Additional file 2: of Pericellular collagen I coating for enhanced homing and chondrogenic differentiation of mesenchymal stem cells in direct intra-articular injection

Figure S2. Ex-vivo adhesion of MSCs suspended in col. I solution. BLIS analysis of MSCs suspended in 10 ng/ml, 500 ng/ml, and 10μg/ml col. I solution on cartilage slices. MSCs pretransfected by luciferase and the total adhered MSC number calculated by luminescent intensity (n = 5 in each group). (JPG 86 kb

Additional file 1: of Pericellular collagen I coating for enhanced homing and chondrogenic differentiation of mesenchymal stem cells in direct intra-articular injection

Figure S1. Concentration of residual collagen I in MSC suspension after PCC. Concentration of col. I detected (n = 5) by Rat Collagen I ELISA Kit (LSBio, USA). (JPG 133 kb

Additional file 3: of Pericellular collagen I coating for enhanced homing and chondrogenic differentiation of mesenchymal stem cells in direct intra-articular injection

Figure S3. In-vivo homing suspended in col. I solution. a, b BLIS analysis of luminescent distribution within left knees of New Zealand rabbits at 2 days after DIAI of luciferase-labeled MSCs or MSCs suspended in 10 ng/ml col. I solution. Red frames indicate cartilage defect (a, n = 3 in each group). Luminescent intensity of total ROI radiance within cartilage defect (red frame) calculated to reveal homing of MSCs (b). a Sagittal frozen sections of cartilage lesion site, red particles are CM-DiI-labeled MSCs (scale bar, 400 μm). (TIF 1614 kb

Gene/protein expression analysis of <i>Cs</i>severin at different life cycle stages of <i>C. sinensis</i>.

<p>(A) Quantitative real-time PCR analysis. The transcription levels of <i>Cs</i>severin at life stages of adult worm, metacercaria and egg were analyzed by means of the 2^−ΔΔCt ratio, with <i>Cs</i> β-actin serving as the internal standard. *: <i>p</i><0.05, the transcription level of <i>Cs</i>severin in egg was statistically higher than that in adult worm and metacercaria. (B) Western blotting analysis. Thirty microgram of total proteins from each life cycle stage were subjected to SDS-PAGE and analyzed. Rat anti-r<i>Cs</i>severin serum was used as primary antibody at a dilution of 1∶100. The same dilution of pre-immune rat serum was used as a negative control, and no corresponding band was observed (not shown).</p

Immunolocalization of <i>Cs</i>severin in adult worm and metacercaria of <i>C. sinensis</i>.

<p>Rat anti-r<i>Cs</i>severin serum was used as primary antibody and red fluorescent Cy3-labeled goat anti-rat IgG as secondary antibody. Slides were observed under white light (pane A, C, E, G, I) or fluorescence microscope (pane B, D, F, H, J). No specific fluorescence was observed in pane B or H which was probed with serum from rat immunized with PBS as negative control. Intensive reddish-orange fluorescences were observed in vitellarium and intrauterine eggs of adult worm (pane D, F, ×50) and oral suck of metacercaria (pane J, ×200). Scattered fluorescences were detected in tegument of adult worm and metacercaria. V, vitellarium. O, oral sucker. T, tegument. E, eggs. W, cyst wall. P, pharynx.</p

Western blotting of r<i>Cs</i>severin.

<p>r<i>Cs</i>severin reacted with sera from naive rats (lane 1), anti-His tag monoclonal antibody (lane 2), sera from <i>C. sinensis</i>-infected rats (lane 3), sera from rats immunized with the <i>Cs</i>ESP (lane 4), sera from rats immunized with r<i>Cs</i>severin (lane 5). <i>Cs</i>ESPs reacted with sera from rats immunized with r<i>Cs</i>severin (lane 6).</p

Apoptosis inhibition of PLC cells with r<i>Cs</i>severin in a concentration-dependent manner.

<p>(A) Flow cytometry analysis of PLC cells treated with PBS, apoptosis inducer (positive control) and 10, 20, 40, and 80 µg/ml r<i>Cs</i>severin for 48 h after induction of spontaneous apoptosis. Representative dot plots of cell apoptosis were showed after AnnexinV/PI dual staining. Apoptotic rate was represented as a percentage of total cell populations. The proportion of dead cells (Annexin V−/PI+), live cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−) and late apoptotic/necrotic cells (Annexin V+/PI+) was respectively measured for comparison. (B) Flow cytometry analysis of L-02 cells with the same treatment. (C) Morphologic changes in apoptotic PLC cells. Following treatment with PBS (negative control) or 80 µg/ml r<i>Cs</i>severin for 48 h, apoptotic nuclei were condensed and brightly stained with Hoechst 33258 then nuclear morphology was photographed and visualized with a Leica DMI4000B (Magnification×400). Each experiment was performed in triplicate.</p

Interaction of r<i>Cs</i>severin with actin.

