Fluozin 3

All breast tumor cells and 293 T cells were purchased from ATCC and maintained as per standard protocols in an incubator with 95% humidity and 5% CO₂ at 37 °C. Cells were cultured in DMEM with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cysteine deficient medium was prepared according to the previous report^{63 (link)}. MCF10A cells were maintained as per a standard protocol of ATCC and cultured in MEGM supplemented with MEGM bullet kit. Erastin, selective HDAC6 inhibitors tubacin, tubastatin A, and CAY10603, Myriocin, and the metal chelator N, N, N′, N′-tetrakis (2-pyridylmethyl) ethylenediamine (TEPN) were obtained from Cayman Chemicals (Ann Arbor, Michigan, US); Z-VAD-FMK, necrostatin-1, and ferrostatin-1 were purchased from Calbiochem Research Biochemicals (Sigma). The molecular probe FluoZin-3 was purchased from Thermo Fisher Scientific. All antibodies used in this study were listed in supplementary table 1.

Intracellular calcium was measured by Fluo4 NW (Molecular Probes), intracellular potassium was measured by FluxOR (Molecular Probes) according to manufacturer’s instructions in human bladder epithelial cells grown in 96-well plates (60,000 cells/well) after exposure to FimCH (5 μg). Extracellular Zn²⁺ was measured by FluoZin-3 (Thermo Fischer Scientific) by addition of 1 μg/ml of indicator salt for 60 minutes prior to FimCH treatment. Fluorescent intensity was measured by Infinite F200 (Tecan) microplate reader at 20 seconds intervals for indicated times.

Cell culture media, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), and fluorescent probes (Calcium Green-5N, Fluo-5N, FluoZin-1, FluoZin-2, FluoZin-3, fura-2, indo-1, Leadmium Green, mag-fura-2, Newport Green DCF, and Rhod-5N) were purchased from Thermo Fisher Scientific. fura-2FF was purchased from TEF Labs. Fetal bovine serum was purchased from Midwest Scientific. All other reagents were purchased from Sigma-Aldrich unless otherwise noted.

We adapted a protocol from the one employed by Inoue and colleagues to monitor Zn²⁺-influx by TRPM7²² . Briefly, TRPM7^−/− HEK293T cells were transiently transfected with pcDNA3.1D/V5-His-TOPO-XTRPM6, pcDNA3.1D/V5-His-TOPO-XTRPM7, pcDNA5/FRT/TO-mTRPM7, or pcDNA5/FRT/TO-hTRPM6 and allowed to express for 24–48 hours. The cells were labeled with the zinc indicator Fluo-Zin-3 following manufacturer’s orders (Thermo Fisher Scientific). The cells were then incubated with HBSS containing 30 μM ZnCl₂ in the presence or absence of 250 μM 2-APB for 5 minutes, and then images were acquired on an inverted Olympus IX70 fluorescence microscope. The fluorescence intensity of the cells was analyzed using the Fiji software package with ROI analysis^{32 (link)}.

The Zn²⁺ influx assay used to characterize TRPM7 channel function has been previously described in detail (Bai et al., 2021 (link)). Briefly, cells were plated into 24-well dishes coated with polylysine to aid comparison between individual samples by fluorescence microscopy. Before labeling, cells were washed with Hanks’ balanced salt solution (HBSS) containing 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 6 mM glucose, 0.44 mM KH₂PO₄, 1.3 mM CaCl₂, 1.0 mM MgSO₄, and 4.2 mM NaHCO₃. Cells were then labeled with the Zn²⁺ indicator FluoZin-3 (2.5 μM) in HBSS following manufacturer instructions (Thermo Fisher Scientific). The cells were then washed once with HBSS and then placed back into HBSS before images were quickly acquired on an inverted Olympus IX70 fluorescence microscope with a 10X phase contrast objective (Olympus Neoplan 10/0.25 Ph, Olympus, Tokyo, Japan). Cells were visually inspected for uneven dye loading prior to imaging. To stimulate Zn²⁺ influx, 30 μM ZnCl₂ in HBSS was introduced to cells. For static measurements, images of cells from the different samples were taken at a specific time point 5 to 10 minutes after the addition of 30 μM ZnCl₂.