Journal: Cell reports

Article Title: The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis

doi: 10.1016/j.celrep.2019.06.007

Figure Lengend Snippet:

Article Snippet: Anti-Ly6G Microbead Kit mouse , Miltenyi Biotec , Cat#130–092–332.

Techniques: Control, Northern Blot, Recombinant, Purification, Adjuvant, Lysis, Staining, Reverse Transcription, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software

Journal: Biology of Reproduction

Article Title: Expression Profiling Reveals Developmentally Regulated lncRNA Repertoire in the Mouse Male Germline 1

doi: 10.1095/biolreprod.113.113308

Figure Lengend Snippet: Subcellular localization of two lncRNAs in adult murine testes by ISH assays. A–D) ISH-based localization of a bidirectional lncRNA (AK082424) in adult murine testes. Brown, punctuated dots representing specific hybridization signals are confined to the nuclei of spermatocytes and round spermatids in seminiferous tubules at stages I–III, VII, and XI. Arrows point to representative positive cells (B–D). Sections hybridized to control probes show no specific hybridization signals (A). Insets show the digitally magnified view of the framed regions. Panels B–D are in the same magnification. Bar = 50 μm. E) Schematic of the localization of lncRNA AK082424 during spermatogenesis in adult murine testes. Cells expressing this lncRNA are framed, and the height of the frame represents relative expression levels. Note that specific hybridization signals are confined to the nuclei of spermatocytes and round spermatids (red dots). Roman numerals (I–XII) mark stages of the seminiferous epithelial cycle, and steps of spermatid differentiation (i.e., spermiogenesis) are labeled with Arabic numerals (1–16). Sc, Sertoli cells; As, single type A spermatogonia; As-pr, paired type A spermatogonia; Aal, aligned type A spermatogonia; A1-4, type A1-A4 spermatogonia; In, intermediate spermatogonia; B, type B spermatogonia; Pl, preleptotene spermatocyte; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; Di, diplotene spermatocyte; M, meiotically dividing. F–I) ISH-based localization of an antisense lncRNA (AK016507) in adult mouse testes. Brown, punctuated dots representing specific hybridization signals are confined mainly to the cytoplasm of spermatocytes and round and elongating spermatids in seminiferous tubules at stages IV, V, VII, X, and XI. Arrows point to representative positive cells (G–I). Sections hybridized to control probes show no specific hybridization signals (F). Insets show the magnified views of the framed regions. Panels F–I are all in the same magnification. Bar = 50 μm. J) Schematic of localization of lncRNA AK016507 during spermatogenesis in adult murine testes. Cells expressing this lncRNA are framed, and the height of the frame represents relative expression levels. Note that specific hybridization signals are mainly confined to the cytoplasm of spermatocytes and round and elongating spermatids (red dots). Labels are the same as in E.

Article Snippet: The labeled cDNAs were hybridized to the Mouse LncRNA Array (version 2.0; 8 × 60 K; Arraystar) using the SureHyb Hybridization Chambers according to the One-Color Microarray-Based Gene Expression Analysis protocol (Agilent).

Techniques: Hybridization, Control, Expressing, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

doi: 10.4049/jimmunol.1400663

Figure Lengend Snippet: (A) IgG subtype analysis. Antibodies were engendered in C57Bl/6 mice against nickel column-purified VACV IMV membrane protein, WR101/H3LΔTM that has been adjuvanted in CpG/ISCOMs or alum, or in PBS alone as a control. Sera were obtained after 14 days and probed against VACV proteome microarrays on to which 8 two-fold serial dilutions of purified WR101/H3L were printed. Specific reactivity to purified WR101/H3L was visualized using fluorescently-tagged secondary antibodies to IgG, IgG1 and IgG2c and signal intensities quantified in a confocal laser scanner; data for a single concentration of printed antigen is shown. (B) Relative proportions of IgG1 and IgG2c derived from data shown in (A). The IgG2a proportion of the total signal is shown above the zero line, and the IgG1 proportion shown below. The IgG response is polarized according to adjuvant. (C) Protection of B6 mice against intranasal (i.n.) challenge of VACV-WR using adjuvanted WR101/H3LΔTM and WR101/H3L. CpG/ISCOMs reduce weight loss and promote recovery compared to alum or PBS. (D) and (E) correlations between nadir body weight (expressed as percentage of original body weight) and titer of IgG2c and IgG1, respectively. Titer was defined from the WR101/H3L titration series on the array at the lowest concentration to give a signal intensity >2000. Liner regression was used to generate the trend lines.

Article Snippet: Biotinylated goat anti-mouse IgG, IgG1 and IgG2c secondary antibodies were purchased from Jackson ImmunoResearch (Cat# 115-065-008, 115-065-205, and 115-065-208, respectively).

