Application of C2IN-C3lim in the early stage of differentiation inhibits osteoclast-formation.

<p>C2IN-C3lim (0.5 and 2µg/mL) was added from day 0 on (A), from day 1 on (B), from day 2 on (C) or at day 0 only with subsequent medium change on day 1 (D). The number of multi-nucleated (at least three nuclei) and TRAP-positive osteoclasts per well (96 well plate) were determined at day 5. </p

Specific and selective uptake of C2IN-C3lim into macrophage-like RAW 264.7 cells.

<p><i>A</i>. ADP-ribosylation status of Rho in RAW 264.7 cells treated with C2IN-C3lim. Cells were incubated with 0.5 or 2 µg/mL of C2IN-C3lim or left untreated for control. The cells were lysed after 6 and 24 h and equal amounts of lysate proteins incubated with fresh C3bot1 and biotin-labelled NAD⁺. The biotinylated, i.e. ADP-ribosylated Rho is shown. Equal amounts of loaded protein were confirmed by Ponceau S staining of the blotted proteins (not shown). <i>B</i>. C2I alone is not taken up into RAW 264.7 cells. Cells were incubated with C2I (2 µg/mL) alone or with C2I (0.4 µg/mL) + C2IIa (0.8 µg/mL) or left untreated for control. After 6 h cells were lysed and equal amounts of lysate proteins incubated with fresh C2I and biotin-labelled NAD⁺. The biotinylated, i.e. ADP-ribosylated actin is shown. Equal amounts of loaded protein were confirmed by Ponceau S staining of the blotted proteins (not shown). <i>C</i>. C2IN-C3lim is not taken up into pre-osteoblastic MC3T3 cells under comparable conditions. Cells were incubated with C2IN-C3lim (5 µg/mL) or with C2IN-C3lim (1 µg/mL) + C2IIa (2 µg/mL) or left untreated for control. After 6 h the cells were lysed and the ADP-ribosylation status of Rho determined as described in A. The biotinylated, i.e. ADP-ribosylated Rho is shown. Equal amounts of loaded protein were confirmed by Ponceau S staining of the blotted proteins (not shown).</p

Effect of C2IN-C3lim on osteoclast-formation.

<p>C2IN-C3lim (0.5 and 2µg/mL) was added to RAW264.7 cells from day 0 on and cells were treated with RANKL. For control, cells were treated with RANKL in the absence of C2IN-C3lim. At day 5, osteoclasts were stained for tartrate-resistant acid phosphatase. Osteoclasts formed in the absence (A) and presence of increasing concentration of C2IN-C3lim (B: 0.5 µg/mL; C: 2 µg/mL) are shown.</p

Uptake of C2IN-C3lim into differentiating osteoclasts and morhophological changes caused by C2IN-C3lim.

<p>RAW264.7 cells which were grown in the presence of RANKL to induce differentiation to osteoclasts. Cells were left untreated for control (A) or treated with C2IN-C3lim (2 µg/mL) at day 0 and 2 (B and C). At day 5, osteoclasts were stained for actin (red), nuclei (blue) and C3 (green) and cells analyzed by phase contrast microscopy (left row) and fluorescence microscopy. </p

C3bot1E174Q-C2I ADP-ribosylates actin in the cytosol of intact J774A.1 and RAW264.7 macrophages.

