Fig. 7: Hsp90 is involved in NudCL2-meidated myosin-9 stability and cell migration.

A549 cells transfected with or without the indicated siRNAs and vectors were treated with 3.78 µM geldanamycin (GA) or DMSO for 48 h and subjected to the following analyses: a A549 cells were harvested and lysed. Immunoprecipitation analyses were carried out using the indicated antibodies. Three percent of the total input is shown. b Western blotting analysis of the expression of myosin-9, LIS1, and Hsp90. β-actin was used as a loading control. c Relative protein levels compared with the control at the same time point of GA treatment in (b) were measured using ImageJ software and shown. d–f Cells were fixed and stained with phalloidin (red) and anti-paxillin (green) antibody. DNA was visualized with DAPI (blue). Scale bar, 20 μm. Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. g, h Transwell migration assays revealed cell motility. Scale bar, 200 μm. Cells that migrated to the undersides of the filters were counted. i Western blotting analysis of the expression of myosin-9, LIS1, Myc-Hsp90, and NudCL2. β-actin was used as a loading control. j ImageJ software was used to quantify protein levels in (i). The relative amounts of myosin-9 and LIS1 compared with the control were calculated and shown. k, l Cells were stained as described in (d). Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. m Western blotting analysis of the expression of myosin-9, LIS1, Hsp90, and Myc-NudCL2. β-actin was used as a loading control. n ImageJ software was used to quantify protein levels in (m). The relative amounts of myosin-9 and LIS1 compared with the control were calculated and shown. o, p Cells were stained as described in (d). Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. q A549 cells transfected with the indicated siRNAs were treated with GA and subjected to western blotting analysis of the expression of myosin-9, LIS1, Hsp90, and NudCL2. β-actin was used as a loading control. r ImageJ software was used to quantify protein levels in (q). The relative amounts of myosin-9 and LIS1 compared with the control were calculated and shown. s, t Cells were stained as described in (d). Cells with lamellipodia were counted, and the number of cellular focal adhesions was plotted. Quantitative data derived from at least three independent experiments are shown as the mean ± SD. More than 100 cells were counted in each experiment. n, sample size. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant (P > 0.05). Student’s t-test. u Working model for the role of NudCL2 in cell migration. NudCL2 stabilizes myosin-9 and LIS1 proteins by Hsp90 and plays an important role in the precise regulation of cell migration. Depletion of NudCL2 leads to myosin-9 and LIS1 degradation and increases single-cell migration but not collective cell migration.