Immunofluorescence Analysis of ABCB1 and ABCG2 Expression

The immunofluorescence assay was performed as previously described (Zhang X. Y. et al., 2016 (link)). After being cultured overnight in 24-well plates, the cells (2 × 10⁴) were treated with VS-4718 for 0, 24, 48, and 72 h. Then cells were fixed in 4% paraformaldehyde for 10 min and permeabilized by 0.1% Triton X-100 for 10 min before blocking with 6% BSA for 1 h. Cells were incubated with monoclonal antibodies ABCB1 (F4, dilution 1:100) and ABCG2 (BXP-21, dilution 1:150) at 4 °C overnight. Alexa Fluor 488 conjugated secondary antibody (1:1,000) was used after washing with iced PBS. DAPI was used to counterstain the nuclei. Immunofluorescence images were collected using an EVOS FL Auto fluorescence microscope (Life Technologies Corporation, Gaithersburg, MD).