Trypsin edta

Treated cells in T25 flasks were detached using trypsin-EDTA (Biosera, Shanghai, China; LMT1706), deactivated with PBS, and centrifuged at 1.100 rpm for 5 min. Supernatants were discarded, and the pellets were resuspended in 250 µL of ice-cold PBS supplemented with protease and phosphatase inhibitors. After centrifugation at 1.100 rpm for 5 min at 4 °C the pellets were resuspended in 200 µL of 5x CPV NP-40 lysis buffer (10 mMTris-HCl, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40 with protease and phosphatase inhibitors). Tubes were incubated on a rotating platform at 4 °C for 10 min and centrifuged strictly at 1.000 rpm for 5 min at 4 °C. The supernatants (cytoplasmic protein fractions) were kept at −80 °C. The pellets were resuspended in 100 µL of RIPA solution, vortexed, and incubated for 60 min on a rotator at 4 °C. Samples were centrifuged to insoluble pellet fraction at 13.000 rpm for 30 min at 4 °C and after the supernatants (nucleus protein fractions) were kept at −80 °C.

Bombyx mori (B. mori, silkworm) cocoons were
purchased from Kozabirlik (Bursa, Turkey). CNFs (<100 ppm iron
content), sodium carbonate (Na₂CO₃), lithium
bromide (LiBr), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), protease
type XIV (>3.5 units/mg), and methanol (CH₃OH) were
purchased
from Sigma-Aldrich. NaCl was purchased from Isolab. Dulbecco’s
modified Eagle’s medium, penicillin–streptomycin, fetal
bovine serum (FBS), and trypsin–EDTA were purchased from Biosera.
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT), dimethyl sulfoxide (DMSO), glutaraldehyde, and hexamethyldisilazane
(HMDS) were purchased from Glentham, Genaxxon Bioscience, Merck, and
Sigma, respectively.

FACS-sorted P19 cells were diluted to 1 cell/100 µL by adding 100 cells into 10 mL DMEM/10% FBS. Then, 100 µL of the cell suspension was distributed into each well of the 96-well plate and incubated for 1 week. The wells with a visible single-cell colony were washed with 100 µL of PBS (Phosphate Buffered Saline) without calcium and magnesium (Lonza, Basel, Switzerland) and then detached by adding 20 µL of trypsin-EDTA (0.25%) (Biosera, Nuaille, France). Next, the cells were split in the same arrangement into two 96-well plates. After one week, the cells reached an approximately 80% confluence and were used for further analysis.

CHO cells were cultured in DMEM containing HAT (hypoxanthine–aminopterin–thymidine; HAT Media Supplement (50×) Hybri-Max™; Sigma-Aldrich, St. Louis, MO, USA) for a week to eliminate preexisting HPRT-mutant cells from the culture. CHO cells were treated with the previously specified inhibitor molecules and exposed to 0–25 mJ/cm² UVB. Cells were cultured for one more week and then harvested with trypsin-EDTA (Biosera, Budapest, Hungary). In the case of each sample, an equal number of cells (1 × 10⁶) were seeded into 100 mm Petri dishes in selective DMEM containing 5 μM 6-thioguanine (6-TG; Sigma-Aldrich, St. Louis, MO, USA). The 6-TG-resistant cells were allowed to form visible clones for 10 days. Clones were washed with PBS, fixed with 100% methanol (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, and stained with May–Grünwald–Giemsa (Molar Chemicals, Halásztelek, HU, Hungary). HPRT-mutant colonies were counted. For the positive control, 10 μM 1-methyl-3-nitro-1-nitrosoguanidine (MNNG; TCI Europe N.V., Zwijndrecht, Belgium) was used.

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.

Human breast (T47D) and cervical (HeLa) cancer cell line were obtained from the cell bank of Pasteur Institute of Iran. Human cervical (HeLa) cancer cell line was used as a negative breast cancer control. Briefly, cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen) and 700 μl penicillin-streptomycin (Biosera). Cells were kept at 37°C in a humidified CO₂ incubator (5% CO₂). Then, cell lines grown in monolayer were harvested by washing the plates once with PBS, pH=7.3, and then the cells were incubated with trypsin/EDTA (Biosera) for 2–5 min at 37°C. Finally, cells were counted using hemocytometer.