<p>(A) The bindings of r<i>Cs</i>severin to F-actin and its fragments were examined using gel overlay assay as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002606#s2" target="_blank">materials and methods</a>. In pane a, actin and its fragments were separated on 12.5% SDS-PAGE. Protein molecular weight markers (M), F-actin at 37°C for 1 h (lane 1), F-actin digested with 0.25% trypsin at 37°C for 1 h (lane 2), purified r<i>Cs</i>severin (lane 3), 0.25% trypsin at 37°C for 1 h (lane 4). In pane b, r<i>Cs</i>severin binding to F-actin and its fragments were examined using gel overlay assay. The membrane was incubated with r<i>Cs</i>severin then rat anti-r<i>Cs</i>severin serum. Protein molecular weight markers (M), F-actin fragments after digesting with 0.25% trypsin at 37°C for 1 h (lane 1), F-actin at 37°C for 1 h (lane 2), purified r<i>Cs</i>severin (lane 3). In pane c, the membrane was incubated with r<i>Cs</i>severin then rat anti-r<i>Cs</i>severin serum (lane 1), the membrane was incubated with BSA then rat anti-r<i>Cs</i>severin serum (lane 2), the membrane was incubated with r<i>Cs</i>severin then pre-immune rat serum (lane 3). (B) The bindings of r<i>Cs</i>severin to cytoskeletal actin filaments of PLC cells by immunocytochemistry. Pane a, cells were coated with r<i>Cs</i>severin before fixed with 4% paraformaldehyde and successively with rat anti-r<i>Cs</i>severin serum and mouse anti-F-actin monoclonal antibody, and then a mixture of FITC (green fluorescence) and Cy3 (red fluorescence)-labeled goat anti mouse/rat IgG. Pane b, cells were coated with r<i>Cs</i>severin after permeabilized with 0.3% Triton X-100 and successively with rat anti-r<i>Cs</i>severin serum and mouse anti-F-actin monoclonal antibody, and then a mixture of FITC (green fluorescence) and Cy3 (red fluorescence)-labeled goat anti mouse/rat IgG. Pane c, cells incubated with monoclonal anti-F-actin antibody without coated with r<i>Cs</i>severin as negative control. Pane d, cells were coated with r<i>Cs</i>severin before fixed with 4% paraformaldehyde, then incubated only with anti-r<i>Cs</i>severin serum after permeabilized with 0.3% Triton X-100 as another negative control. The images were taken under a LSM 710 Zeiss confocal microscope, with an oil immersion objetive (63×, 1.40 numerical aperture).</p

Far-UV CD spectra of r<i>Cs</i>severin in the absence and presence of Ca²⁺.

<p>Spectral changes of CD for PBS–r<i>Cs</i>severin solutions (1 µM in 10 mM) on the addition of the 1 µM calciumion (upper row) and 1 µM EDTA (lower row) conditions demonstrated r<i>Cs</i>severin could bind to Ca²⁺.</p

Effect of r<i>Cs</i>severin on mitochondrial membrane potential in PLC cells.

<p>(A) Following treatment with PBS (negative control) or 80 µg/ml r<i>Cs</i>severin for 48 h, representative dot plot showed the changed MMP by flow cytometry after the labeling of fluorescent probe with JC-1. Reduction of mitochondrial membrane potential was demonstrated by the change in JC-1-derived fluorescence from red (JC-1 aggregates, representing high potential) to green (JC-1 monomer, representing low potential). (B) The quantitative MMP from each group was marked by the intensity ratio of red fluorescence over green fluorescence by flow cytometry. The data are expressed as mean ± SD from three independent experiments. * means <i>p</i><0.05, there is statistic difference (ANOVA/Dunnett's T3 test) between 80 µg/ml r<i>Cs</i>severin group and PBS group (negative control). (C) Typical fluorescence photomicrograph of <i>in situ</i> JC-1 staining output by laser scan confocal microscopy (Magnification×100).</p