Fig. 3: Pre-incubation with hemin increases proximity-labeling efficiency.

A Hemin pre-incubation stimulates the peroxidase activity of the HyPro and HyPro2 enzymes. Note that negative control reactions (Ctrl) pre-incubated with 20–50 μM, but not lower concentrations of hemin, show background-level peroxidase activity⁴³. B Quantification of the peroxidase activity data from (A) across 3 independent experiments. Data were Ctrl background-subtracted, normalized to experiment-specific averages, presented as mean ± SD and compared by a two-sided t-test assuming unequal variance. C Proximity labeling of ACTB transcription sites by HyPro2 in low-viscosity buffer (LVB) without hemin pre-incubation (top row) and in trehalose-containing buffer with 5 μM hemin pre-incubation (bottom row). ACTB transcription sites (arrowheads) are magnified 3 × in close-ups. Main images and close-ups, individual optical sections. Scale bars, 5 μm. Both conditions were imaged using identical microscopy settings. Note that labeling intensities are comparable, but signal diffusion (filled arrows in the “no hemin and LVB” close-up) is effectively suppressed in the “5 μM hemin and trehalose” condition. Also note that hemin and trehalose suppress the nonspecific background staining occasionally detected in LVB (open arrows in the “no hemin and LVB” main image). The experiment was repeated 3 times, with similar results. D Maximum-normalized intensity profiles of RNA-FISH and HyPro-FISH signals along the direction indicated by large arrowheads in (C). HyPro-FISH signal spreads beyond RNA-FISH in “no hemin and LVB” but not in “5 μM hemin and trehalose”.