Alexa 633 coupled phalloidin

The following primary antibodies were used: rat anti-radixin (R21, gift from S. Tsukita, WB 1:50, ICC 1:50); rabbit anti-radixin (Sigma-Aldrich, #R3653, IHC 1:200); rabbit anti-radixin (Abcam, EP 1862Y, #ab52495, IHC 1:200); mouse anti-γ-adaptin (BD Biosciences, #610386; WB 1:5,000); mouse anti-ezrin (Abcam, #ab4069, clone 3C12, WB 1:1,000); mouse anti-neuN (Millipore, clone A60, #MAB377, IHC 1:1,00). The following secondary antibodies were used: peroxidase-conjugated donkey anti-rabbit (Dianova, Hamburg, Germany, #711-036-152, WB 1:10,000); peroxidase-conjugated donkey anti-rat (Dianova, #712-036-153, WB 1:10,000); peroxidase-conjugated donkey anti-mouse (Dianova, #715-036-151, WB 1:10,000); IRDye 800CW goat anti-rabbit (LI-COR, IgG, #926-32211, WB 1:10,000); IRDye 680RD goat anti-mouse (LI-COR, IgG, #926-68070, WB 1:10,000); Alexa-488 goat anti-mouse (Dianova, #115-545-146, IHC 1:500); Cy3 donkey anti-rabbit (Dianova, #711-166-152, IHC 1:500); Cy3 donkey anti-rat (Dianova, #712-166-150, IHC 1:500); Atto488-labelled FluoTag-X4 anti-rabbit nanobody (NanoTag, IgG, #N2404, IHC 1:200). Alexa-633-coupled phalloidin (Thermo Scientific, #A22284) or Tritc-coupled phalloidin (Tebu-bio, #PHDR1) was used to visualize actin-containing stereocilia. Diamidino-2-phenylindole (DAPI, Sigma, #D9542) was used to stain the nucleus.

Cells were seeded at 1 × 10⁴ cells on an 8-well slide chamber under differentiation conditions for 5 to 7 days. After fixation with 4% PFA/PBS for 10 min and permeabilization with 0.2% Triton X-100 for 1 min, cells were incubated with monoclonal mouse anti β-tubulin (Sigma) at 4 °C overnight. The structure of intercellular β-tubulin and F-actin was visualized using a Leica TCS SP8 confocal microscope by triple-labeling with Alexa 563-coupled donkey anti-mouse secondary antibody (Thermo Fisher, Waltham, MA), Alexa 633-coupled phalloidin (Thermo Fisher), and DAPI respectively. The number of filopodia per cell and cells with nuclear lobulation were counted in at least 50 cells for each cell type. For nuclear area and protrusion analysis, images were processed using Image Pro plus software (Media Cybernetics, Rockville, MD). Fluorescent line scans of β-tubulin and F-actin were performed using the Leica TCS SP8 software along the vertical line in protrusions for YFP and YFP-WT cells and marginal fibrillary structures for YFP-Y64C and YFP-G12V. Line scans were conducted for at least 20 cells in each cell type.