Pureblu hoechst 33342

The DLD-1 cells were seeded at a density of 1 × 10 ${}^5$ cells/3.5 mL (9.5 cm ${}^2$ ), the next day (day 0) decitabine (1 µM) or azacitidine (4 µM) was added, then the cells were cultured for 10 days (first passage after 3 days) and seeded at a density of 2 × 10 ${}^4$ cells/0.35 mL (0.7 cm ${}^2$ ) to take microscopic images on day 13 (Figure A5). The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min under standard cell culture conditions and stained with 2 µM PureBlu Hoechst 33342 (Bio-Rad) for 15 min at RT. Microscopic images were taken by Olympus IX81 inverted fluorescence microscope (Olympus, Tokyo, Japan).

After 1-h exposure to NPs, the cells were stained with 20 nM PureBlu Hoechst 33342 in DMEM (BioRad, Hercules CA, USA) for 5 min at 37°C and washed thrice with PBS (2.5% FBS). Cells were then fixed with 4% paraformaldehyde/PBS (Sigma-Aldrich) for 30 min at 37 °C and washed. SEM images were obtained using a Hitachi S-3400N scanning electron microscope equipped with a Thermo Scientific UltraDry EDS detector. SEM images are provided with a description of the measurement conditions. Microscopic images were taken using an AxioObserverZ1 inverted fluorescence wide-field microscope (Carl Zeiss, Oberkochen, Germany) with an LD40×/0.4 Korr Ph2 objective. A BF Condenser (NA = 0.4) was used to measure bright field images, while the upconverted emission of NaGdF₄:Yb³⁺,Er³⁺ was captured using a 975 nm laser diode (Spectra-Laser, Opole, Poland) excitation with a customized optical setup. Filter cube was composed of FF750-SDi02 dichroic and FF01-945 emission filter to cut the 976-nm excitation and transmit visible radiation. CCD AxioCam MRc5 (Carl Zeiss) and EMCCD Rolera EM-C2 (QImaging, Surrey, BC, Canada) as well as ZEN2011 (Carl Zeiss) software were used to document images.

A549 cells were transduced with CellLight Golgi‐RFP (Thermo Fischer Scientific, C10593) at a concentration of 40 particles per cell and cultured for 24 h. Subsequently, the cells were treated with vehicle or BFA (0.1 μg·mL⁻¹) for 6 h, and then, nuclei were stained with PureBlu Hoechst 33342 (Bio‐Rad, Hercules, CA, USA, 135‐1304). After fixation in 4% paraformaldehyde, fluorescence images were acquired using a BZ‐9000 fluorescence microscope (KEYENCE, Osaka, Japan).