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1 Supplementary information Epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer Je-In Youn, Vinit Kumar, Michelle Collazo, Yulia Nefedova, Thomas Condamine, Pingyan Cheng, Alejandro Villagra, Scott Antonia, Judith C. McCaffrey, Mayer Fishman, Amod Sarnaik, Pedro Horna, Eduardo Sotomayor, Dmitry I. Gabrilovich
2 a Bone marrow Spleen Blood b Spleen Bone marrow 4T1 LLC c Granulocytic 9 Monocytic Granulocytic 5 Monocytic 3 1 4T1 LLC Naïve 2 wk 3 wk 4 wk Naïve 2 wk 3 wk 4 wk Naïve 2 wk 3 wk 4 wk Naïve 2 wk 3 wk 4 wk Monocytic Granulocytic Control K ras/cc1 Supplemental Figure 1. Expansion of PMN-MDSC and M-MDSC in different types of cancer. a proportion of MDSC populations (PMN-MDSC-granulocytic; M-MDSC monocytic) in different sites during tumor progression ( after tumor inoculation); b absolute number of MDSC; LLC- Lewis lung carcinoma, 4T1 mammary adenocarcinoma, c proportion of MDSC in mice with K-ras/CC1 spontaneous lung cancer
3 Day 1 Day 2 Day 3 Day 4 Day 5 BrdU 1.3% 18.9% 24.9% 21.4% 22.% M-MDSC DAPI BrdU.2%.1%.1%.1%.6% PMN-MDSC DAPI Supplemental Figure 2. Proliferation of M-MDSC and PMN-MDSC in vitro. BrDU taining sorted BM MDSC cultured with GM-CSF and tumor explant supernatants for 5 days. Cells proliferation was evaluated after 4-h pulse with BrDU.
4 a b <FITC-A>: Ly6C Ly6C <FITC-A>: F4/8 F4/ <APC-A>: CD11c CD11c <PE-A>: Ly6G Ly6G <PE-A>: Gr-1 Gr <PE-A>: Gr-1 Gr-1 Supplemental Figure 3. Differentiation of M-MDSC in vitro. Differentiation CD11b + Ly6C hi Ly6G - M-MDSC from BM of 14 old k-ras/cc1 double transgenic mice. Cells were cultured for 5 days with 1 ng/ml GM-CSF with EL-4 TES. a The phenotype of gated CD11b + cells. b. Wright-Giemsa staining of the cells. Scale bar = 1 μm.
5 No primary antibody Spleen from WT mice Spleen from RB-KO mice Supplemental Fig. 4. Immunofluorescence staining of Rb in splenocytes. Rb1 staining of splenocytes from naïve wild-type and Rb1 KO mice (red fluorescence) and counterstained with DAPI. Scale bar = 1 µm.
6 Monocytes M-MDSC high high low low Supplemental Figure 5. Gating strategy for sorting of high and low monocytes and M-MDSC
7 Healthy donor SSC-A CD14 CD11b CD15 Cancer patient SSC-A CD33 SSC-A CD14 CD11b CD15 Supplemental Figure 6. Typical example of the analysis of cells in peripheral blood. Gating strategy of PMN-MDSC and M- MDSC in the blood of cancer patients and cells with the same phenotype in peripheral blood of healthy donor.
8 Healthy donor Healthy donor +TCM MM Patient MM Patient +TCM CD66b CD11b Supplementary Figure 7. Differentiation of CD14 + BM cells. Differentiation of CD14 + cells (more than 7% HLA-DR + ) sorted from BM of healthy donor or multiple myeloma patients and cultured for 5 days with GM-CSF with or without tumor-conditioned media from PCI-3 head and neck tumor cell line. Two experiments with the same results were performed.
