Fig. 2: Characterization of the photoswitchable intein, PS Intein.

a Positive and negative controls compared to PS Intein, all applied to reconstitution of mCherry. “no 2xVVD” is a constitutively active positive control without the double VVD insert. “no 2xVVD/C1A” is a negative control with the catalytic Cys1 substituted with Ala. mCherry fluorescence signals normalized to co-expressed mEGFP were analyzed. Mean values for individual experiments and SD are shown (n = 3). Statistical analysis of dark and illuminated sample pairs was performed using an unpaired t-test. Statistical analysis between the negative control dark “no 2xVVD/C1A” sample with the dark and illuminated PS Intein samples was performed using a two-way ANOVA with multiple comparisons. Significance levels are represented as ****P < 0.0001, **P < 0.01, while “ns” indicates non-significance. Exact P values are as follows: no 2xVVD insert dark vs. no 2xVVD insert light, P = 0.78514; no 2xVVD/C1A dark vs. no 2xVVD/C1A light, P = 0.14239; PS Intein dark vs. PS Intein light, P = 1.98  × 10⁻⁸; no 2xVVD/C1A dark vs. PS Intein dark, P = 0.0082; no 2xVVD/C1A dark vs. PS Intein light, P = 4.06747 × 10^-5. b Microscopy analysis of PS intein applied to reconstitution of mCherry corresponding to the sample in (a). Dark and illuminated samples are shown. c The influence of illumination times on PS Intein mediated reconstitution of mCherry in HeLa cells. The timeline for experiments is presented at the top. Cells were analyzed as in (a). d Design of PS Intein-mediated light-induced intracellular targeting of mCherry. MycA1 NLS signal was inserted at the N-terminus. Super-PKI-2-NES-msfGFP was inserted between IntN and IntC. Illumination results in reconstitution of mCherry with attached NLS. e Microscopy analysis of HeLa cells expressing the construct shown in (d). Arb. units, arbitrary units. Source data are provided as a Source Data file.