TNTs participate in the fusion of osteoclast precursors. (A) Human monocytes isolated from blood were differentiated into osteoclasts (hOC) and analyzed on days 3, 6, and 10. Representative super-resolution microscopy images: F-actin (phalloidin, white) and nuclei (DAPI, cyan). Scale bar, 20 µm. Image in Fig. 1 A (day 10, right) is reused in Fig. S3 B. (B) Same experiment as in A with osteoclasts derived from the murine HoxB8 immortalized cell line (mOC) on days 3, 5, and 7. (A and B) White arrowheads show TNTs and pink arrowheads show zipper-like structures. (C) Left panels: Super-resolution microscopy images of TNTs with colored-coded Z-stack of F-actin (phalloidin) staining of 3 day-hOC or 3 day-mOC from 0 µm (substrate, dark blue) to 15 µm (yellow). Scale bar, 20 µm. See Videos 1 and 2. Right panels: Quantification of the percentage of cells forming thick and thin TNTs in hOCs and mOCs after immunofluorescence analysis (see Materials and methods), from one representative differentiation out of 3. n > 250 cells per condition, means ± SEM are shown. (D) Representative immunofluorescence analysis showing thin (white arrowheads) and thick TNTs (orange arrowheads): F-actin (phalloidin, white), nuclei (DAPI, cyan), and microtubules (α-tubulin, orange). Scale bar, 10 µm. (E) Bright-field confocal images from a time-lapse movie of hOCs fusing from a TNT (hour:min). See also Videos 3, 4, and 5. Dashed green and red lines delineate the nuclei before cell fusion and dashed orange lines after fusion. Arrowhead shows a TNT-like protrusion. Scale bar, 10 µm.