Figure 3. CMD exclusion of GFP-PODXL is dependent on NHERF1. (A) NHERF1 was knocked down in GFP-PODXL cells (NHERF1 shRNA) using a retroviral shRNA system. Control cells expressing GFP-PODXL (vector) were retrovirally transduced with a puromycin resistance plasmid without an shRNA sequence. Cell lysates were blotted with anti-NHERF1 and antiezrin antibodies. Molecular mass is indicated in kilodaltons. (B) Live imaging of cells 4 d after seeding revealed that control cells (vector) exclude GFP-PODXL from the CMD but NHERF1 shRNA cells do not. Exclusion was quantified as in Fig. 1 C, and the percentage of cells excluding GFP-PODXL from the CMD appears on each image. 148/230 control cells (vector) excluded GFP-PODXL from the CMD, whereas very few cells expressing NHERF1 shRNA (8/186) excluded GFP-PODXL. Images were collected with identical microscope settings to Fig. 1 C. (C) Knocking down NHERF1 allowed GFP-PODXL to enter primary cilia. Cells were imaged live 12 d after seeding on filters. (top row) Z stacks of the apical membrane of control (vector) or NHERF1 shRNA live cells expressing GFP-PODXL were projected into a single image. Images were collected with identical microscope settings to Fig. 1 C, but at lower digital zoom. (bottom row) Single confocal sections above the apical membrane were taken from the z stacks in the top row, and image brightness was increased. Visible cilia are labeled with arrowheads. (D) 12 d after seeding, NHERF1 shRNA and control cells (vector) were fixed and stained with phalloidin and antiacetylated tubulin. GFP-PODXL is found in the CMD of NHERF1 shRNA cells, but actin is still excluded from the CMD, and primary cilia appear to grow normally. (E) 100/214 control cells (vector) had a cilium detected by antiacetylated tubulin after fixation, whereas only 15/208 live cells had a GFP-PODXL–positive cilium. After NHERF1 knockdown, similar numbers of cilia could be detected using each method (74/194 fixed and 57/145 live). Images in B and D are projections of z stacks. Error bars represent standard deviation between experiments. Bars, 10 µm.
Figure 3.

CMD exclusion of GFP-PODXL is dependent on NHERF1. (A) NHERF1 was knocked down in GFP-PODXL cells (NHERF1 shRNA) using a retroviral shRNA system. Control cells expressing GFP-PODXL (vector) were retrovirally transduced with a puromycin resistance plasmid without an shRNA sequence. Cell lysates were blotted with anti-NHERF1 and antiezrin antibodies. Molecular mass is indicated in kilodaltons. (B) Live imaging of cells 4 d after seeding revealed that control cells (vector) exclude GFP-PODXL from the CMD but NHERF1 shRNA cells do not. Exclusion was quantified as in Fig. 1 C, and the percentage of cells excluding GFP-PODXL from the CMD appears on each image. 148/230 control cells (vector) excluded GFP-PODXL from the CMD, whereas very few cells expressing NHERF1 shRNA (8/186) excluded GFP-PODXL. Images were collected with identical microscope settings to Fig. 1 C. (C) Knocking down NHERF1 allowed GFP-PODXL to enter primary cilia. Cells were imaged live 12 d after seeding on filters. (top row) Z stacks of the apical membrane of control (vector) or NHERF1 shRNA live cells expressing GFP-PODXL were projected into a single image. Images were collected with identical microscope settings to Fig. 1 C, but at lower digital zoom. (bottom row) Single confocal sections above the apical membrane were taken from the z stacks in the top row, and image brightness was increased. Visible cilia are labeled with arrowheads. (D) 12 d after seeding, NHERF1 shRNA and control cells (vector) were fixed and stained with phalloidin and antiacetylated tubulin. GFP-PODXL is found in the CMD of NHERF1 shRNA cells, but actin is still excluded from the CMD, and primary cilia appear to grow normally. (E) 100/214 control cells (vector) had a cilium detected by antiacetylated tubulin after fixation, whereas only 15/208 live cells had a GFP-PODXL–positive cilium. After NHERF1 knockdown, similar numbers of cilia could be detected using each method (74/194 fixed and 57/145 live). Images in B and D are projections of z stacks. Error bars represent standard deviation between experiments. Bars, 10 µm.

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