Chloropromazine

The ethidium bromide accumulation and efflux assays were measured by florescence intensity (48 (link), 89 (link)) with minor modifications. Briefly, mid-log-phase cultures were washed with PBS containing 0.05% Tween 80 (PBST) and then stained with 2 µg/ml ethidium bromide (Sigma). ethidium bromide (1 µg/ml) was used for accumulation assays with efflux inhibitors, including chloropromazine (10 µg/ml; Sigma), verapamil (100 µg/ml; Sigma), reserpine (6 µg/ml; Sigma), or carbonyl cyanide m-chlorophenyl hydrazone (1 µg/ml; Sigma). For the ethidium bromide efflux assay, bacteria were washed with PBST and then incubated with 2 µg/ml ethidium and 100 µg/ml verapamil for 60 min. After the bacteria were washed twice with PBST, efflux activity was measured as the decay ratio of fluorescence intensity. For Nile red uptake staining, mid-log-phase cultures were washed with PBS and then stained with 20 µM Nile red (Sigma) (90 (link)). In all assays, the cells were incubated in 96-well plates, and analysis was performed at the indicated time points by excitation at 544 nm and emission at 590 nm on a FLUOstar OPTIMA microplate reader (BMG Labtech). All data were normalized to the time zero reading of each well. All experiments were repeated at least three times and similar results were obtained. Representative results are shown in Fig. 7A and B.

The commercially available trastuzumab (T)-DM1 was purchased from Hoffmann-La Roche Inc. (Basel, Switzerland), and the BS trastuzumab-DM1 was manufactured by Shanghai Hengrui Pharmaceutical Co., Ltd. (Shanghai, China) as previously detailed¹⁶ (link). The ADC DX-006 was kindly provided by Hangzhou DAC Biotech Co., Ltd. (Hangzhou, China). Chloropromazine, E64d, CA-074 methyl ester (CA-074-ME), pepstatin A, aprotinin and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bafilomycin A1 was purchased from Selleck Chemicals (Houston, TX, USA). Ansamitocin P-3 was purchased from Medchem Express (Monmouth Junction, NJ, USA). Cell Counting Kit-8 (CCK-8) was purchased from Beyotime (Nanjing, China). Monoclonal antibody against HER2 (Cat#AB16901) was purchased from Abcam (Cambridge, MA, USA). Rhodamine Red™-X (RRX) AffiniPure Donkey Anti-Mouse IgG (H + L) was purchased from Jackson ImmunoResearch Laboratories Inc. (Philadelphia, PA, USA). Acetonitrile (ACN, HPLC-grade) was purchased from Merck (Darmstadt, Germany). Deionized water was prepared by Milli-Q system (Millipore, Billerica, MA, USA). All other reagents and solvents were purchased from Sigma–Aldrich and of analytical grade.