Kif6, but not Kif9, displays IFT-like movements along DMTs. Quantification results are presented as mean ± SD and subjected to unpaired two-tailed Student’s t test. (A) Experimental scheme. Cultured mEPCs were infected with adenovirus at day −1 to express GFP-tagged Ift81, Kif6, or Kif9 and live imaged during days 5–8 by using GI-SIM to record the motilities of these proteins in cilia. (B) Expression levels of GFP-tagged proteins in mEPCs harvested at day 7. Immunoblotting was performed using antibodies against GFP, Kif6, Kif9, and Ift81, respectively. The asterisk indicates the non-specific band closed to Ift81-GFP. (C–E) Motilities of Ift81-GFP (C), Kif6-GFP (D), and Kif9-GFP (E) puncta in motile cilia. Images were recorded at 25 frames per sec (fps). The trafficking trajectories of GFP-positive puncta projected using the first 200 frames from Videos 1, 2, and 3 were shown as projected images. Typical trajectories framed by dashed red lines in the projected images were magnified by 300% so that the cilia could be roughly outlined (dashed green lines). Corresponding representative image sequences of these cilia are presented, in which typical, clearly traceable GFP-positive puncta are denoted to show their anterograde (arrowheads) or retrograde (arrows) movements. The corresponding kymographs were also presented. (F and G) Quantification results of velocities and frequencies from the indicated numbers of particles (F) and cilia (G). Antero: Anterograde transport; Retro: Retrograde transport; s: second. Source data are available for this figure: SourceData F2.