Detection of the concentration of lithium in water
The water samples were obtain from the places that were shown in Fig.1e. The concentration of lithium in water samples was detected with K-Lite8F (Cornley, Meizhou, China) according to the manual. To evaluate the effects of lithium batteries to water, two disable Apple 6s plus batteries and two disable Huawei P20 pro batteries were used in these study. Each battery was placed in a beaker, soaked in ultrapure water, and then the battery is punctured with a clean needle, then added ultrapure water to 2 liter. After three days, the polluted water was filtered by filter paper. The concentration of lithium in these samples was detected with K-Lite8F (Cornley) as well.
Cell culture and treatment
Human AC16 cardiomyocyte line was cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) penicillin and streptomycin (Gibco). Cells were grown in a humidified atmosphere of 5% CO2 at 37°C. LiCl (Sinopharm Chemical Reagent, Shanghai, China) or Li2SO4 (Sinopharm Chemical Reagent, Shanghai, China) was reconstituted in ddH2O. AC16 cells were incubated with LiCl or Li2SO4 at different concentrations (0.2mmol/L, 1mmol/L, 5mmol/L or 25mmol/L) for 48 hours as specified in figure legends.
Cell Viability Assay
The cell viability was tested with CellTiter-Lumi™ Luminescent Cell Viability Assay Kit (Beyotime, Nantong, China). The AC16 cells were seeded into 96-well plates (1×103 cells/well) . After 48 hours treatment with Control (ddH2O), LiCl, NaCl or Li2SO4 at different concentrations (0.2mmol/L, 1mmol/L, 5mmol/L or 25mmol/L), the cells were assessed. 100 µl CellTiter-Lumi™ reagent was added into each well of the plate. Then the plate was incubated at 37˚C for 10 min. The luminometer was subsequently recorded with SpectraMax M5 plate reader (Molecular Devices).
The cell viability was measured via the Cell Counting Kit-8 (CCK-8) assay as well. AC16 cells were seeded into 96-well plates (1×103 cells/well) . After 48 hours treatment with Control, LiCl, NaCl or Li2SO4 at different concentrations (0.2mmol/L, 1mmol/L, 5mmol/L or 25mmol/L), the cells were assessed. 10 µl CCK-8 reagent (Dojindo, Kumamoto, Japan) was added into each well of the plate. Then the plate was incubated at 37˚C for 2 h. The absorbance at 450 nm was subsequently recorded with SpectraMax M5 plate reader (Molecular Devices).
Cell Apoptosis Assay
AC16 cells were treated with Control, 5mmol/L LiCl or 2.5mmol/L Li2SO4 for 48 hours. To quantify the cell apoptotic degree, the harvested cells were stained with annexin V‐FITC/PI Cell Apoptosis Kit (Keygen, Nanjing, China) according to the manufacturer's instructions. After incubation for 30 min at 4 °C, the cells were analyzed using FCM (FACS Canto; BD Biosciences).
EdU proliferation assay
5-Ethynyl-2’-deoxyuridine (EdU) is a synthetic thymidine analog which can incorporate into newly synthesized DNA during S phase. Therefore, EdU detection can be used for tracking DNA replication directly . The immunofluorescence staining of EdU was performed with BeyoClick™ EdU-555 proliferation kit (Beyotime) followed the Kit manual. Briefly, cells were cultured in 24-well plates, fixed in 4% paraformaldehyde after 48 hours treatment with Control, 5mmol/L LiCl or 2.5mmol/L Li2SO4, and permeabilized with 0.2% Triton X-100 for 15 min. Cells were counterstained with Hoechst for 5 min, then were washed with and imaged in PBS. At last, the image were taken using fluorescence microscopy (Nikon).
Cultured AC16 cells were lysed in strong RIPA buffer containing Halt Protease Inhibitor Cocktails (Thermo, Waltham, MA). Protein concentrations were measured using a BCA protein assay kit (Pierce, Rockford, IL). Primary antibodies targeting proliferating cell nuclear antigen (PCNA) (ab29, abcam, Cambridge, UK), tumor protein p53(TP53) (ab1101, abcam), CYCLIN E (ab33911, abcam), glycogen synthase kinase 3 beta (Gsk3β) (ab32391, abcam), GSK3β (P-Ser9) (ab131097, abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab9485, abcam) were incubated with proteins overnight at 4°C, followed by incubation with the appropriate HRP (horseradish peroxidase) conjugated secondary antibodies(BA1038 and BA1039, Boster, Wuhan, China). Detection of HRP was performed using the ECL assay kit (Beyotime) and an ImageQuant LAS 4000 mini (GE, Boston, Unite States).
GraphPad Prism 7.0 software was used for statistical analysis. Statistical significance between two groups was determined using an unpaired two-tailed Student’s t test. Data are presented as mean ± SD (standard deviation) or mean ± SEM (standard error of the mean) as indicated in the figure legends. P values were considered statistically significant if P < 0.05.