As2o3

T24 cells were seeded into 96-well plates (2 × 10⁴ cells/well) and cultured for 12 h. Next, T24 cells were incubated with As₂O₃ (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) at 0, 10, and 20 μmol/L for 6 h. After washing, T24 cells were incubated with 10% CCK-8 (Dojindo Molecular Technologies, Inc., Minato-ku, Tokyo, Japan) and optical density measured using a xMark Microporous Plate Absorption Spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
The apoptotic rate of T24 cells was detected using an Annexin V, 633 Apoptosis Detection Kit (Dojindo Molecular Technologies), following the kit instructions. T24 cells were seeded into 6-well plates (5 × 10⁵ cells/well) and cultured for 12 h. Next, cells were incubated with As₂O₃ (Sigma-Aldrich) at 20 μmol/L for 6 h, then incubated with Annexin V, followed by propidium iodide (PI) buffer for 15 min at 25°C in a dark room. Subsequently, apoptotic cells were quantified using a NovoCyte 1040 flow cytometer (ACEA Biosciences, Inc., Zhejiang, China).

The BEAS-2B cell line was obtained from the American Type Culture Collection. Cells were maintained in 5% CO₂ at 37°C in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum(FBS, Life Technologies/Gibco), 100 U/mL penicillin, and 100 ug/mL streptomycin (Life Technologies/Gibco). Cell culture flasks used should be pre-coated with a mixture of 0.01mg/ml fibronectin, 0.03 mg/ml bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in DMEM. For arsenic chronic treatment, 1 × 10⁵ cells were seeded into 6-cm dishes for 12 h and maintained in 0.25 μM As₂O₃ (Sigma) for 48-72 h per passage. This process was continued for about 10 weeks (20 passages) and 20 weeks (40 passages). For arsenic acute stimulate, 5 μM As₂O₃ (Sigma) was co-cultured with BEAS-2B cells with or without miR-100 inhibition for 0 h, 6 h, 12 h, and 24 h, respectively.

Cells were harvested with trypsin-EDTA (Biosera, Budapest, Hungary) and then seeded in six-well plates for the hypoxanthine phosphoribosyltransferase (HPRT) gene mutation assay or 24-well plates for all other experiments. At ~80% confluence, cells were pretreated with 25 μM ABT-888 (PARP1 inhibitor, veliparib, Selleckchem, Houston, TX, USA), 10–50 μM resveratrol (Abcam, Cambridge, UK), 5–25 μM spironolactone (Selleckchem, Houston, TX, USA), or 0.5–4 μg/mL As₂O₃ (Sigma-Aldrich, St. Louis, MO, USA) solution. In the case of veliparib treatment, we chose the concentration that caused marked inhibition of PARP1 protein, according to our previous [29 (link)] and current experiments (Figure S9). For the other chemicals, we identified three different concentrations due to their more complex and not fully understood mode of action—based on prior published data [26 (link),27 (link),30 (link),31 (link),32 (link)]. As₂O₃ was dissolved in 1 M NaOH (Sigma-Aldrich, St. Louis, MO, USA) and diluted in Dulbecco’s phosphate-buffered saline (DPBS; Biosera, Budapest, Hungary). Other chemicals were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA). Pretreated cells were incubated for 120 min at 37 °C before UVB irradiation.