<p><i>A.</i> J774A.1 cells were incubated with C3bot1E174Q-C2I (0.5 µg/mL, 2 µg/mL, 4 µg/mL), C3bot1E174Q-C2I+C2IIa (0.5 µg/mL+1 µg/mL, 2 µg/mL+4 µg/mL, 4 µg/mL+8 µg/mL), C2I alone (0.5 µg/mL, 2 µg/mL, 4 µg/mL) or left untreated. Cells were lysed and lysates incubated for 30 min at 37°C with C2I (300 ng) and biotin-labelled NAD⁺ (10 µM) to ADP-ribosylate actin, which was not ADP-ribosylated by the toxins in the intact cells. Samples were subjected to SDS-PAGE, blotted and biotinylated (i.e. ADP-ribosylated) actin was detected with streptavidin-peroxidase. Comparable amounts of total protein in the lanes were confirmed by Ponceau S staining (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054517#pone.0054517.s003" target="_blank">Figure S3</a>). <i>B.</i> Concentration-dependent intoxication of J774A.1 cells with C3bot1E174Q-C2I, C3bot1E174Q-C2I+C2IIa or C2I+C2IIa. Cells were incubated for 6 h with 0.1, 0.3, 1, 3 or 10 µg/mL of the respective or left untreated for control. Subsequently, cells were lysed and further treated as described in A. Intensity of the bands showing ADP-ribosylated actin was determined by densitometry and is given as percentage of actin from untreated control cells (mean±S.D.; n = 3). Comparable protein loading was confirmed by Ponceau S staining (not shown). <i>C.</i> Concentration-dependent intoxication of RAW264.7 cells with C3bot1E174Q-C2I. Cells were incubated for 6 h with 0.1, 0.3, 1, 3 or 10 µg/mL C3bot1E174Q-C2I or left untreated for control. Cells were lysed and treated as described in B. Intensity of ADP-ribosylated actin was determined by densitometry and is given as percentage of actin from untreated control cells (mean±S.D.; n = 3). Comparable protein loading was confirmed by Ponceau S staining (not shown). <i>D</i>. Effect of bafilomycin A1 on intoxication of J774A.1 cells. Cells were pre-treated with 300 nM bafilomycin A1 (Baf) at 37°C and after 30 min C3bot1E174Q-C2I (3 µg/mL) or for control C2I (200 ng/mL)+C2IIa (400 ng/mL) was added to the medium. For control cells were left untreated. Cells were incubated for further 6 h, lysed and further treated as described in A. Intensity of the bands showing ADP-ribosylated actin was determined by densitometry and is given as percentage of actin from untreated control cells (mean±S.D.; n = 3; * = p≤0.5). <i>E.</i> F-actin staining of RAW.264.7 cells treated with C2 toxin or C3bot1E174Q-C2I. Cells seeded in a 96 well plate were treated for 24 h with either C2 toxin as a control (400 ng/mL C2IIa+200 ng/mL C2I) or with C3bot1E174Q-C2I (4 µg/mL) or left untreated. Cells were fixed, permeabilized and F-actin was stained with phalloidin-Alexa-591 and fluorescence detected at 513 nm with an ELISA reader (mean±S.D.; n = 3; * = p≤0.5, *** = p≤0.005).</p

Effect of C3bot1, C3lim and C2IN-C3lim on RAW 264.7 cells.

<p><i>A</i>. Effect of C3bot1, C3lim and C2IN-C3lim on the morphology of RAW 264.7 cells grown in 12 well plates. Each of the proteins were added in final concentrations of 0.2 µg/mL (= 4 nM C2IN-C3lim, 8 nM C3bot1 or C3lim), 0.6 µg/mL (= 12 nM C2IN-C3lim, 24 nM C3bot1 or C3lim), 2 µg/mL (= 40 nM C2IN-C3lim, 80 nM C3bot1 or C3lim), and 6 µg/mL (= 120 nM C2IN-C3lim, 240 nM C3bot1 or C3lim) into the medium and cells were incubated in the presence of the proteins and pictures were taken from the cells after 24 h. For control, cells were left untreated. The morphology of control cells and C2IN-C3lim-treated cells is shown in the left panel. The percentages of cells displaying the typical “C3-morphology” were calculated from six individual pictures (right panel). The values were given as mean ± S.D. (n = 6). <i>B</i>. Effect of C2IN-C3lim on the proliferation of RAW 264.7 cells. Cells were incubated in 96 well plates with 0.6 and 2 µg/mL C2IN-C3lim or left untreated for control. After 24, 48 and 72 h pictures from the cells were taken and the total number of cells determined from 3 different pictures (left panel). The values were given as mean ± S.D. (n = 3). Significance was determined by student’s t-test for cells treated with the respective C3 protein against untreated (*** = p<0.001; ** = p<0.01; * = p<0. 1; n.s. = not significant). Alternatively, RAW 264.7 cells grown in 96 well plates were incubated with 0.6 and 2 µg/mL C2IN-C3lim or left untreated for control. After 24, 48 and 72 h the amount of viable cells was determined by MTT test (mid panel). The values are given in percent from control cells as mean ± S.D. (n = 3). Right panel: The values for 72 h C2IN-C3lim are given in percent from the values after 24 h C2IN-C3lim-treatment with the respective concentration of C2IN-C3lim. <i>C</i>. Effect of C3bot1E174Q on the morphology of RAW 264.7 cells. RAW 264.7 cells grown in 96 well plates were incubated for up to 72 h with 20 µg/mL (= 800 nM) C3bot1E174Q or left untreated for control. Pictures from the cells were taken and the percentages of cells displaying “C3-morphology” calculated from three individual pictures. The values were given as mean ± S.D. (n = 3). Significance was determined by student’s t-test for cells treated with C3bot1E174Q against untreated (n.s. = not significant).</p

