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1 Supplementary information: Sterile inflammation as a factor in human male infertility: Involvement of Toll like receptor 2, biglycan and peritubular cells C. Mayer 1, M. Adam 1,2, L. Glashauser 1, K. Dietrich 1, J.U. Schwarzer 3, F.-M. Köhn 4, L. Strauss 2, H. Welter 1, M. Poutanen 2, A. Mayerhofer 1* 1 Biomedical Center (BMC), Cell Biology, Anatomy III, Ludwig-Maximilians-Universität (LMU), D Planegg, Germany 2 Turku Center for Disease Modeling and Department of Physiology, Institute of Biomedicine, University of Turku, FL Turku, Finland 3 Andrology-Center, D Munich, Germany 4 Andrologicum, D Munich, Germany PAM and BGN do not significantly affect ATP levels Cellular ATP levels of HTPCs, as a measure of cellular viability and cell number, were studied using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Mannheim, Germany) as described 1. Unpaired t-test (in case of PAM) or ANOVA (BGN) was employed (Prism, GraphPad Software (version 4.0a), Inc., San Diego, CA, USA) and a probability value of p < 0.05 was considered significant. Results of ATP-measurements in HTPCs after 24 h in the absence (control) or presence of PAM or BGN revealed that neither treatment significantly affected ATP-levels (Supplementary Figure 1). Individual measurements, means and SD are shown (AU: arbitrary units). Experiments were repeated three times using cells derived from
2 2 different patients with comparable results. Supplementary Figure 1 Immunohistochemical detection of claudin-11, CD-68 and BGN in human testes, claudin-11 in WT and AROM+ mouse testes Additional immunohistochemical studies were performed. The method was performed (n=4 human testicular sections) as described 2,3 using the following commercial antibodies and antisera: mouse anti-cd-68 (1:100, DAKO, Hamburg, Germany); rabbit anti-claudin-11, (#NBP , 1:100, Novus, Littleton, CO, USA); rabbit anti-bgn (#HPA003157; 1:500, Sigma, Deisenhofen, Germany). As shown in Supplementary Figure 2A, immunostaining for claudin-11, a robust marker for Sertoli cell tight junctions, was readily detected, although CD68-positive macrophages were found in the immediate vicinity, as seen in a consecutive section. Some macrophages, expressing CD68 also stained for BGN, as seen in the two
3 3 consecutive sections in Supplementary Figure 2B. This may indicate that several testicular sources of BGN exist, including macrophages, a result in line with studies in rodents 4,5. Furthermore, claudin-11 mrna levels were determined in testes of the 5 month old WT and AROM+ mice, when we observed elevated levels of BGN and TRL2. We used qpcr, as described 2 (see also main text). Primers employed for mouse claudin-11 were as follows: forward primer 5 -TCC TTA TTC TGC TGG CTC TCT-3 and reverse primer 5 -TCC AAA TGA CTG TGC ATC CC-3 and L19. Results shown in Supplementary Figure 2C revealed that expression levels do not differ (Mann-Whitney test; Prism, GraphPad Software). Supplementary Figure 2
4 4 References: 1 Saller, S. et al. Dopamine in human follicular fluid is associated with cellular uptake and metabolism-dependent generation of reactive oxygen species in granulosa cells: implications for physiology and pathology. Human reproduction 29, , doi: /humrep/det422 (2014). 2 Welter, H. et al. Angiotensin II regulates testicular peritubular cell function via AT1 receptor: a specific situation in male infertility. Molecular and cellular endocrinology 393, , doi: /j.mce (2014). 3 Blohberger, J. et al. Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions? Cell death & disease 6, e1685, doi: /cddis (2015). 4 Moreth, K., Iozzo, R. V. & Schaefer, L. Small leucine-rich proteoglycans orchestrate receptor crosstalk during inflammation. Cell cycle 11, , doi: /cc (2012). 5 Babelova, A. et al. Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X receptors. The Journal of biological chemistry 284, , doi: /jbc.m (2009). Additional information: Full length gels and blots. Note that the cropped areas displayed in the composite manuscript figures are marked in red. Original gels for Figure 1:
5 5 Original Blots for Figures 2 and 3: Original Blots for Figure 6:
CD31 5'-AGA GAC GGT CTT GTC GCA GT-3' 5 ' -TAC TGG GCT TCG AGA GCA GT-3'
Table S1. The primer sets used for real-time RT-PCR analysis. Gene Forward Reverse VEGF PDGFB TGF-β MCP-1 5'-GTT GCA GCA TGA ATC TGA GG-3' 5'-GGA GAC TCT TCG AGG AGC ACT T-3' 5'-GAA TCA GGC ATC GAG AGA
a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53
1 2 3 4 5 6 7 8 9 10 Supplementary Figure 1. Induction of p53 LOH by MADM. a) Primary cultures derived from the pancreas of an 11-week-old Pdx1-Cre; K-MADM-p53 mouse revealed increased p53 KO/KO (green,
Abbreviations: P- paraffin-embedded section; C, cryosection; Bio-SA, biotin-streptavidin-conjugated fluorescein amplification.
