Fig. 1: EGFP-actin incorporation pattern during stereocilia widening.

a Diagrams of inner hair cell (IHC) stereocilia, oriented en face in a 3D view and as a side view. b A single P6 IHC imaged at timepoints (from 2 to 18 h) after transfection with EGFP-actin. Left panels are 3D reconstructions oriented en face except for the final image, which is a top-down view (scale bar represents 5 μm). Right panels are side views of stereocilia made from x-z reslices (scale bar represents 1 μm). The outline of a row 1 stereocilium is traced by yellow dashed lines and the cuticular plate is denoted by blue dashed lines. The lowest panel is an x-y slice showing stereocilia in cross-section mid-way down row 1 stereocilia. The inset (1 ×1 μm), a magnified region marked by a red box, demonstrates peripheral EGFP-actin localization around row 1 stereocilia. c P5 IHC 18 h post EGFP-actin transfection imaged by expansion microscopy, stained with antibodies against β-actin (ACTB) (magenta) and EGFP (green). Panels show a 3D reconstruction (Scale bar represents 10 μm), a x-z reslice through the center of stereocilia, and a top-down view of x-y slice (Scale bars represent 2 μm). Yellow and blue arrows indicate row 1 and row 2, respectively. d Lattice SIM images of EGFP-actin or EGFP-FSCN2 (green and grey) from P5 IHCs 18 h post transfection with phalloidin-stained F-actin (magenta). Scale bar represents 5 μm. b–d The experiments were repeated three times with similar results.