Podosomes from LPL Nb-expressing cells are dynamically unstable.

<p>(A) Stable THP-1 cells constitutively expressing the F-actin binding peptide LifeAct-mCherry and EGFP-tagged LPL Nb5/9, GSN Nb11/13, CapG Nb4 or EGFP were differentiated into macrophages by addition of PMA for 3 days. A 2D reconstruction of podosome turnover in the cells was acquired by time-lapse confocal imaging of life cells for 20 minutes. Actin is shown in red and stable cells in green. The arrow marks the podosome of which the relative actin intensity profile, described in C and D, was determined. Bar: 10 µm. (B) Representation of the average lifetime of podosomes for the different constructs. The lifetime of 120–150 podosomes in 8–12 cells was determined per condition in 3 independent experiments. Error bars represent mean ± SEM. Unpaired t-tests were performed to observe statistical differences between cells expressing EGFP and cells expressing the nanobodies. Statistically significant differences are denoted. (C) Representation of the relative actin intensity profile of a podosome as a function of time for CapG Nb4 (solid line, black), EGFP (dashed line, black), GSN Nb11 (solid line, green), GSN Nb13 (dashed line, green), LPL Nb5 (dashed line, red) and LPL Nb9 (solid line, red). Relative actin intensity profiles were generated for 10–15 podosomes in 5–10 cells per condition. The chosen examples are representative of the generated profiles. (D) Boxplot of the actin intensities (shown in C) for the different constructs. Mann-Whitney Rank Sum tests were performed to observe statistical differences in the distribution of the intensities between cells expressing EGFP and cells expressing nanobodies (ns: non significant p>0.05; * p<0.05; ** p<0.01; *** p<0.001).