANOVA with Dunnett’s post hoc test: p = 0.019 for cell area in KO versus WT (*), p = 0.007 for roundness in KO versus WT (**). c , d TREM2 KO pMac migrate slower towards C5a, but not ADP, compared with WT. Cell migration measured by transwell assay. Migration of pMac towards 30 μM ADP or 3 nM C5a is compared to unstimulated migration over 6 h ( c ), and inhibitors used to unmask the contribution of purinergic receptors P2RY1 (3 μM MRS2179), P2RY12 (30 μM PSB0739), and P2RY13 (10 μM MRS2211) to ADP-induced migration ( d ). Data is expressed as the percentage of migrated cells and was normalised to average migration for the harvest. Means ± SEM, for N = 4 harvests. 2-way ANOVA with Dunnett’s post hoc test. Black annotations compare stimulation to unstimulated control, or ADP + purinergic inhibitors to the ADP-only control. Grey annotations compare R47H or KO versus WT for each stimulation. * p

    Article Snippet: TREM2 functions at the cell surface; therefore, proteins were purified by cell surface biotinylation and TREM2 detected by Western blotting, with comparison to the input cell homogenate (Fig. b).

    Techniques: Migration, Microscopy, Staining, Software, Transwell Assay