*4.4. CPD-Specific Enzyme-Linked Immunosorbent Assay (ELISA)*

A CPD-specific ELISA was performed as previously described by Boros et al. [82]. Genomic DNA was extracted by an Invitrogen™ PureLink™ Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) 24 h after the UVA irradiation. Flat-bottomed 96-well plates were coated with 0.003% protamine-sulfate and incubated at 37 ◦C to completely dry. DNA was denatured at 100 ◦C for 10 min, then immediately chilled on ice for 15 min. Denatured DNA was distributed to wells in triplicate (15 ng DNA to each well) and incubated at 37 ◦C overnight. Plates were washed with PBS (Biosera, Budapest, Hungary) containing 0.05% Tween-20 (Amresco, Solon, OH, USA) (PBS-T) and incubated with 150 µL/well 5% FBS at 37 ◦C for 30 min to prevent nonspecific antibody binding. After washing plates three times with PBS-T, anti-CPD monoclonal antibody (clone TDM-2, dilution 1:1500, Cosmo Bio Co., Ltd., Tokyo, Japan) was added to each well. Plates were incubated at 37 ◦C for 60 min. After washing three times, HRP-conjugated anti-mouse IgG secondary antibody (dilution 1:3000, Bio-Rad, Hercules, CA, USA) was added and plates were incubated at 37 ◦C for 30 min. Plates were washed three times with PBS-T and once with 150 µL/well citrate-phosphate buffer (0.51% C6H8O7.H2O (Sigma-Aldrich, St. Louis, MO, USA) and 0.73% Na2HPO<sup>4</sup> (Sigma-Aldrich, St. Louis, MO, USA) in distilled water; pH 5.0). Substrate solution (0.04% o-phenylenediamine (Sigma-Aldrich, St. Louis, MO, USA) and 0.006% H2O<sup>2</sup> (Sigma-Aldrich, St. Louis, MO, USA) dissolved in citrate-phosphate buffer with H2O<sup>2</sup> added to the solution, when o-phenylenediamine was completely dissolved) was added to each well. Plates were incubated until the appropriate color intensity appeared. To stop the enzyme reaction, 50 µL/well 2 N H2SO<sup>4</sup> (Sigma-Aldrich, St. Louis, MO, USA) was added to each well. Absorbance was measured at 492 nm using an Epoch Microplate Spectrophotometer (BioTek, Budapest, Hungary).

#### *4.5. HPRT Gene Mutation Assay*

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/cm<sup>2</sup> 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 <sup>×</sup> <sup>10</sup><sup>6</sup> ) 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.

## *4.6. Apoptosis Assay*

Cell viability was measured 48 h after UVB irradiation using Alexa Fluor 488–conjugated Annexin V/propidium iodide (PI) dual staining (apoptosis assay, Alexa FluorTM 488 Annexin V/Dead Cell Apoptosis Kit, Thermo Fisher Scientific, Waltham, MA, USA). The supernatant of the cells was collected, living cells were harvested with trypsin-EDTA and added to the supernatant. To avoid the loss of apoptotic cells, cell culture media was not changed between UVB exposure and viability measurement. Cells were labeled according to the manufacturer's instructions. Cells were analyzed by flow cytometry using a FACS Calibur (Becton Dickinson, San Jose, CA, USA) flow cytometer and CellQuestPro software 5.2 (Becton Dickinson, San Jose, CA, USA). Fluorescence intensity was measured in the FL-1 (for Annexin V) and FL-3 (for PI) channels. For data evaluation, FlowJo 10.6.2. (Becton Dickinson, San Jose, CA, USA) flow cytometry software was used.

## *4.7. Cell Cycle Analysis*

Cell cycle progression was quantified 1, 3, and 6 days after UVB irradiation. Cells were harvested with trypsin-EDTA, washed with DPBS, and fixed with ice-cold 80% ethanol (VWR, Radnor, PA, USA). Equal numbers of cells were centrifuged at 3500 rpm, for 5 min and re-suspended in 50 µL DPBS containing 0.2 mg/mL RNase A (Sigma-Aldrich, St. Louis, MO, USA), 0.1 µL Triton-X 100 (Amresco, Solon, OH, USA), and 5 mg/mL PI (Thermo Fisher Scientific, Waltham, MA, USA). Samples were incubated at 37 ◦C for 45 min and then supplemented with 0.5% bovine serum albumin (BSA; VWR, Radnor, PA, USA). Cell cycle progression was analyzed by flow cytometry with an FACS Calibur and fluorescence was measured on the *x*-axis in the FL2-A channel. Doublet discrimination was performed for single-cell analysis. FlowJo software was used for analyzing the data.
