1. Upregulation of Inflammatory Cytokines and Oncogenic Signal Pathways Preceding Tumor Formation in a Murine Model of T-Cell Lymphoma in Skin 
Xuesong Wu, Ryan E. Sells, Sam T. Hwang 
Journal of Investigative Dermatology 
Volume 131, Issue 8, Pages (August 2011)
DOI: /jid
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
MBL2 ear skin tumors form in immunodeficient but not immunocompetent mice. (a) MBL2 cells were injected (4 × 105 cells per ear) into the ear skin of C57BL/6 or SCID/Beige mice (5 mice per group; experiment was repeated >5 times). Inoculated ears were observed twice weekly for 1 month. Representative pictures of C57BL/6 (left) and SCID/Beige (right) mouse ears at 21 days after MBL2 cell inoculation are shown. (b) Kinetics of MBL2 tumor growth in mouse ears. At different time points (three mice per time point), the ears were processed to determine the luciferase level (arbitrary relative light unit (RLU)).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
DNFB promotes the formation of tumors in C57BL/6 mice. (a) DNFB (or vehicle) was applied only once after MBL2 cell inoculation. Ears were examined after 48hours or 2 weeks later. Bar=100μm. (b) Ear thickness of mice following vehicle, DNFB, and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment was measured 2 weeks after tumor inoculation. (c) Clobetasol propionate cream (0.05%) treatment was given for 5 consecutive days, either just following MBL2 cell inoculation or 10 days later. Mice were killed 2 weeks after tumor cell inoculation with measurement of ear thickness (graph) and documentation of tumor histology by hematoxylin and eosin (H&E) staining (Supplementary Figure S2 online).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
DNFB promotes MBL2 cell survival through the changes of apoptosis and proliferation. (a) DNFB or vehicle was topically applied to ears injected with MBL2 cells that were labeled in vitro with CellTracker CMRA before ear skin inoculation (A). Single-cell suspensions were created from pooled ear skins (n=4 per group) at 48hours for analysis by flow cytometry (B). Based on gating in B, percentages of apoptotic (Annexin V+/7-AAD–), dead (Annexin V+/7-AAD+), and live cells (Annexin V-/7-AAD–) in CMRA-labeled tumor cells are shown graphically (C). (b) Detection of proliferating cell nuclear antigen (PCNA) by western blot at indicated time points from extracts from vehicle-treated (lanes 1 and 3) and DNFB-treated (lanes 2 and 4) MBL2 (lanes 1 and 2) and MBL2-Luc (lanes 3 and 4) inoculated ears.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Inflammatory infiltrates in DNFB-induced tumor microenvironment are predominantly composed of neutrophils and macrophages. (a) Topical 0.5% DNFB (10μl) or vehicle (10μl) was applied once over the injection site of C57BL/6 ear skin after CMRA-labeled MBL2 cell inoculation. After 2 days, single-cell suspensions were stained for inflammatory markers and analyzed by flow cytometry. Infiltrating leukocytes were identified by CD45 exclusively of CMRA+ tumor cells. Infiltrating leukocytes were analyzed for the expression of Gr-1 (neutrophil), F4/80 (macrophage), and CD3 (T lymphocytes). (b) SCID/Beige mice were inoculated with MBL2 cells and treated with DNFB or vehicle (see Materials and Methods). Ear thickness (graph, n=5) was used to assess tumor growth shown in the graph. Histology of MBL2-inoculated SCID/Beige ears by hematoxylin and eosin (H&E) staining. Bar=200μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Depletion of macrophages, but not neutrophils, blocks the formation of tumors in DNFB-treated C57BL/6 mice. (a) Following neutrophil or macrophage depletion (see Materials and Methods), MBL2-free mice were treated with DNFB (see Figure 2) and subsequently analyzed for effectiveness of the depletions at day 3. Ear cell suspensions were analyzed with anti-Gr-1 and anti-F4/80 mAb. (b) Following 2 days of anti-Gr-1 or clodronate treatment, mouse ears were inoculated with MBL2 cells and subsequently treated with DNFB. At week 2, tumors were assessed by ear thickness and histology (Supplementary Figure S3 online). (c) Identification of macrophages with anti-CD163 staining in representative plaque-stage mycosis fungoides (MF; upper) and tumor-stage MF (lower) biopsies (red: anti-CD3; green: anti-CD163; white dotted line marks epidermal basement membrane; bar=100μm). PBS, phosphate-buffered saline.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
DNFB treatment activates signal transducer and activator of transcription 3 (STAT3), NF-κB, and IL-1β signaling pathways. (a) MBL2 (lanes 1 and 2) and MBL2-Luc (lanes 3 and 4) cells were inoculated into C57BL/6 ear skin and treated with DNFB or vehicle. After 2 days, whole-ear extracts were prepared for the detection of the indicated (phospho-) proteins by western blot. Lanes 1 and 3, extracts from vehicle-treated, tumor cell-inoculated ear skin; lanes 2 and 4, extracts from DNFB-treated, tumor cell-inoculated ear skin. (b) Recombinant IL-1β (50ng per mouse) was co-injected with MBL2 cells subcutaneously (s.c.) in flank skin of mice. Tumor growth was assessed 2 weeks after inoculation with MBL2 cells. Two control groups included mice injected with MBL2 cells only and those treated with topical DNFB after inoculation (2–6 mice per group).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions