Fig. S1 A. week 4 week 6
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1 Fig. S1 Trabecular Number Trabecular Thickness number/mm mm SKG-c SKG-A mm Trabecular Spacing D. week 4 week 6 Figure S1. MicroCT analysis of SKG mice shows significant changes in proximal tibia trabecular parameter consistent with osteopenia. SKG mice at 14 weeks arthritis (SKG-A, gray bars), are compared to healthy litter mate controls (SKG-c, black bars). (A) Trabecular number and (B) trabecular thickness are significantly decreased, and (C) trabecular spacing is correspondingly increased in arthritic mice, p.4 for all comparisons, Student s t-test. Osteoclast resorptive activity measured by serum c-telopeptide (CTX) produciton is increased at 4 and 6 weeks after zymosan injection, n=5 per group (D).
2 Fig. S2 SKG-c SKG-A % total BM p=.18 SKG-c SKG-A TRAP+ MN cells/well - Ly6Chi lo Ly6Chi Figure S2. Peripheral OC differentiation increases in SKG Arthritis. Peripheral blood cells were collected from SKG mice 8 weeks after induction of arthritis (SKG-A) or from healthy SKG littermates (SKG-c), mononuclear cells were isolated by Ficoll gradeint centrifugation and plated at 1x15 cells/ well in 96 well plates in 5% CMG and 25ng/mL RANKL and cultured for 7 days. Results are representative of 2 independent experiments. -/lo Ly6Chi bone marrow increase as early as 1 week after zymosan induction of arthritis. and Ly6C staining of B22-CD3Ter119- gated bone marrow from mice 1 week after zymosan injection (SKG-A) or control PBS injected mice (SKG-c) demonstrates increased, n=5. Results are representative of 3 independent experiments. OC precursor activity is found in both the - and hi Ly6Chi bone marrow cells. The bone marrow CD45R- CD3- -/lo Ly6Chi population was further subdivided into - and lo cells and purified by fluorescence activated cell sorting. Triplicate wells
3 Fig. S3.2 SKG/SKG SKG/wt wt/wt.15 BV/TV BV/TV mm Figure S3. Female SKG mice are osteopenic compared to heterozygous and wild-type litermates. MicroCT analysis of week old female mice from SKG het x SKG het parents shows trabecular osteopenia of SKG/SKG mice (light gray bars, n=6) compared to SKG het (dark gray bars, n=3) and wild-type mice (black bars, n=5) at both (A) proximal tibia and (B) distal femur, p.5, p.2, Student s t-test. (C) Cortical thickness, however, is not signficantly changed at this age.
4 Fig. S4 1 4 BALB/c CD3 - B22 - Ter119 - BALB/c % Ly6C C57BL/6 CD3 - B22 - Ter119 - input -/lo Ly6C hi (98%) + Ly6C hi (99%) + Ly6C int (98%) C57BL/6 (98%) % Ly6C TNF-Tg CD3 - B22 - Ter119 - input -/lo Ly6C hi (97%) + Ly6C hi (94%) + Ly6C int (96%) TNF-Tg (99%) % D. 1.1 x1 6 /femur Ly6C input -/lo Ly6C hi (99%) + Ly6C hi (97%) + Ly6C int (95%) (97%) C57BL/6 TNF-Tg Fig. S4. -/lo Ly6C hi are the primary osteoclast precursor population independent of strain. Representative dot plots of and Ly6C stained CD3 - B22 - Ter119 - bone marrow and multinucleated TRAP + cells cultured from individual bone marrow populations plated in triplicate for 3-4d in MCSF and 25ng/mL RANKL demonstrates that -/lo Ly6C hi cells are also the primary osteclast precursors in (A) BALB/c (12.5X1 3 cells/well) (B) C57BL/6 mice (2.5X1 3 cells/well) and (C) 2 week old TNF-Tg mice with arthritis (2.5X1 3 cells/well). The percent of in total bone marrow is noted on the dot plot, and the purity of each sorted population is stated in parenthesis on the x-axis of the graph panel. Results are representative of 2 or more replicates. (D) The number of bone marrow is significantly increased in arthritic TNF-Tg mice compared to age matched wild-type controls, p=.3.
5 Fig. S5 -/lo Ly6C hi d8 MCSF culture % of max F4/ Alexa-488 D /lo Ly6C hi d8 GMCSF culture E. F. Ter119 - CD3 - B22 - -/lo Ly6C hi CD11c 1 2 FL2-H: FL2-CD11c 1 1 Ki isotype G MHCII I-A/E H. 5 p=.3 cpm lo Ly6C hi CX3CR1 + lo Ly6C h i CX3CR1 neg control 5 μm HU Figure S5. -/lo Ly6C hi CD117 + are multipotent in vitro and like circulating are predominantly quiescent. (A-C) In the presence of MCSF, sorted -/lo Ly6C hi CD117 + differentiate into hi F4/8 + macrophages with phagocytic activity. (A) After 8d culture in MCSF, -derived cells are essentially all hi F4/8 +. (B) -derived macrophages phagocytose Alexa-488 labelled zymosan A and become Alex-488+ (solid line) compared to untreated cells (solid gray); uptake is blocked by preincubation with cytochalasin D (dashed line). (C) -derived macrophages from (B) stained with rhodamine-phalloidin demonstrating intracellular Alexa-488 zymosan A particles, 2X. (D-E) Sorted -/lo Ly6C hi CD117 + differentiate into CD11c + dendritic cells in the presence of GMCSF. (D) cultured in GMCSF for 8d and stimulated with LPS for 4h differnentiate into CD11c + cells that express MHCII + in response to LPS. (E) Rhodamine-phalloidin staining of the cells in (D) demonstrates typical dendritic mophology, 2X. (F) Staining for Ki-67 demonstrates that the -/lo Ly6C hi population is predominantly quiescent on isolation. (G) 3 H thymidine uptake shows that -/lo Ly6C hi CX 3 CR1 + proliferate in response to MCSF in contrast to the CX 3 CR1 - subpopulation. (H) Treatment with (HU) hydroxyurea greatly reduces TRAP + multinucleated OC differentition from -/lo Ly6C hi, suggesting that proliferation is required for OC differentiation from bone marrow.
6 Fig. S6 serum CTX lymph node T-cells 5 ng/ml % IL serum TNFα skin score D. 7.5 serum IL-1β 15 pg/ml pg/ml 35 serum IL pg/ml CD4 + E Figure S6. Co-adoptive transfer of -/lo Ly6Chi does not alter serum CTX, nor decrease inflammatory cytokines. (A) Serum CTX measuered by ELIS (B) Despite amelioration of inflammatory arthritis, serum levels of TNFα, IL-6 and IL-1 are not significantly reduced in mice receiving co-adoptive transfer of. (C) IL-17 producing lymp node T-cells are decreased in the group, but serum IL-17 levels are unchanged (data not shown), (D) Co-transfer of significantly exacerbates inflammatory skin lesions compared to adoptive transfer of SKG T-cells alone, p=.3 Student s t-test. The Treg group had no skin abnormalities. (E) Represenative images of skin pathology shows shows acanthosis, hyperkeratosis and dermal infiltrates that are more pronounced in the group comparted to the group. H&E stain, 4X.
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Supplemental Materials and Methods Mice Female C57BL/6 (B6, I-E null, H-2 b ), BALB/c (H-2 d ) + ), FVB/N (H-2 q, I-E null, CD45.1 + ), and B6D2F1 (H-2 b/d ) mice were purchased from the Animal Resources
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and follicular helper T cells is Egr2-dependent. (a) Diagrammatic representation of the
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SUPPLEMENTARY FIGURE 1
SUPPLEMENTARY FIGURE 1 A LN Cell count (1 ) 1 3 1 CD+ 1 1 CDL lo CD hi 1 CD+FoxP3+ 1 1 1 7 3 3 3 % of cells 9 7 7 % of cells CD+ 3 1 % of cells CDL lo CD hi 1 1 % of CD+ cells CD+FoxP3+ 3 1 % of CD+ T
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D CD8 T cell number (x10 6 )
IFNγ Supplemental Figure 1. CD T cell number (x1 6 ) 18 15 1 9 6 3 CD CD T cells CD6L C CD5 CD T cells CD6L D CD8 T cell number (x1 6 ) 1 8 6 E CD CD8 T cells CD6L F Log(1)CFU/g Feces 1 8 6 p
SUPPLEMENTARY INFORMATION
a. Smo+/+ b. Smo+/+ 5.63 5.48 c. Lin- d. e. 6 5 4 3 Ter119 Mac B T Sca1 Smo+/+ 25 15 2 o BMT 2 1 5 * Supplementary Figure 1: Deletion of Smoothened does not alter the frequency of hematopoietic lineages
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SUPPLEMENTARY METHODS Histological analysis. Colonic tissues were collected from 5 parts of the middle colon on day 7 after the start of DSS treatment, and then were cut into segments, fixed with 4% paraformaldehyde,
Conv. R GF Col. GF. Sham Ovx. E. coli. Veh. E. coli. E. coli. Veh. E. coli
B A Uterus weight (mg) 5 5 Body Weight (g) - - Uterus Weight (mg) - - Supplemental Figure. Effects of prolide and treatment with probiotics on uterus weight and body weight. Data are expressed as ean +
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SUPPLEMENTARY INFORMATION
DOI: 1.138/ncb3355 a S1A8 + cells/ total.1.8.6.4.2 b S1A8/?-Actin c % T-cell proliferation 3 25 2 15 1 5 T cells Supplementary Figure 1 Inter-tumoral heterogeneity of MDSC accumulation in mammary tumor
a 10 4 Link et al. Supplementary Figure 1 Nature Immunology: doi: /ni.1842 Cells per mouse ( 10 5 ) TRPV2KO anti-gr1 anti-gr anti-f4/80
a 10 4 WT 10 4 TRPV2KO 10 3 10 3 anti-gr1 10 2 10 1 anti-gr1 10 2 10 1 10 0 10 0 10 1 10 2 10 3 10 4 anti-f4/80 42.3 45.2 10 0 10 0 10 1 10 2 10 3 10 4 anti-f4/80 10 4 10 4 40 42.5 anti-cd11b 10 3 10 2
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Supplemental Figure S1. RANK expression on human lung cancer cells.
Supplemental Figure S1. RANK expression on human lung cancer cells. (A) Incidence and H-Scores of RANK expression determined from IHC in the indicated primary lung cancer subgroups. The overall expression