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. 2018 Dec 21;12(3):462–474. doi: 10.1016/j.tranon.2018.11.014

Figure 4.

Figure 4

L-plastin induces [Ca2+]i oscillations and sustained NFATc1 translocation in osteoclast precursors. (A-B) Fura2-loaded RANKL-primed RAW 264.7 cells were imaged at baseline, 50 μg/ml recombinant L-plastin (rLPL) or same amount of media, vehicle (Veh) was added to control for mechanical perturbations, and changes in [Ca2+]i were imaged for additional 60-120 seconds. (A) Top: representative [Ca2+]i recordings (top, 10- second bar indicates time scale) in seven individual cells per experimental condition. Bottom: For each cell, the average variation in [Ca2+]i, measured as a standard deviation of [Ca2+]i, levels was determined and presented in ascending order. (B) Average [Ca2+]i (top) and variation (standard deviations of [Ca2+]i level, bottom). Data are means ± SEM, N = 33-63 cells/condition, from four independent experiments, **P < .01, ***P < .001 compared to baseline, assessed by ANOVA and post hoc Bonferroni test. (C) NFATc1 localization was assessed by immunofluorescence (green); nuclei were stained with DAPI (blue). RAW264.7 cells cultured for 2 days untreated (−) or primed with RANKL (50 ng/ml). The cells were washed and incubated for 2 hours in the fresh medium without RANKL (2), with RANKL (50 ng/ml, 2+), with L-plastin (rLPL, 10 μg/ml), or with L-plastin (10 μg/ml) following pretreatment with 25 μM BAPTA-AM (rLPL+BA). Left: representative images, 10-μm scale bar applies to all images. Right: average percentage of cells with nuclear NFATc1 from total number of cells scored (>50 cells per condition). Data are means ± SEM, N = 2-4 independent experiments, *P < .05 compared to NC, assessed by ANOVA and post hoc Bonferroni test, #P < .05 for compared to rLPL vs. rLPL+BAPTA by Student's t test. (D) RAW 264.7 cells were cultured untreated or primed with RANKL (50 ng/ml) for 2 days and then cultured for additional 2 days without treatment, with RANKL (50 ng/ml), or pretreated with 25 μM of BAPTA or its vehicle and treated with L-plastin (rLPL). Average numbers of osteoclasts formed in indicated conditions (numbers 2 and 5 indicate for how many days cells were exposed to RANKL). Data are means ± SEM, N = 3-6 independent experiments, **P < .01, compared to NC, #P < .05 compared to rLPL by ANOVA.