Additional file 1: of Blockade of TGF-β/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

Figure S1. The molecular structure of HPH-15. (PDF 7 kb

Additional file 2: of Blockade of TGF-β/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

Figure S2. HPH-15 treatment did not affect the growing of the mice. The body weight changes of mice treated with vehicle or HPH-15 were assessed every 3 days. All values represent mean Âą SEM; n = 5 in each group. (PDF 6 kb

Additional file 4: of Blockade of TGF-β/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

Figure S4. The inverse proportional change of Ly6Chi macrophages and CD206+ M2 macrophages from the inflammation stage (day 7) to the fibrotic stage (day 21). The single-cell suspension obtained from the back skin of bleomycin-injected C57BL/6 mice on day 7 and day 21 was stained with the mAbs against CD45, CD11b, Ly6C, and CD206. Stained samples were analyzed using the FACSCanto II system. Data were analyzed using FlowJo software version 7. All values represent mean ± SEM. n = 3 at each time point. ** p ≤ 0.01. (PDF 49 kb

Additional file 3: of Blockade of TGF-β/Smad signaling by the small compound HPH-15 ameliorates experimental skin fibrosis

Figure S3. Every-other-day injection of bleomycin for 2 weeks induced significant skin fibrosis. Back skin of NaCl- or bleomycin-injected mice was harvested on day 14 and stained with H&E. Scale bar = 100 Îźm. n = 3 in each group. (PDF 80 kb

Specificity and epitope of anti-CD44 fully human mAb.

<p>(A) MV1 (left), MV5 (middle) and GV5 (right) were compared for the reactivity with HEK293F cells expressing human CD44R1-GFP (upper) or human CD44s-GFP (lower) by FCM. (B) Reactivity of antibodies against various GST-fused recombinant CD44 proteins (R1a and R1b; Δex5-v8-v9-v10-Δex16, v8, v9, v10 and ex5) fused to GST was determined by ELISA. Difference in the length of Δex5 and Δex16 between R1a and R1b recombinant proteins is described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029728#s3" target="_blank">Materials and Methods</a>”.</p

Fine specificity of a class-switched anti-CD44R1 fully human IgG mAb (GV5) revealed by FCM.

<p>GV5, Cetuximab and Trastuzumab were compared for the reactivity with HUVEC, NHEK and HCT116 by FCM (histograms) with an Accuri C6 flow cytometer (A). Reactivity of GV5 with various human cells was analyzed by FCM, and depicted as histograms (B) using a BD-LSR flow cytometer and as ΔMFI (C) using an Accuri C6 flow cytometer.</p

Internalization of CD44R1, CDC and ADCC by GV5 fully human mAb.

<p>(A) HSC3 cells were cultured with or without GV5 (10 µg/ml) for 12 h, and were immunostained with GV5 followed by FITC-conjugated donkey anti-human IgG (H+L). Cell-surface CD44R1 proteins in these cells were detected by FCM. Experiments were repeated three times, and similar results were obtained. (B) After HSC-3 cells and sera for complement and mAb were mixed and incubated in each well of U-bottomed 96-well plate for 1 h, DAPI was added to each well. Percentages of DAPI-stained cells (dead cells) were calculated by FCM. Experiments were repeated three times, and similar results were obtained. (C) HSC-3 cells (1.5×10⁵) were mixed with effector cells of splenocytes (3×10⁶ or 9×10⁶) from KNS nude mice, which were pre-cultured overnight with lL-2, with or without mAb, in each well of U-bottomed 96-well plate for 5 h, and PI was added to each well. Cytotoxicity was analyzed using an Accuri flow cytometer. <b>R1</b>, HSC3 human target cells; <b>R2</b>, mouse effector cells; <b>R3</b>, PI-stained dead cells in <b>R1</b>; <b>R4</b>, PI-unstained living cells in <b>R1</b>. (D) Cell death (%) of HSC-3 cells by mouse effector cells (E/T = 0, 20 or 60) with or without GV5 was plotted. E/T means effector to target ratio.</p

Immunoperoxidase staining of human xenografts developed in athymic mice with an anti-CD44R1 fully human IgG mAb (GV5).

<p>Tissue sections of human ME180 tumors developed in athymic mice were fixed with cold acetone, and were sequentially incubated with primary mAb, species-specific biotinylated anti-human IgG Fcγ, ABC reagent and substrate solution containing DAB and H₂O₂. Nuclei were stained with hematoxylin. Upper or lower panels respectively show low or high magnification of the stained tissues.</p

Therapeutic effect of anti-CD44R1 fully human mAb in the tumor neutralization model.

<p>Tumor neutralization model was adopted. ME180 human tumor cells (1.0×10⁶) with or without mAb (50 µg/site) in 200 µl of PBS were subcutaneously inoculated into the right dorsal flank of each animal. The size of each tumor formed was periodically measured, and tumor volume (mm³) was calculated by the formula 0.4×(length)×(width)². Results were analyzed statistically by two-way ANOVA tests with repeated measures.</p

ADCC activity of anti-CD44R1 fully human mAb against human tumor cells using human lymphocytes.

<p>HSC-3 cells were labeled with Calcein-AM, and cells (2×10⁵) were mixed with effector cells of human PBL (5×10⁵ or 2×10⁶), which were pre-cultured overnight with IL-2 with or without mAb in each well of U-bottomed 96-well plate for 7 h. Cytotoxicity was evaluated by the release of Calcein-AM by dead tumor cells into the medium, and results were automatically recorded using a Terascan VP microfluorocytometer at 1 h intervals for 7 h. Results were analyzed statistically by two-way ANOVA tests with repeated measures (A) and by two-sided Student's <i>t</i> tests (A and B). Spots and vertical bars respectively show means and standard errors.</p