9 relative expr of Hdac Expression of hdac2 scramble HDAC2 sirna HDAC1 sirna Relative exp of Hdac Expression of Hdac1 scramble HDAC1 sirna HDAC2 sirna Supplementary Figure 8. Silencing of HDAC-1 and HDAC-2 in MDSC with sirna. Expression of hdac1 and hdac2 in MDSC transfected with 1 nm of sirna specific for HDAC1 or HDAC2 or control (scramble) sirnas. Expression was evaluated by qrt-pcr in triplicates. Each experiment was performed twice.
10 Supplementary Table 1. Changes in the expression of Rb associated transcripts between M-MDSC and PMN- MDSC M-MDSC/PMN-MDSC Exp.1 Exp. 2 Genes Retinoblastoma binding protein 7 (Rbbp7) Retinoblastoma-like 1 (p17) Retinoblastoma binding protein 8 (Rbbp8) Retinoblastoma binding protein 4 (Rbbp4) Retinoblastoma 1 (Rb1) M-MDSC and PMN-MDSC were isolated from spleens of EL-4 TB mice. Two micrograms of total RNA served as the mrna source for microarray analysis using Affymetrix gene chips. Scanned output files were analyzed using the Affymetrix GeneChip Operating Software. Signal intensity was scaled to an average intensity of 5, prior to comparison analysis. Probe sets that yielded a change with a p-value less than.2 were identified as changed. A log base 2 transformation was applied to the data. Each array was normalized (centered) by extracting the median over the entire array. The table shows only selected data of transcripts associated with Rb that showed significant changes in two performed experiments.
Supplemental figure 1: Phenotype of IMC and MDSC after purification. A. Gating
Supplemental Figure Legend: Supplemental figure 1: Phenotype of IMC and MDSC after purification. A. Gating strategy for mouse MDSC. CD11b + Ly6C high Ly6G - cells are defined as M-MDSC. CD11b + Ly6C low
Development of CD11b-IRF8 transgenic and MTAG double transgenic mice The CMV-
Supplemental Data Development of CD11b-IRF8 transgenic and MTAG double transgenic mice The CMV- IRF8 transgenic mouse was previously developed by cloning complementary DNA, containing the IRF-8 coding
Supplementary Figure 1: Analysis of monocyte subsets and lineage relationships. (a) Gating strategy for definition of MDP and cmop populations in BM
Supplementary Figure 1: Analysis of monocyte subsets and lineage relationships. (a) Gating strategy for definition of MDP and cmop populations in BM of Cx3cr1 GFP/+ mice related to Fig. 1a. MDP was defined
Interferon- -producing immature myeloid cells confer protection. against severe invasive group A Streptococcus infections. Supplementary Information
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Supplemental Information Inventory
Cell Stem Cell, Volume 6 Supplemental Information Distinct Hematopoietic Stem Cell Subtypes Are Differentially Regulated by TGF-β1 Grant A. Challen, Nathan C. Boles, Stuart M. Chambers, and Margaret A.
Rorα is essential for nuocyte development
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The transcrip-on factor NR4A1 (Nur77) controls bone marrow differen-a-on and survival of Ly6C monocytes
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a KYSE270-CON KYSE270-Id1
a KYSE27-CON KYSE27- shcon shcon sh b Human Mouse CD31 Relative MVD 3.5 3 2.5 2 1.5 1.5 *** *** c KYSE15 KYSE27 sirna (nm) 5 1 Id2 Id2 sirna 5 1 sirna (nm) 5 1 Id2 sirna 5 1 Id2 [h] (pg per ml) d 3 2 1
ClustalW algorithm. The alignment statistics are 96.9% for both pairwise identity and percent identical sites.