Immunofluorescence microscopy of C3bot1E174Q-C2I-treated J774A.1 and epithelial cells.

<p><i>A.</i> J774A.1 cells grown on coverslips were incubated at 37°C with C3bot1E174Q-C2I (4 µg/mL), with C3bot1E174Q (4 µg/mL) or left untreated. <i>B.</i> Epithelial Vero and HeLa cells were incubated at 37°C with C3bot1E174Q-C2I (4 µg/mL) or left untreated. After 6 h, cells were fixed, permeabilized and stained with an antibody against C3bot and a secondary antibody coupled to Alexa 488 (green). The actin filaments were visualized using phalloidin-Alexa 594 (red) and nuclei were stained with Hoechst (blue). The cells were embedded in ProLong Gold antifade and analyzed by immunofluorescence microscopy.</p

Effect of C3bot1E174Q-C2I on primary cultured macrophages.

<p>Monocytes from human blood were differentiated for 7 days. The resulting macrophages were treated for 6 h at 37°C with C3bot1E174Q-C2I (4 µg/mL) or for control with C2I+C2IIa (200+400 ng/mL) or left untreated. Cells were lysed and lysates incubated for 30 min at 37°C with C2I (300 ng) and biotin-labelled NAD⁺ (10 µM) to ADP-ribosylate unmodified actin. Comparable amounts of lysate protein were confirmed by SDS-PAGE and Coomassie staining (not shown). The biotinylated (i.e. ADP-ribosylated) actin was detected by Western blotting with streptavidin-peroxidase. <i>B.</i> Intensity of the bands showing ADP-ribosylated actin was determined by densitometry and is given as percentage of actin from untreated control cells (mean±S.D.; n = 3; * = p≤0.5, ** = p≤0.05). <i>B.</i> Comparable amounts of total protein in the lanes were confirmed by Ponceau S staining and anti-Hsp90 Western blotting. <i>B.</i> Morphology of the cells described in A after 6 h. <i>C.</i> HeLa cells were incubated with C3bot1E174Q-C2I (4 µg/mL)+C2IIa (8 µg/mL) or with C3bot1E174Q-C2I alone (4 µg/mL). For control cells were left untreated or were incubated with C2I alone (4 µg/mL). After 6 h of incubation at 37°C all cells were washed, incubated with an antibody against C2I for 5 min at 4°C to remove non-internalized C2I and C2I fusions, washed again and pictures were taken.</p

C3bot1E174Q-C2I ADP-ribosylates actin <i>in vitro</i>.

<p>Lysate from J774A.1 cells (20 µg) was either left untreated for control (con) or incubated for 10 min at 37°C with either C3bot1E174Q-C2I (0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 10 ng/mL) or C2I (0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 10 ng/mL) in the presence of biotin-NAD⁺ (10 µM). Samples were subjected to SDS-PAGE, blotted and biotinylated (i.e. ADP-ribosylated) actin was detected with streptavidin-peroxidase.</p

Confocal fluorescence microscopy of C3bot1E174Q-C2I-treated J774A.1 cells.

<p>J774A.1 cells grown on coverslips were incubated at 37°C with C3bot1E174Q-C2I (4 µg/mL) or left untreated as a negative control. After 3 h as well as 6 h cells were fixed, permeabilized and the toxin was stained with a primary antibody against C3bot and visualized using an Alexa 488-coupled antibody (green). The actin cytoskeleton was visualized using phalloidin-Alexa 594 (red). Slides were mounted with Fluoromount-G containing DAPI (blue) and analyzed by confocal microscopy.</p