Fig. 1. Scheme of the TetOne-ATOH1-t2a-TagBFP2 lentiviral plasmid used to generate a stable iPSC line with induced expression of the ATOH1-T2A-TagBFP2 fusion gene. The ATOH1-T2A-TagBFP2 gene is controlled by the TRE3GS inducible doxycycline-dependent promoter containing seven repeats of the tetO operator sequence. The plasmid also comprises the Tet-On 3G (TRE3GS promoter transactivator) and PuroR (puromycin resistance gene) genes controlled by the hPGK (promoter of human gene PGK1) and SV40 constitutive promoters, respectively. Standard components of lentiviral plasmids that are essential for correct and effective assembly of functional viral particles in the packaging cells and that ensure high expression of transgenes (5'LTR/3'LTR — long terminal repeats, RRE — Rev viral protein binding site (Rev response element); cPPT/CTS — central polypurine tract/central termination sequence; WPRE — Woodchuck hepatitis virus post–transcriptional regulatory element; SV40p(A) — SV40 transcription terminator with a poly(A) signal; AmpR — ampicillin resistance gene) are highlighted in gray

Fig. 2. Expression of the TetOne-ATOH1-t2a-TagBFP2 construct in iPSCs. А. Scheme to generate a stable iPSC line carrying the ATOH1 gene controlled by the TRE3G inducible promoter. F — TagBFP fluorescent protein. B. Cells 24 h after induction of expression by doxycycline. C. After freezing and thawing the majority of cells become incapable of doxycycline-dependent expression (the only cell in the field of view showing a bright TagBFP2 expression signal after adding doxycycline is marked with arrow)

Fig. 3. Immunofluorescence analysis of simultaneous class III β-tubulin (TUBB3) and TagBFP2 expression in the cells on day 4 of differentiation after induction with TetOne-ATOH1-t2a-TagBFP2. А. Red signal — rabbit anti-TUBB3 (Affinity) antibody was used along with the goat anti-rabbit IgG Alexa Fluor 568 (ThermoFisher) secondary antibody. B. Blue signal — TagBFP2 fluorescence