Cl 1000 ultraviolet crosslinker

UVC radiation resistance was assessed using JSC-093350089 WT and CW12015. Both strains were cultivated at 28°C on GMM agar plates by seeding 1 × 10⁷ spores per Petri dish (D = 10 cm). Spores were collected after 5 days of growth and counted. An equal amount of spores were resuspended in 5 ml of GMM agar and poured onto Petri dishes consisting of 20 ml GMM agar. Mycelia-containing plates were exposed to varying doses of UVC radiation in triplicate using a CL-1000 Ultraviolet Crosslinker (UVP, Inc.).

Purified PTCHD1 was incubated for 30 min on ice with 6 μM inclusion bodies of PhotoClick cholesterol (β-methyl-cyclodextrin and hex-5’-ynyl 3β-hydroxy-6-diazirinyl-5α-cholan-24-oate, Avanti Polar Lipids). The samples were irradiated with UV light at 365 nm using a CL-1000 Ultraviolet crosslinker (UVP, Upland, CA, USA) and incubated for 1 h at RT in click reagent solution (100 μM 5-TAMRA-Azide, 1 mM CuSO4, 1 mM tris(2-carboxyethyl)phosphine hydrochloride, and 100 μM tris(benzyltriazolylmethyl)amine). The reaction was stopped with 10 mM EDTA, and excess click reagents were precipitated with 4 x volume of ice-cold acetone before centrifugation for 10 min at 4 °C, 13,000× g RPM. The pellet was washed twice with cold MeOH, re-centrifuged, and air-dried before resuspending in 2 x Laemmli buffer. An SDS-PAGE gel was run, and the bands were visualised with a Cy3/TAMRA/Rhodamine filter set (Ex/Em ~540/568 nm). A no cholesterol reaction was used to ensure that 5-TAMRA-Azide does not bind non-specifically to PTCHD1 or the GDN micelles. Further controls omitting the purified protein, the UV radiation required for cross-linking, or excitation at 546 nm required for the click reaction were used to ensure the cholesterol probe specifically interacts with PTCHD1.

Cells were exposed to UVB (50 J/m²) in a CL-1000 Ultraviolet Crosslinker (UVP). For localized UV irradiation, cells were irradiated through an isopore-polycarbonate membrane (3-μm pore diameter) (Millipore) using UVC (200 J/m²) in a Stratalinker 1800 (Stratagene). For UV-induced CPDs anti-thymine dimer antibody KTM53 (Kamiya Biomedical Company, MC-062) was used followed by incubation with Alexa Fluor 488 donkey anti-mouse antibody (Invitrogen, A-21202). Nuclei were counterstained with DRAQ5 (Cell Signaling Technology, #4084). For XPG detection cells were stained with anti-XPG (Sigma Aldrich, X1629) and developed with Cy3-AffiniPure F(ab’)2 fragment goat anti-rabbit IgG (Jackson ImmunoResearch, 111–166–003). Fluorescence images were obtained with a ConfoCor 2 fluorescence microscope (Carl Zeiss Microscopy).

The cells or transfected cells were irradiated with UVC (254 nm) using a UV lamp CL-1000 Ultraviolet Crosslinker (UVP, Upland, CA). Before UV irradiation, the culture media was removed, cells were irradiated at 20–60 J/m², and fresh media was added to the plates immediately after irradiation. Cells were then cultured at 37°C for various periods. For cisplatin treatment, cells were grown to 80% confluency, and cisplatin (Sigma) was dissolved in DMSO and added to the medium at 25 µM (MCF-7 and MDA-MB-231) or 10 µM (R1) concentration for 24 h. The DMSO vehicle served as the control.

Media was removed from each well and replaced with 10 μl DPBS to ensure the cells did not dry out, but not so much that the UV wasn’t able to penetrate. The plate lid was removed, mock-treated wells covered with foil, and a total dose of 25 J/m² UV delivered to exposed wells in a UVP CL-1000 Ultraviolet crosslinker. Media was immediately replaced and plates returned to the incubator.

The 10 g TiO₂ solution was first bubbled with nitrogen for 30 min to remove the oxygen dissolved in the solution. Then, the solution was mixed with 0.039 g (0.009 mol) AAm and 2.079 g (0.021 mol) DMAA under magnetic stirring for 30 min at 20 °C. The mixed solution was cooled down to 0 °C in an ice water bath. 0.1 wt %. KPS and 8 μL TEMED were then added with another 30 min stirring. The obtained solution was poured into a glass tube (diameter 11 mm and length 50 mm) or a glass mold (150 mm × 75 mm × 3 mm) with the cover up to avoid contact with the oxygen. To facilitate the in situ free-radical polymerization, the hydrogel was put in a UV chamber (UVP CL-1000 Ultraviolet Crosslinker, Upland, CA, USA)) with light intensity 37 W/m² (five 8-Watt light bulbs with 254 nm wave length) for 30 min.