Primer sequences used for quantitative RT-PCR.

<p>Primers were designed with the help of Beacon Designer software (Bio-Rad, Hercules, CA, USA) and provided by Invitrogen.</p

GalXM-induced apoptosis of BW5147 cells.

<p>(<b>A</b>) BW5147 and BW5147 (T200⁻) cells (both 1×10⁶/ml) were incubated for 18 h in the presence or absence (NS) of PHA (10 µg/ml) or GalXM (10 µg/ml). After incubation, cells were collected by cytospin and stained by Hemacolor. GalXM-treated cells exhibited altered morphology, surface blubs and nuclear fragmentation (arrows). N = cell nucleus; original magnification 40x. In selected experiments, cells were incubated for 18 h in the presence or absence (NS) of PHA (10 µg/ml), mAb to CD3 (1 µg/ml) or GalXM (10 µg/ml). After incubation, the percentage of cells undergoing apoptosis was evaluated by PI staining and analyzed using FACScan flow cytometry. Data are expressed as fold increase of percentage of apoptotic cells (<b>B</b>), or shown as FACScan histograms from one representative experiment out of seven with similar results (<b>C</b>). *, <i>p</i><0.05 (treated <i>vs</i> untreated, n = 7). Error bars denote s.e.m.</p

IL-17A production and caspase-3 activation.

<p>CD3⁺ T cells (5×10⁶/ml, A or 1×10⁶/ml, B and C) were activated for 30 min in the presence or absence (NS) of soluble anti-CD3 (3 µg/ml) and anti-CD28 (3 µg/ml) mAbs (A, B and C) or rhTGF-β1 (5 ng/ml) and rhIL-6 (20 ng/ml) in mAb to CD3 (B and C) or mAbs to CD3 plus CD28 (A) pre-coated 48-well culture plates, washed, and subsequently incubated for 72 h (A) or 18 h (B and C) in the presence or absence of GalXM (10 µg/ml). In A, after incubation, IL-17A levels were evaluated in culture supernatants by specific ELISA assay. *, <i>p</i><0.05 (Control and RA, n = 7; GalXM treated <i>vs</i> untreated cells). In B, after incubation, cells were labelled with PE anti-active caspase-3 and Alexa Fluor anti-IL-17A mAbs and analysed using FACScan flow cytometry. The percentage of double positive cells is shown as dot plots (B) or bar graph (C). *, <i>p</i><0.05 (Control and RA, n = 7; GalXM treated <i>vs</i> untreated cells). The results reported in the bar graphs are the mean ± SEM.</p

CDRs sequences.

<p>CDRs sequences.</p

Phospho-Akt activation and TNF-α production in PM stimulated with V_HCDR3.

<p>PM (3×10⁶/ml) were stimulated for 1 h in the presence or absence (NS) of V_HCDR3, LPS, NC or SP (all at 10 µg/ml). After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to pAkt and Akt; pAkt was normalized against Akt (<b>A</b>) *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 5). PM (5×10⁶/ml) were stimulated for 18 h as above described. After incubation, TNF-α level was evaluated in culture supernatants by specific ELISA assays. (<b>B</b>) *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 5).</p

TNF-α induced TLR-4 expression in PM stimulated with V_HCDR3.

<p>PM (5×10⁶/ml) were cultured for 1 h in the presence or absence (NS) of V_HCDR3, LPS or NC (all 10 µg/ml). After incubation, TNF-α level was evaluated in culture supernatants by specific ELISA assay (<b>A</b>). *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 7). PM (1×10⁶/ml) were cultured for 1 h with V_HCDR3, LPS or NC (all 10 µg/ml), in the presence or absence (NS) of mAb to TNF-α (0.5 µg/ml). After incubation, permeabilized cells were reacted with RPE-labelled mAb to TLR-4 and analyzed by FACScan flow cytometry. Values represent the percentage of positive cells (<b>B</b>) *, <i>P</i><0.05 (V_HCDR3 plus mAb to TNF-α treated <i>vs</i> V_HCDR3 treated cells, n = 7). PM (3×10⁶/ml) were cultured for 1 h as above described. After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to TLR-4 and actin. TLR-4 production was normalized against actin (<b>C</b>) *, <i>P</i><0.05 (V_HCDR3 plus mAb to TNF-α treated <i>vs</i> V_HCDR3 treated cells, n = 5). For testing the expression level of TLR-4 gene, PM (1×10⁶/ml) were cultured for 1 h as above described. After incubation, total RNA was isolated and analyzed for mRNA expression with RT-PCR. Transcript copy numbers were determined by qPCR using cDNA as a template. Copy numbers were normalized against the copy number of the GADPH gene (<b>D</b>). *, <i>P</i><0.05 (V_HCDR3 plus mAb to TNF-α treated <i>vs</i> V_HCDR3 treated cells, n = 5). Error bars, s.e.m.</p

