Kclo4

Ultrapure water (resistivity
> 18.2 MΩ·cm, Millipore Milli-Q) was used for all experiments
in this work. Prior to each experiment, all cell compartments were
cleaned by storing them in a potassium permanganate solution (1 g
L^–1 KMnO₄ (Fluka, ACS reagent) in 0.5
M H₂SO₄ (Fluka, ACS reagent)) overnight. The
solution was subsequently drained, and the cell compartments were
rinsed with a dilute piranha solution (1:3 v/v of H₂O₂ (Merck, Emprove exp)/H₂SO₄) to remove
residual KMnO₄ and MnO_x. Afterward,
the cell compartments were cleaned by repetitively rinsing and boiling
with Milli-Q water to remove all inorganic contaminants. Electrolytes
were prepared from LiClO₄ (Sigma-Aldrich, ≥99.99%
trace metal basis), NaClO₄ (Sigma-Aldrich, ≥99.99%
trace metal basis), KClO₄ (Sigma-Aldrich, ≥99.99%
trace metal basis), NaClO₄ (Sigma-Aldrich, ≥99.99%
trace metal basis), H₂SO₄ (Merck, Suprapur,
96%), PdSO₄ (Sigma-Aldrich, 99.99% trace metal basis),
and HClO₄ (Sigma-Aldrich, Ultrapure, 70%). In this work,
a Pt wire (0.5 mm diameter, MaTecK, 99.9%) was used as the counter
electrode, a reversible hydrogen electrode (RHE) was used as the reference
electrode, and all of the potentials were corrected for ohmic drop
and controlled with an Autolab PGSTAT302N potentiostat.

Both TPC-1 and SW1736 cells (7 × 10⁴/500 µL) were seeded using each DMEM high glucose medium and RPMI1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin in 24-well plates. After 24 h incubation, medium was replaced to Opti-MEM medium and treated both scrambled siRNA and GLI1 siRNA, and then incubated for 48 h in a CO₂ incubator at 37 °C with 5% CO₂. Following incubation, the cells were washed with pre-warmed Hank’s balanced salt solution (HBSS; HyClone) containing 0.5% BSA (bHBSS). Next, 37 kBq carrier-free I-125 (Perkin Elmer Life Science, Waltham, MA, USA) and 100 μM sodium iodide (NaI, specific activity of 740 MBq/mM, Sigma) were added and the cells incubated for 30 min in a humidified incubator at 37 °C with 5% CO₂. To prevent I-125 uptake, the cells were treated with 50 μM potassium perchlorate (KClO₄, Sigma), which is a competitive inhibitor of iodide transport, for 30 min before adding I-125. After incubation, the cells were washed twice with chilled bHBSS and lysed with 500 μL of RIPA buffer. Radioactivity was measured using a Cobra II gamma counter (Canberra Packard, Mississauga, ON, Canada). Uptake values were normalized with the protein concentration, as determined by BCA protein assay kit. Results are presented as count per minute (cpm)/μg.

Water sample analysis was performed using an Ion Chromatography system (IC-1100, Dionex) with a separation column − Ion Pac AS 16 (2 × 250 mm and 4× 250 mm), guard column − Ion Pac AG 16 (2 × 50 mm and 4 × 50 mm) and an anion self-regenerating suppressor ASRS 300 (4 mm). The Ion Pac AS 16 column is specific for ClO₄⁻ ion with a lower detection limit of 2 ppb (μg/L). This method for ClO₄⁻ detection in drinking water is recommended according to USEPA methods 314.0 and 314.1. The eluent used was 50 mM Sodium hydroxide (NaOH, Fluka) at a flow rate 1.5 mL/min. The injection volume was 1000 μL. Calibration standards of ClO₄⁻ was prepared with high purity KClO₄ (Sigma Aldrich) by diluting 1000 mg/L primary standard. All solutions were prepared in ultra-pure milliQ water (Millipore).