Fig. 1: An Xfect-based protein transfection method can introduce β-Galactosidase (β-Gal) into budding yeast cells.

a Effects of spheroplastization on protein transfection. Saccharomyces cerevisiae S288c (Sc) and Candida utilis NBRC0988 (Cu) cells (Zymolyase −), or their spheroplasts (Zymolyase +), were incubated with β-Gal in the presence (+) or absence (−) of Xfect™ Protein Transfection Reagent (Takara Bio Inc., Shiga, Japan), and the β-Gal activity was measured by a colorimetric method. Bars and error bars, respectively, represent the mean and standard deviation from five independent experiments (n = 5) and one-tailed Welch’s t-test was applied (*p < 0.05, **p < 0.01, ***p < 0.001). Experiments were performed in triplicates. b Effects of trypsin digestion of membrane-absorbed β-Gal on β-Gal quantification. β-Gal was introduced to Sc and Cu cells as in (a), which were subsequently treated with (trypsin +) or without (trypsin −) 1% trypsin in phosphate-buffered saline (PBS, pH 7.4) in order to digest membrane-absorbed β-Gal and β-Gal/Xfect complexes. The activity of intracellular β-Gal was determined as in (a). Bars and error bars, respectively, represent the mean and standard deviation from six independent experiments (n = 6) and one-tailed Welch’s t-test was applied (*p < 0.05). Experiments were performed in triplicates. c Fluorescent-based detection of β-Gal activities inside Sc and Cu living cells. β-Gal- (Xfect −) or β-Gal/Xfect-mixed (Xfect +) cells, with (citrate +) or without (citrate −) subsequent β-Gal inactivation by citrate buffer (pH 4.0), were incubated with SPiDER-βGal of the indicated concentration, and their images were acquired using the fluorescence microscopy BZ-X700. The scale bar is 5 µm, and a representative image for each experiment is shown. d Quantitative comparison of intracellular β-Gal activity between Sc and Cu. Variability in the intracellular fluorescence signals between Sc and Cu cells were determined using citrate-treated cell images (c) and Hybrid cell-counting tool. The data of Sc (1.0 µM SPiDER-βGal) and Cu (0.2 µM SPiDER-βGal) are shown as the box plots (n = 120, three fields per replicate). The center line is the median, bounds are the 25th and 75th percentiles, and whiskers are ±1.5 IQR. Error bars indicate SD from cell counts. Asterisk indicate significant differences analyzed using one-tailed Welch’s t-test, *p < 0.05.