Additional file 1: Figure S1. of Mitosis in circulating tumor cells stratifies highly aggressive breast carcinomas

Common recognizable Cytologies of CTCs in Mitosis isolated from breast cancer patients with all the “standard” CTC stains from Fig. 1. Figure S2. Common recognizable Cytologies of CTCs in Mitosis isolated from breast cancer patients with all the “standard” CTC stains from Fig. 1. Figure S3. Kaplan-Meier estimates of probabilities of Overall Survival of the patient subpopulations based on receptor status from Fig. 2a (n=33). Figure S4. Box plot of total number of CTCs in each patient versus mitotic CTCs for each patient. Figure S5. CTC counts and Mitotic CTC counts for each patient sample in relation to time of filtration after blood draw. Table S1. Patient subpopulations classified by stage, receptor status and treatment. (PDF 748 kb

Immunogenicity of LukS-Mut9 with different adjuvants in mice.

<p><b><i>A</i></b>) Total Ab titers determined by ELISA for individual mouse sera (EC50; i.e. dilution of serum with 50% maximal signal on ELISA plates coated with wild type LukS-PV). <b><i>B</i></b>) Neutralization determined in HL-60 toxin neutralization assay using wild type LukS-PV and LukF-PV toxins. Percent neutralization of wild type toxin is shown at 1∶100 dilution of serum from vaccinated mice (sera pooled from 5 mice in each group). Doses used: antigens: 10 ug; Al(OH)3∶34 µg, AlPO4∶70 µg, GLA-SE: 20 ug, and CpG: 10 µg/mouse.</p

Imunogenicity and protective efficacy of LukS-Mut9 and LukF-Mut1.

<p><b><i>A)</i></b> Antibody titers of mice immunized with lukS-Mut9 or LukF-Mut1 towards the homologous wild type antigens or the combination of both mutants towards each antigen. <b><i>B)</i></b> Protection from lethal challenge by active immunization with LukS-Mut9 and LukF-Mut1 along with GLA-SE in USA300 bacteremia model.</p

Inhibition of oligomerization by anti LukS-PV polyclonal antibody.

<p><b><i>A)</i></b> Lane1: Marker; lanes 2–8:, 30 µg/ml of LukS-PV was incubated for 30 min at RT with anti-LukS-PV rabbit polyclonal antibodies (pAbs) at 2-fold decreasing concentrations (1.1 mg/ml to 0.017 mg/ml) and then equal concentration of LukF-PV subunit was added; lane 9: LukS+LukF without pAbs; lane10: LukS+LukF+pAbs without MPD; lane 11: pAbs+MPD only. <b><i>B)</i></b> Inhibition of oligomeric band formed by LukS-PV+hlgB by anti-LukS-PV pAb. Lane1: Marker; lanes 2–8∶30 ug/ml of LukS-PV was incubated for 30 min at RT with anti-LukS-PV rabbit polyclonal antibodies (pAbs) at 2-fold decreasing concentrations (1.1 mg/ml to 0.017 mg/ml) then equal concentration of HlgB subunit was added; lane 9: LukS-PV+HlgB without pAbs; lane10: LukS-PV+HlgB+pAbs without MPD; lanes 11–14 LukS-PV+HlgB along with naïve rabbit IgG (1.1 mg/ml to 0.14 mg/ml), and lane 15: rabbit anti-LukS-PV pAbs +MPD only.</p

SDS-PAGE and Western blot of mutant and wild type forms of LukS-PV and LukF-PV.

<p><b><i>A)</i></b> LukS-PV wt and mutants 2–9, <b><i>B)</i></b> LukF-PV wt and mutants 1–3. Upper panels: coomassie staining; Lower panels: Western blot.</p

LukS-Mut9 antisera cross neutralize PMN lytic activity induced by other leukocidins.

<p>Inhibition by LukS-Mut 9 immunized sera of PMN lytic activity induced by <i>S. aureus</i> strain 8325-4 (PVL-neg) supernatant (<b><i>A</i></b>), USA300 (PVL-pos) Supernatant (<b><i>B</i></b>), purified PVL (S+F subunits) (<b><i>C</i></b>), and purified Hlg (B+C subunits) (<b><i>D</i></b>).</p

LukS-Mut9 induces cross reactive antibodies.

<p>Serum samples after 4 immunizations with LukS-Mut9 were tested for ELISA titers against WT LukS-PV (<b><i>A</i></b>) and HlgC (<b><i>B</i></b>).</p

Passive immunization with rabbit anti-LukS-PV antibodies protects against lethal challenge with USA 300. Fractions indicate survivors/total number of mice.

<p>Passive immunization with rabbit anti-LukS-PV antibodies protects against lethal challenge with USA 300. Fractions indicate survivors/total number of mice.</p

Homologous and heterologous oligomerization of wt and mutant LukS-PV and LukF-PV.

<p>Individual subunits as indicated above the panel were co-incubated overnight at a concentration of 30 µg/ml with 40%MPD and analyzed by SDS-PAGE without boiling. Lane 1: molecular weight Marker; Lanes 2–5: different combinations of wild type (wt) or mutant leukocidins as shown above the panel.</p

List of LukS-PV and LukF-PV mutants showing increased molecular energy in the heterodimer model.

<p>List of LukS-PV and LukF-PV mutants showing increased molecular energy in the heterodimer model.</p