Figure 1.
Annexin V and PCI binding to Jurkat cells.
Untreated and Ct (6µM Camptothecin 24 h, in full RPMI medium at 37°C) Jurkat cells were incubated with 75 nM Annexin V-EGFP and/or 250 nM PCI-Cy3 for 1 h, RT, in Annexin V binding buffer, containing 1.8 mM Ca2+ or in PBS (Ca2+ free) and analysed by flow cytometry. Data (A) show representative dot plots from experiments performed in presence of calcium, analysed by FCS Express 4 Flow Cytometry Software (De Novo Software), bar graphs (B) represent quantification of Annexin V-, PCI- and double positive cells in Ca2+ containing (open bars) and Ca2+-free conditions (filled bars) of two independent experiments showing means and range.
Figure 2.
Confocal microscopy of PCI binding to untreated and Ct cells.
Jurkat cells (A) and U937 cells (B) were incubated with 250 nM PCI-Cy3 (red) and in some experiments also with 75 nM Annexin V-FITC (Aii, Bi, green). Nuclear staining was done with Hoechst (blue). Confocal microscopy pictures show Ct Jurkat cells (Ai) and untreated Jurkat cells (Aii) in two different magnifications (40×, n.a. 1.3 oil objective; 100×, n.a. 1.3 oil objective). Data in (B) show z-stack galleries of 1µM slice thickness representing a Ct U937 cell (Bi) and an untreated U937 cell (Bii) (63×, n.a 1.4 oil objective). The scale bars represents a length of 5µm. Picture processing was done in LSM Image browser software (Carl Zeiss).
Figure 3.
PCI binding to U937 cells is differentiation dependent.
U937 cells were differentiated with 20–72 h. Panel (A): Expression of macrophage surface markers CD14 and CD11b indicating time dependent differentiation. At 0, 24 and 72 h of differentiation U937 cells were incubated with 75 nM Annexin V-FITC and/or 100 nM PCI-Cy3 for 1 h, RT, in Hepes buffer with 3% BSA±2.5 mM Ca2+. Time course data in (B) represent PCI and Annexin V binding of two independent experiments showing means and range. U937 cells, treated with PCI-Cy3 (red) and Annexin V-FITC (green) in presence of Ca2+ were analysed by confocal microscopy. Pictures (C) show 72 h differentiated U937 cells. Nuclear staining was done with Hoechst (blue). The scale bar represents a length of 5µm (63×, n.a. 1.4 oil objective).
Figure 4.
Colocalization of PCI-Cy3 and Annexin V-FITC on Ct-Jurkat cells.
Ct treated Jurkat cells were incubated with 250-Cy 3 and 75 nM Annexin V-FITC and analysed for FRET signal. Calculations and corrected pictures (fire) were processed with ImageJ software using the PixFRET software as described in “Materials and Methods”, signal intensity is indicated by the color spectrum displaying intense signals in yellow. The figure shows representative pictures of two independent experiments. The scale bar shows the length of 5µm (40×, n.a. 1.3 oil objective).
Figure 5.
Localisation and effect of PCI in efferocytosis.
Flow cytometry dot plot graphs of the phagocytosis model using 24-SE and Ct Jurkat cells stained with CFDA-SE. Double positive cells represent macrophages containing engulfed apoptotic cells (A). Bar graphs show the quantifications (as described in “Materials and Methods”) of the phagocytosis assay, after substraction of 4°C control (surface bound apoptotic cells). Jurkat cells were preincubated with the indicated concentrations of protein for 1 h at 37°C, without washing protein away, Jurkat cells were added to the U937 cells and incubated for 24 h on 37°C or 4°C. The graph represents means and SEM of three independent experiments each performed in duplicates. Statistics was evaluated by one-way ANOVA and as post test the Dunnett's multiple comparison. * = P<0.05 (B). For the results in (C) 24 h and 72 h differentiated U937 cells were used and either the macrophages or the Ct Jurkat cells were preincubated with proteins. After washing, phagocytosis was performed for 24 h at 37°C or 4°C. Data represent means and SEM after substraction of 4°C of four independent experiments, each performed in duplicates. Fluorescence microscopy pictures show untreated Jurkat cells (Di) and Ct Jurkat cells (Dii) both stained for beta3 integrin and secondary Alexa 488 antibody (green) incubated with 50 nM PCI-Cy3 (red) for 2 h. The third picture (Diii) represents 24 h PMA-differentiated THP-1 macrophages, stained with the macrophage antibody 25F9-FITC (green) and incubated with Ct Jurkat cells and PCI-Cy3 simultaneously for 2 h at RT, as described in “Materials and Methods”. Nuclear staining was done with Hoechst (blue), 10×, n.a. 0.4; 20×, n.a.0.4; 40×, n.a. 0.8. Arrows indicate apoptotic particles, covered with PCI-Cy3.
Figure 6.
Phagocytosis of untreated and activated platelets.
The graphs (A) represent phagocytosis and (B) shows surface binding of untreated and ADP (20µM) activated platelets by human blood derived monocytes with or without 300 nM PCI. Platelets were stained with CMFDA, activated with ADP and phagocytosis was conducted with monocytes for 60 minutes. Surface bound platelets, detected by CD61-Alexa647 were substracted from the total monocyte associated platelets to gain the count of phagocytosis. The bar graphs represent mean and standard deviation of 5 experiments, each performed in duplicates. Statistical significance was calculated with the two tailed paired Student's T-test,* = P<0.05.
Figure 7.
Surface markers of activated platelets.
The graphs show the influence of PCI on platelet activation. Platelets were incubated with 300-FITC to detect PS surface exposure (n = 15), PAC-1 antibody to determine GPIIb/IIIa activation (n = 10) and anti-CD62P-Alexa647 for surface expression of P-selectin (n = 10). Bar graphs represent mean and standard deviation. Statistical significance was calculated with the two tailed paired Student's T-test, * = P<0.05.
Figure 8.
Phagocytosis inhibition of PCI binding microspheres.
The graphs represent phagocytosis of microspheres by U937 macrophages. Either the microspheres or the macrophages were preincubated with buffer (control), 100 nM PCI or 100 nM AT3. After washing away unbound protein coculture was performed for indicated time periods at 37°C or 4°C. The phagocytosis index is calculated by the multiplication of the percent phagocytosing macrophages with the mean fluorescent intensity of ingested microspheres. The 4°C values were substracted and timelines were normalized to the control showing mean and SEM of three independent experiments, each performed in duplicates. Statistical significance was evaluated by one way ANOVA and the Dunnett's multiple comparison test. * = P<0.05.
Figure 9.
Upregulation of PCI expression during U937 macrophage differentiation.
The bar graph (A) of RT PCR data shows PCI expression during differentiation of U937 cells treated with 20 nM PMA for indicated time points. The expression rate was normalized to GAPDH house-keeping gene. Data represent means and range of 2 experiments. Statistics was evaluated by one-way ANOVA and as post test the Dunnett's multiple comparison. * = P<0.05. Graph (B) shows PCI antibody binding to U937 cells during differentiation. Data represent antibody binding after substraction of the proper isotype control to the surface of PMA treated cells (open triangles) or treated with DMSO control (filled triangles) of two independent experiments, showing means and range.