Techniques: Nickel Column, Purification, Membrane, Control, Concentration Assay, Derivative Assay, Adjuvant, Titration

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: T cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and non-virion association *

doi: 10.4049/jimmunol.1400663

Figure Lengend Snippet: A. Scatter plots of IgG signals by microarray vs. corresponding T cell responses from representative IFNγ ELISPOT. Cut-offs (hashed lines); for T cells defined as mean+2.5SD of control antigens (n=35; SDS-IBs proteome n = 225); antibody targets antigens defined as described in Table 2 (n=26; proteome on array n=194). Different temporal expressions indicated by symbols, as defined by Yang et al (32) using cluster analysis of viral mRNAs at 0.5, 1, 2 and 4h infection time points: E1.1=early subcluster 1; E1.2=early subcluster 2; PR=post-replicative. B. Bar charts of antibody signals (average of 6 mice) and T cells (averages of spot-forming cells/106 on d8 and d13, as shown in Fig 4). The first 65 antigens are ranked by antibody signal and thereafter by T cell response. C. Pie charts showing proportions of reactive antibody and CD4 T cell targets, compared to the whole VACV-WR proteome. Each protein was classified into a functional category according to Yang et al (2010) (32). Note: most of the pseudogenes were omitted from the arrays, so the proteome pie charts for IgG and for the T cell screens differ slightly.

Article Snippet: Biotinylated goat anti-mouse IgG, IgG1 and IgG2c secondary antibodies were purchased from Jackson ImmunoResearch (Cat# 115-065-008, 115-065-205, and 115-065-208, respectively).

Techniques: Microarray, Enzyme-linked Immunospot, Control, Infection, Functional Assay

Journal:

Article Title: A crucial role for the putative Arabidopsis topoisomerase VI in plant growth and development

doi: 10.1073/pnas.152337599

Figure Lengend Snippet: Identification of BIN5 and BIN3 genes. (A) bin5 chromosome walk. The bin5 mutation was mapped to a 46-kb region on BAC F9G14. Complementation constructs B and C, both containing ORF20, rescued the bin5 mutant phenotypes, indicating that ORF20 is mutated in bin5. (B) BIN5/AtSPO11-3 gene and bin5 mutation. BIN5 was identified as AtSPO11-3, one of three Arabidopsis homologs of archaebacterial topoisomerase VI subunit A (Top6A) (23, 25). BIN5 contains two exons and DNA sequencing of bin5 mutant DNA revealed a single base pair addition at nucleotide position +1035 with respect to the putative translation start site (ccg agt gat to ccc gag tga t). A phylogenetic distance tree shows that BIN5 is most closely related to AtSPO11-2 and archaebacterial TOP6A than to nematode, mouse, fly, fungus, and yeast SPO11. Numbers in branch nodes indicate the number of times that the group of sequences to the right of the node occurred of 100 bootstrap replicates. Numbers are only given when 70 or more occurrences were observed. (C) BIN3/AtTop6B gene and mutations. BIN3 was identified by plasmid rescue with a T-DNA-tagged allele, bin3-1. BIN3/AtTOP6B gene contains 18 introns and 19 exons that encode a predicted polypeptide of 670 aa (25). Phylogenetic distance tree shows relationship of BIN3/AtTOP6B, archaebacterial TOP6B, and prokaryotic homologs of Aeropyrum pernix, Archaeoglobus fulgidus, Pyrococcus horikoshii, and Pyrococcus abyssi.

Article Snippet: Genes that are down-regulated by at least 2-fold in both bin3 and bin5 compared with wild-type control were identified using GENESPRING 4.2 (Silicon Genetics, Redwood City, CA) and listed in Table 2, which is published as supporting information on the PNAS web site, www.pnas.org . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Gene no. Gene Col bin3 bin5 bri1-116 −BL +BL −BL +BL −BL +BL −BL +BL At4g38850 SAUR-AC1 442 2260 161 449 105 476 223 128 At4g38860 SAUR-AC1-like 652 2808 99 834 264 975 285 208 At3g15540 IAA19 980 1958 301 535 372 472 360 438 At2g14900 GASA3 10567 13722 4754 6042 2698 5365 7635 7332 At5g57560 TCH4 19164 26329 14615 15691 12952 16996 5823 5046 At2g26710 BAS1 922 2527 589 1286 954 1150 627 669 At2g31730 Similar to LeER33 1939 2820 1311 1670 1549 1561 1023 1306 At1g65310 Putative xyloglucan endoglucanase 1296 2431 302 394 419 287 274 397 At1g04610 YUCCA3 1372 1639 288 452 532 446 593 653 At5g13870 EXGT-A4 1025 1885 280 267 189 450 637 644 At4g20780 Putative Ca-dependent protein kinase 5508 6829 1223 1225 1077 1265 2532 2130 At2g40610 Putative expansin 3945 7978 615 2767 1011 2070 1178 802 At4g28780 Pro-rich APG like protein 2263 4792 861 2559 531 1995 1373 859 At1g21820 Unknown 2227 4069 1305 1617 1760 1982 1846 1851 At2g32560 Unknown 2534 4380 1235 1817 1516 1519 1366 1769 At2g36220 Unknown 1425 3803 1488 800 1034 1391 1311 1435 At4g09890 Unknown 1497 2725 340 465 388 464 582 657 At4g01950 Unknown 1327 2921 893 1802 946 1465 319 239 Open in a separate window RNA from seedlings treated without or with BL was used for microarray experiments using Affymetrix arabidopsis gene chip .

Techniques: Mutagenesis, Construct, DNA Sequencing, Plasmid Preparation