Dmi4000b fluorescent microscope

For each treatment, a 300 µL cell suspension (10⁵ cells/mL) was seeded in an Eight-well Glass Chamber Slide (#80806, Ibidi, Grafelfing, Germany). Bufalin (40 nM) was added the next day and the medium was discarded after a specific time point (24 h for HCT116 and DLD1, and 48 h for SW480 and HCT15). The slides were then washed with cold PBS 3 times and fixed with neutral-buffered formalin for 10 min. The cell membrane was then permeabilized with 0.1% Triton-X (#KGF011, Keygen, Nanjing, Jiangsu, CN) for 10 min, washed with cold PBS 3 times and incubated with β-catenin primary antibody (aAb32672, Abcam, Cambridge, UK) diluted in 10% donkey serum (#SL050, Solarbio, Beijing, CN) overnight at 4 °C. The following day, the primary antibody was removed and the cells were washed with cold PBS 3 times. FITC-conjugated secondary antibody (donkey anti-rabbit IgG; #ab150073, Abcam, Cambridge, UK) diluted in 10% donkey serum was then added and incubated in darkness for 1 h. Next, the wells were washed 3 times with cold PBS and DAPI counterstaining was performed according to manufacturer instructions (#C1005, Beyotime, Shanghai, CN). Photos were taken at a magnification of 40x with a Leica DMI4000 B fluorescent microscope (Wetzlar, Germany). DAPI and FITC images were merged using ImageJ.

EA.hy926 cells were cultured on coverslips, treated with bolus addition of H₂O₂ or/and 7, 8-DHF for 24 hrs and stained with an antibody against NF-κB (1:1000) and a secondary FITC-conjugated antibody (1:200). Cellular fluorescence was observed under the Leica DMI 4000B fluorescent microscope.

HeLa cells were treated with different concentrations of inhibitors for 16 hours. For all immunofluorescence experiments, cells were fixed in -20 °C methanol for 5 minutes or 3.7% (v/v) formaldehyde in phosphate-buffered saline (PBS) for 20 minutes. For LC3B staining, cells were fixed in -20 °C methanol. For EEA1 and LAMP1 staining, cells were fixed in PBS+ formaldehyde. Cells were then permeabilized with TBSTx (0.1% Triton X-100), blocked in AbDil (TBSTx + 2% BSA + 0.1% NaN3), and probed with primary and secondary antibodies diluted in AbDil. Cells were rinsed thoroughly with TBSTx and mounted by anti-fade prolong Gold with DAPI. Images were taken using a Leica DMI4000B fluorescent microscope. All experiments were repeated at least three times). MEC-2, OCI-AML-2, OCI-AML-3 and MV4-11 cells were treated with serially-diluted PI3KD/V-IN-01 for 6/12 hours. Hela cells were starved with EBSS for 2 hours, then treated with 25μM HCQ and serially diluted PI3KD/V-IN-01 for 1 hour. Cells were lysed in lysis buffer. P62, LC3B, and GAPDH antibodies were used for immunoblotting.

HeLa cells were plated in 24-well plate 24 hours before experiment. Cells were treated with 5 μg/ml Acridine Orange (A3568, Life Technology) at 37°C for 15 minutes and washed with PBS for three times. To evaluate the effect of NH₄Cl on vesicle pH, cells were treated with different concentrations of NH₄Cl for 10 minutes before fluorescent microscope acquisition. Illuminated by 488-nm laser beam, the red fluorescence indicates acidity while the green fluorescence indicates alkalinity by I3 Channel equipped in Leica DMI4000B fluorescent microscope.