Identity Human Mouse Supplemental Figure 1. Amino acid sequence alignment of -MyHC. The alignment was created using the ClustalW algorithm. The alignment statistics are 96.9% for both pairwise identity
SI Appendix. Tumor-specific CD8 + Tc9 cells are superior effector than Tc1 cells for. adoptive immunotherapy of cancers
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Autocrine Complement Inhibits IL10-Dependent T-Cell Mediated. Antitumor Immunity to Promote Tumor Progression
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Nature Biotechnology: doi: /nbt.4086
Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3 p815 p815-hcd2 Ag (-) anti-cd3
Supplementary Figure 1. Characterization of the POP2 transcriptional and post-transcriptional regulatory elements. (A) POP2 nucleotide sequence
1 5 6 7 8 9 10 11 1 1 1 Supplementary Figure 1. Characterization of the POP transcriptional and post-transcriptional regulatory elements. (A) POP nucleotide sequence depicting the consensus sequence for
88.7 <GFP-A>: EGFP GFP. Null. % of Cells! <APC-A> B220!
B. C. 1 Cell Number (X18) A. % of Cells % of Max 8 Con 6 4 88.7 1 13 14 : EGFP 4 6 4 1 WT 8 % of% ofcells Max 6 6 4 G. 1 13 B Hdac3-/- 14 15 1 13 14 15 Ter119 Ly6C Wild type WT 4
Supplementary Figure 1. Xbp1 deficiency does not alter hematopoietic cellularity.
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Supplemental Information. The TRAIL-Induced Cancer Secretome. Promotes a Tumor-Supportive Immune. Microenvironment via CCR2
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Supplemental Table S1. RT-PCR primers used in this study
Supplemental Table S1. RT-PCR primers used in this study -----------------------------------------------------------------------------------------------------------------------------------------------
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION ARTICLE NUMBER: 16108 DOI: 10.1038/NMICROBIOL.2016.108 The binary toxin CDT enhances Clostridium difficile virulence by suppressing protective colonic eosinophilia Carrie A. Cowardin,
Nature Immunology: doi: /ni Supplementary Figure 1
Supplementary Figure 1 Validation of the monoclonal antibody to mouse ACKR1 and expression of ACKR1 by BM hematopoietic cells. (a to d) Comparison of immunostaining of BM cells by anti-mouse ACKR1 antibodies:
(A-B) P2ry14 expression was assessed by (A) genotyping (upper arrow: WT; lower
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SUPPLEMENTARY FIG. S2. Expression of single HLA loci in shns- and shb 2 m-transduced MKs. Expression of HLA class I antigens (HLA-ABC) as well as
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Macrophage colony stimulating factor induces macrophage proliferation and survival through a pathway involving DAP12 and β-catenin Karel Otero, Isaiah R Turnbull, Pietro Luigi Poliani *, William Vermi
To determine the effect of N1IC in the susceptibility of T cells to the tolerogenic effect of tumorassociated
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Supplementary Table-1: List of genes that were identically matched between the ST2 and
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Supplementary Figures
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Table S1. Antibodies and recombinant proteins used in this study
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a Lamtor1 (gene) b Lamtor1 (mrna) c WT Lamtor1 Lamtor1 flox Lamtor2 A.U. p = LysM-Cre Lamtor3 Lamtor4 Lamtor5 BMDMs: Φ WT Φ KO β-actin WT BMDMs
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MLN8237 induces proliferation arrest, expression of differentiation markers and
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Supplementary Material & Methods
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Flow cytometry Stained cells were analyzed and sorted by SORP FACS Aria (BD Biosciences).
Mice C57BL/6-Ly5.1 or -Ly5.2 congenic mice were used for LSK transduction and competitive repopulation assays. Animal care was in accordance with the guidelines of Keio University for animal and recombinant
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isolated from ctr and pictreated mice. Activation of effector CD4 +
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7-amino actinomycin D (7ADD) was added to all samples 10 minutes prior to analysis on the flow cytometer in order to gate 7AAD viable cells.
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Supplemental Figure 1
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Nature Immunology: doi: /ni Supplementary Figure 1
Supplementary Figure 1 Sox2 localizes in neutrophils of mouse spleen. (a) Microscopy analysis of Sox2 and MPO in wild-type mouse spleen. Nucl, nucleus. (b) Immunohistochemistry staining of Sox2 and MPO
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Supplemental Figure 1
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