Supplementary Table 1. Sequence of primers for real time PCR. Gene Forward primer Reverse primer S25 5 -GTG GTC CAC ACT ACT CTC TGA GTT TC-3 5 - GAC TTT CCG GCA TCC TTC TTC-3 Mafa cds 5 -CTT CAG CAA GGA
SUPPLEMENTARY DATA. Supplementary Table 1. Primer sequences for qrt-pcr
Supplementary Table 1. Primer sequences for qrt-pcr Gene PRDM16 UCP1 PGC1α Dio2 Elovl3 Cidea Cox8b PPARγ AP2 mttfam CyCs Nampt NRF1 16s-rRNA Hexokinase 2, intron 9 β-actin Primer Sequences 5'-CCA CCA GCG
Supplementary Figure 1 a
Supplementary Figure a Normalized expression/tbp (A.U.).6... Trip-br transcripts Trans Trans Trans b..5. Trip-br Ctrl LPS Normalized expression/tbp (A.U.) c Trip-br transcripts. adipocytes.... Trans Trans
Supplementary Table 2. Conserved regulatory elements in the promoters of CD36.
Supplementary Table 1. RT-qPCR primers for CD3, PPARg and CEBP. Assay Forward Primer Reverse Primer 1A CAT TTG TGG CCT TGT GCT CTT TGA TGA GTC ACA GAA AGA ATC AAT TC 1B AGG AAA TGA ACT GAT GAG TCA CAG
c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP
Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative
Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.
Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons
Figure S1. Analysis of genomic and cdna sequences of the targeted regions in WT-KI and
Figure S1. Analysis of genomic and sequences of the targeted regions in and indicated mutant KI cells, with WT and corresponding mutant sequences underlined. (A) cells; (B) K21E-KI cells; (C) D33A-KI cells;
Description of Supplementary Files. File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables
Description of Supplementary Files File Name: Supplementary Information Description: Supplementary Figures and Supplementary Tables Supplementary Figure 1: (A), HCT116 IDH1-WT and IDH1-R132H cells were
McAlpine PERK-GSK3 regulates foam cell formation. Supplemental Material. Supplementary Table I. Sequences of real time PCR primers.
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Supplementary Materials
Supplementary Materials 1 Supplementary Table 1. List of primers used for quantitative PCR analysis. Gene name Gene symbol Accession IDs Sequence range Product Primer sequences size (bp) β-actin Actb gi
SUPPLEMENTARY FIGURES AND TABLES
SUPPLEMENTARY FIGURES AND TABLES Supplementary Figure S1: CaSR expression in neuroblastoma models. A. Proteins were isolated from three neuroblastoma cell lines and from the liver metastasis of a MYCN-non
SUPPLEMENTARY RESULTS
SUPPLEMENTARY RESULTS Supplementary Table 1. hfpr1- Flpln-CHO hfpr2-flpln-cho pec 50 E max (%) Log( /K A) Log( /K A) N pec 50 E max (%) Log( /K A) Log( /K A) n ERK1/2 phosphorylation fmlp 9.0±0.6 80±7
Supplementary Appendix
Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Sherman SI, Wirth LJ, Droz J-P, et al. Motesanib diphosphate
Supplementary Figure 1. ROS induces rapid Sod1 nuclear localization in a dosagedependent manner. WT yeast cells (SZy1051) were treated with 4NQO at
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Supplementary Figures
Supplementary Figures Supplementary Figure 1. H3F3B expression in lung cancer. a. Comparison of H3F3B expression in relapsed and non-relapsed lung cancer patients. b. Prognosis of two groups of lung cancer
Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1
Supplementary Figure 1 U1 inhibition causes a shift of RNA-seq reads from exons to introns. (a) Evidence for the high purity of 4-shU-labeled RNAs used for RNA-seq. HeLa cells transfected with control
Harald Welter, Ph.D., a Frank M. K ohn, M.D., b and Artur Mayerhofer, M.D. a
Mast cells in human testicular biopsies from patients with mixed atrophy: increased numbers, heterogeneity, and expression of cyclooxygenase 2 and prostaglandin D2 synthase Harald Welter, Ph.D., a Frank
Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most
Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most differentially expressed between human synovial fibroblasts
Toluidin-Staining of mast cells Ear tissue was fixed with Carnoy (60% ethanol, 30% chloroform, 10% acetic acid) overnight at 4 C, afterwards
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Supplementary Information
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Supplementary Figure 1: Hsp60 / IEC mice are embryonically lethal (A) Light microscopic pictures show mouse embryos at developmental stage E12.
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Supplementary Document
Supplementary Document 1. Supplementary Table legends 2. Supplementary Figure legends 3. Supplementary Tables 4. Supplementary Figures 5. Supplementary References 1. Supplementary Table legends Suppl.
BHP 2-7 and Nthy-ori 3-1 cells were grown in RPMI1640 medium (Hyclone) supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL
1 2 3 4 Materials and Methods Cell culture BHP 2-7 and Nthy-ori 3-1 cells were grown in RPMI1640 medium (Hyclone) 5 supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine, and 100 U/mL 6 penicillin-streptomycin.
Supplementary Figure 1
Supplementary Figure 1 3 3 3 1 1 Bregma -1.6mm 3 : Bregma Ref) Http://www.mbl.org/atlas165/atlas165_start.html Bregma -.18mm Supplementary Figure 1 Schematic representation of the utilized brain slice
SUPPLEMENTAL FIGURE 1
SUPPLEMENTL FIGURE 1 C Supplemental Figure 1. pproach for removal of snorns from Rpl13a gene. () Wild type Rpl13a exonintron structure is shown, with exo in black and intronic snorns in red rectangles.
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Mitosis. Single Nano Micro Milli Macro. Primary. PCNA expression
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