Click it alexa fluor 594

HeLa shCtl cells were transiently transfected with siControl and siCSB. ShHBO1 cells were transiently transfected with siControl. Normal human 48BR fibroblasts were transiently transfected with siControl, siHBO1 or siUVsS-A. UVsS-A Kps3 fibroblasts were transiently transfected with siControl or siHBO1. At 48 h after transfection, cells were subjected to RRS assays were performed. In brief, HeLa and fibroblast cells were irradiated with 9 and 12 J m⁻² ultraviolet, cultured for 12 h, and then cells labelled with 5-ethynyluridine (EU) for 2 h. Incorporation of EU was visualized using Click-it Alexa Fluor 594 or Fluor 488 according to the manufacturer’s instructions (Invitrogen).

Unscheduled DNA synthesis (UDS) was measured following UV-C irradiation (16 J/m²) of C5RO primary fibroblasts grown on 24-mm cover slips and transfected with siRNA. Irradiated cells were incubated for 2 h in the presence of 0.1-mM 5-ethynyl-29-deoxyuridine (EdU; Invitrogen) after UV irradiation. Recovery of RNA synthesis (RRS) was performed in siRNA-transfected HeLa or U20S cells 16 h after UV-C irradiation. Unirradiated and irradiated cells were incubated for 2 h in the presence of 0.1-mM 5-ethynyl-uridine (EU). EdU and EU incorporation was visualized using Click-iT Alexa Fluor 594 according to the manufacturer's protocol (Invitrogen). UDS and RRS levels were quantified by measuring and averaging fluorescence intensities for >100 cells with ImageJ software of images obtained with a Zeiss LSM700 confocal microscope.

EdU at a concentration of 3 μg/mL was added to the tissue slice culture medium 2 h before fixation. Simultaneous TUNEL and EdU staining was performed as described previously [35 (link)]. Briefly, tissue sections were deparaffinized in xylene followed by rehydration in graded alcohols and then blocked with PBS 3% bovine serum albumin (BSA). TUNEL reaction was performed using an In Situ Cell Death Detection Kit (Roche Life Sciences, Penzberg, Germany), after which the sections were incubated with Click-iT Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA) cocktail buffer for 30 min.

An EdU (5‐ethynyl‐20‐deoxyuridine) assay was conducted using a Click‐iT Alexa Fluor 594 according to the manufacturer's protocol (Thermo Fisher Scientific). DPSCs or DPSCs transfected with lentiviral‐mediated shRNA were seeded on glass coverslips and cultured with 100 ng/mL rhBMP2 (R&D Systems) or 25 ng/mL rhFGF8b (R&D Systems) every day. On day 7 of culture, DPSCs were incubated with 10 μM EdU for 1 hour. Then, the cells were fixed in PFA, permeabilized with 0.1% Triton X‐100 and stained with Click‐iT Alexa‐594 dye‐conjugate for 30 minutes as instructed. Samples were co‐stained with Hoechst 33342 to visualize nuclei. Images were acquired using a LSM780 confocal microscope (Zeiss).

For EdU incorporation assays, the Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Assay Kit (ThermoFisher, catalog no. C10646) was used according to manufacturer’s protocol. In short, transfected K562s or TRE3G U2OS cells were treated with AraC for 3 h with 10 μM EdU added during the last hour. Cells were collected, and soluble proteins were extracted with a potassium phosphate buffer (10 mM potassium phosphate, pH 7.4, 0.1% Ipgal-CA-630, 10 mM NaCl, 5 mM MgCl₂) as described (33 ). Cells were fixed by addition of 250 μl of potassium phosphate buffer containing 10% paraformaldehyde to give a final concentration of 2.5% paraformaldehyde for 1 h on ice and washed with ice-cold PBS. Cells (1 ×10⁶/sample) were stained for EdU with ClickIT Alexafluor 594 (ThermoFisher, catalog no. C10330) and DNA with FxCycle Violet (ThermoFisher, catalog no. F10347), according to manufacturer’s protocol. Cells were analyzed on a FACSCanto X SORP (BD Biosciences). Data were analyzed with FlowJo 10.6.1 (©Becton Dickinson and Company). EdU positive cells were gated to determine the EdU-positive population.