Fig. 4: Optimization of taccalonolide-fluorescein probes.

a Visualization of unprotected (6), acetyl-protected (5), and pivaloyl-protected (8) probes in live SK-OV-3 cells 5 h after the addition of 5 µM probe either before (left) or after (right) medium containing probe was removed and replaced by fresh medium. b Co-localization of 8 (green) with β-tubulin immunofluorescence (orange) in fixed HCC1937 and HeLa cells after 10 µM probe addition for 6 h. c, d Pivaloyl-protected taccalonolide-fluorescein probes with decreasing linker sizes (left to right) were added to SK-OV-3 human ovarian cancer cells at a concentration of 5 µM c or 0.5 µM d and incubated for 5 h prior to imaging. The same image acquisition and processing conditions were used for each image to directly compare relative fluorescence properties (top rows) along with brightfield images of the field (bottom row). e A no-wash fluorogenic labeling system for cellular tubulin employing the dipivaloyl-protected taccalonolide probe Flu-tacca-7 (11), which is cleaved to generate Flu-tacca-8 upon cellular entry where it can bind cellular microtubles. Scale bars = 50 µm for all images. Source data are provided as a Source Data file.