Glass bottomed cell culture dishes

Immunohistochemistry (IHC) and immunofluorescence (IF) were performed as described previously^{61 (link)} using various antibodies (Supplementary Fig. S1). For immunocytochemistry (ICC), HepG2 cells were seeded on glass-bottomed cell culture dishes (NEST Biotechnology, Jiangsu, China) and incubated for 24 h under hypoxic conditions. Then, cells were fixed in 4% paraformaldehyde for 30 min and washed three times with PBS. Next, the cells were incubated in PBS containing 0.25% Triton X-100 for 10 min and blocked with blocking solution (3% bovine serum albumin, 0.25% Triton X-100 in PBS). Cells were treated for 30 min followed by incubation overnight at 4 °C with a diluted primary antibody against GLUT1 in blocking solution. The next day, the cells were washed three times with PBS and incubated with diluted secondary antibody (1:200, Invitrogen, Oregon, USA) in blocking solution for 1 h. After washing the cells in PBS, Hoechst 33258 was used to stain the nuclei. Images were acquired using an Olympus BX53 microscope with Olympus Cell Sens software (Carl Zeiss Microscopy, GmbH, Jena, Germany). Quantification of fluorescence in microscopic images stained with GFP (Green) and DsRed (Red) was carried out using IMT i-Solution software (Martin Microscope Company, Easley, USA).

Cells were seeded in glass-bottomed cell culture dishes (NEST, 20 mm, 4 × 10⁵ cells per dish). For SARS-CoV-2 entry experiments, Huh7 cells were seeded in 24-well plates (1 × 10⁵ cells per well) and infected with SARS-CoV-2 at an MOI of 10. For the colocalization experiments, Huh7 cells were treated with 1 µM RBD labeled with Alexa Fluor 555 (RBD-AF555) at the indicated time point. The cells were washed three times with PBS and treated with 0.1% Triton X-100 (Solarbio, T8200) for 15 min. Then, the cells were incubated with 1% bovine serum albumin (Sigma-Aldrich, A7906) and the appropriate primary antibodies for 2 h at 37 °C before being washed and incubated simultaneously with fluorescein isothiocyanate– or tetramethylrhodamine-conjugated secondary antibodies. Finally, the cells were treated with a Hoechst 33342 (Sigma-Aldrich, B2261) solution for 3 min and analyzed under a confocal microscope (CLSM; Leica SP8).

Autophagic flux is a typical indicator of dynamic autophagic activity, which can be evaluated using a GFP-mRFP-LC3B adenoviral vector transfection assay [24 (link)–26 (link)]. Briefly, the MOVAS cells were plated on glass-bottomed cell culture dishes (Nest, China) and infected with adenoviral vectors containing GFP-mRFP-LC3B (HanBio, China) for 24 h, according to the manufacturer's instructions. The cells were then transferred to fresh medium and incubated for 24 h. Subsequently, the cells were observed via LSCM to confirm transfection efficiency, and autophagic flux was determined by evaluating the numbers of yellow fluorescent protein (YFP) and red fluorescent protein (RFP) puncta. Yellow puncta, reflecting colocalization of RFP and green fluorescent protein (GFP) fluorescence signals, indicated the presence of autophagosomes, while free red puncta (RFP only) marked autolysosomes where acidic pH quenched GFP fluorescence.

The biofilms were cultured and treated with AKBA as described above in glass-bottomed cell culture dishes (NEST, Wuxi, China). After incubation at 37 °C for 24 h, the dishes were washed with PBS to remove unattached cells. Then, the dishes were stained with 500 µL PBS in the presence of 10 µM SYTO-9/PI (Thermo Fisher Scientific, Waltham, MA, USA) in the dark for 30 min. And the stained biofilms were observed by a CLSM (Zeiss LSM 800, Jena, Germany) with excitation/emission wavelengths of 488 nm/550 nm (SYTO9) and 540 nm/620 nm (PI), respectively (Zhang et al. 2023 (link)).

Cellular uptake of CHA-SME loaded with Cou-6 was qualitatively and quantitatively examined by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Briefly, Caco-2 cells were seeded into 12-well plates or glass-bottomed cell culture dishes (NEST Biotechnology, Wuxi, China) at 2×10⁵ cells/well and incubated for 24 hours. Then, the cells were incubated with Cou-6-labeled CHA-SME at 37°C for different periods. For CLSM, the cells were rinsed thrice with cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (PFA), stained with tetramethylrhodamine (TRITC) phalloidin (YEASEN Biotech, Shanghai, China) and 4′,6-diamidino-2-phenylindole (DAPI), and observed by CLSM (TCS SP8X; Leica Microsystems, Wetzlar, Germany). For flow cytometry, the cells were washed thrice with cold PBS, harvested, and analyzed using a flow cytometer (Accuri C6; BD Biosciences, San Jose, CA, USA).

PC3 cells were seeded on glass-bottomed cell culture dishes (NEST Biotechnology, Shanghai, China) and incubated for 24 h. The culture medium was then changed to 2DG containing culture medium and incubated for 48 h. Cells were fixed with 10 % paraformaldehyde for 30 min and washed three times with PBS. To increase permeabilization, cells were incubated in 0.25 % Triton X-100-containing PBS for 10 min. After washing the cells with a PBS blocking solution (3 % bovine serum albumin, 0.25 % Triton X-100 in PBS), cells were treated for 30 min followed by incubation overnight at 4 °C with diluted primary antibody against microtubule-associated protein 1 light chain-3B (LC3B; 1:200, Cell Signaling Technology, Danvers, MA, USA) or p62 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) in blocking solution. The next day, the cells were washed three times with PBS then incubated with diluted secondary antibody (1:500, Invitrogen, Oregon, USA) in blocking solution for 1 h. After washing the cells in PBS, Hoechst 33258 was used to stain the nucleus. The cells were analyzed using a Carl Zeiss LMS710 confocal microscope (Carl Zeiss, Gottingen, Germany).