Phospho-IkBα activation and TNF-α gene expression in PM stimulated with V_HCDR3.

<p>PM (3×10⁶/ml) were stimulated for 1 h in the presence or absence (NS) of wortmannin (4 nM), V_HCDR3, LPS or NC (all at 10 µg/ml). After incubation, cell lysates were subjected to Western blotting. Membranes were incubated with Abs to pIkBα and IkBα; pIkBα was normalized against IkBα. (<b>A</b>) *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 5); †, <i>P</i><0.05 (wortmannin-treated <i>vs</i> wortmannin-untreated cells, n = 5). For testing the expression level of TNF-α gene, PM (1×10⁶/ml) were cultured for 1, 6 and 18 h as above described. After incubation, total RNA was isolated and analyzed for mRNA expression with RT-PCR. Transcript copy numbers were determined by qPCR using cDNA as a template. Copy numbers were normalized against the copy number of the GADPH gene (<b>B</b>). *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 5). Error bars, s.e.m.</p

Segregation of CD45 GalXM-induced on BW5147 cells.

<p>Fluorescence microscopy analysis of BW5147 and BW5147 (T200⁻) cells incubated for 2 h in the presence or absence (NS) of GalXM (<b>A</b>) or GalXM-FLUOS (green) (<b>B</b>) (both 10 µg/ml). After incubation, cells were labelled with RPE mAb to CD45 (red) and then examined under fluorescent light microscopy in the presence of DAPI (blue). Note the CD45 segregation in BW5147 cells treated with GalXM (<b>A</b>). The colocalization of GalXM with CD45 and the receptor segregation on BW5147 cells was demonstrated in the merged image of panel B (<b>B</b>). Original magnification 100x.</p

Phospho-STAT3 activation and IL-17A production.

<p>PBL (5×10⁶/ml) were activated for 30 min with soluble anti-CD3 (3 µg/ml) and anti-CD28 (3 µg/ml) mAbs, washed, and subsequently incubated for 2 or 18 h in the presence or absence of GalXM (10 µg/ml) or FLLL31 (5 µmol/L). After incubation, cell lysates were analysed by Western blotting. Membranes were incubated with anti-pSTAT3 and anti-STAT3 Abs. Actin was used as an internal loading control (A). Normalization was shown in panel B. Culture supernatants were collected to test IL-17A levels by specific ELISA assay (C). *, <i>p</i><0.05 (Control and RA, n = 7; GalXM treated <i>vs</i> untreated cells). The results reported in the bar graph of panel C are the mean ± SEM.</p

TNF-α and IL-6 production by PM and PMN stimulated with human and/or mouse CDRs and mouse V_HCDR3 uptake by different cell populations.

<p>PM (<b>A</b>) or PM and PMN (<b>B</b>) (both 5×10⁶/ml) were cultured in the presence or absence (NS) of human and/or mouse CDRs, LPS, or NC (all 10 µg/ml) for 18 h. After incubation, TNF-α and IL-6 levels were evaluated in culture supernatants by specific ELISA assays. *, <i>P</i><0.05 (treated <i>vs</i> untreated cells, n = 7). DC, PM, PMN, and T cells (all 1×10⁶/ml) were incubated for 1 h in the presence or absence (NS) of b-V_HCDR3 or b-NC (both 10 µg/ml). After incubation, permeabilized cells were reacted with FITC-labelled mAb to biotin and analyzed by FACScan flow cytometry. Data are reported as the percentage of positive cells (<b>C</b>). *, <i>P</i><0.05 (b-V_HCDR3 treated <i>vs</i> untreated cells, n = 5). Error bars, s.e.m.</p