Figure 4. Loss of leupaxin and reduced kindlin-3 expression result in increased podosomal paxillin phosphorylation and decreased podosome lifetime. (A) IF stainings of +/+ RAW cells and leupaxin−/− RAW cells for vinculin, (green), paxillin (red), and actin (white/blue in merge) Scale bar, 10 µm. (B) IF stainings of +/+ and leupaxin−/− RAW cells for vinculin (green), phospho-paxillin Y31 (red), and actin (white/blue in merge). Scale bar, 5 µm. (C) Quantification of vinculin, total paxillin, and phospho-paxillin Y31 recruitment to podosome clusters assessed by measuring fluorescence intensity (MFI) of confocal images. MFIs of +/+ cells were set to 1. In each independent experiment, five podosome regions in each of at least 10 cells were measured. n ≥ 6. (D) Cumulative distribution of podosome lifetime in +/+ and leupaxin−/− RAW cells. 10–30 podosomes were measured per cell. At least two cells were analyzed in each of eight independent experiments. (E) Control RAW cells and different clones of leupaxin−/− RAW cells were cotransfected with WT paxillin-Cherry or a non-phosphorylatable mutant paxillin-Cherry (paxillin 2YF) and LifeAct-GFP. Podosome lifetime was assessed and blotted as cumulative distribution. 10–30 podosomes were measured per cell. At least 2 cells were analyzed in each of 5 different dishes. (F) IF stainings of +/+ preosteoclasts untreated or treated with Na3VO4 and K3n/− preosteoclasts for phosphorylated paxillin (red). Vinculin (green) and actin (white/blue in merge) served as control stainings. Scale bar, 10 µm. (G) Quantification of the recruitment of vinculin, paxillin and phospho-paxillin Y31 to podosome clusters of +/+ preosteoclasts untreated or treated with Na3VO4 and K3n/− preosteoclasts by measuring MFI of confocal images. MFIs of untreated +/+ cells were set to 1. At least five podosome regions in each of at least 10 cells were measured per experiment. n ≥ 5. (H) Control and K3n/− preosteoclasts were transfected with LifeAct-GFP, left untreated or treated with Na3VO4, or cotransfected with WT paxillin-Cherry or a nonphosphorylatable mutant paxillin-Cherry (paxillin 2YF). The cells were imaged for 10 min with a 15-s time interval. Podosome lifetime was assessed and blotted as cumulative distribution. 10–30 podosomes were measured per cell. Two to five cells were analyzed per condition in each of six different dishes. Dotted white lines mark cell borders.
Figure 4.

Loss of leupaxin and reduced kindlin-3 expression result in increased podosomal paxillin phosphorylation and decreased podosome lifetime. (A) IF stainings of +/+ RAW cells and leupaxin−/− RAW cells for vinculin, (green), paxillin (red), and actin (white/blue in merge) Scale bar, 10 µm. (B) IF stainings of +/+ and leupaxin−/− RAW cells for vinculin (green), phospho-paxillin Y31 (red), and actin (white/blue in merge). Scale bar, 5 µm. (C) Quantification of vinculin, total paxillin, and phospho-paxillin Y31 recruitment to podosome clusters assessed by measuring fluorescence intensity (MFI) of confocal images. MFIs of +/+ cells were set to 1. In each independent experiment, five podosome regions in each of at least 10 cells were measured. n ≥ 6. (D) Cumulative distribution of podosome lifetime in +/+ and leupaxin−/− RAW cells. 10–30 podosomes were measured per cell. At least two cells were analyzed in each of eight independent experiments. (E) Control RAW cells and different clones of leupaxin−/− RAW cells were cotransfected with WT paxillin-Cherry or a non-phosphorylatable mutant paxillin-Cherry (paxillin 2YF) and LifeAct-GFP. Podosome lifetime was assessed and blotted as cumulative distribution. 10–30 podosomes were measured per cell. At least 2 cells were analyzed in each of 5 different dishes. (F) IF stainings of +/+ preosteoclasts untreated or treated with Na3VO4 and K3n/− preosteoclasts for phosphorylated paxillin (red). Vinculin (green) and actin (white/blue in merge) served as control stainings. Scale bar, 10 µm. (G) Quantification of the recruitment of vinculin, paxillin and phospho-paxillin Y31 to podosome clusters of +/+ preosteoclasts untreated or treated with Na3VO4 and K3n/− preosteoclasts by measuring MFI of confocal images. MFIs of untreated +/+ cells were set to 1. At least five podosome regions in each of at least 10 cells were measured per experiment. n ≥ 5. (H) Control and K3n/− preosteoclasts were transfected with LifeAct-GFP, left untreated or treated with Na3VO4, or cotransfected with WT paxillin-Cherry or a nonphosphorylatable mutant paxillin-Cherry (paxillin 2YF). The cells were imaged for 10 min with a 15-s time interval. Podosome lifetime was assessed and blotted as cumulative distribution. 10–30 podosomes were measured per cell. Two to five cells were analyzed per condition in each of six different dishes. Dotted white lines mark cell borders.

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