C10339

The P7 wild-type mice were treated with EdU (50 mg/kg) for 4 h (Supplementary Fig. 2D), whereas the P15 control and sVEGFR3 expressing mice were treated for 20 h (collected at P16; Supplementary Fig. 8F–H). The ear pinna were collected and prepared as described above. The EdU was labeled with Click-it technology according to the protocol provided by the manufacturer (Invitrogen C10339) followed by LYVE1 staining.

To analyze the cell proliferation in stria at embryonic day E16.5 and E18.5, a single dose of EdU (100 µL/10 g of body weight, C10339, Invitrogen) was administered to timed pregnant females. The cochleae of the neonatal pups were dissected at P0. For the postnatal cell proliferation analyses a single dose of EdU (250 µg/10 g of body weight) was administered subcutaneously to the pups at P0, P3, P6 and P14. These pups were euthanized 6 h after the administration of EdU and their cochleae were extracted. EdU detection was performed according to the manufacturer’s instructions with a ClickiT EdU Alexa Fluor 594 Imaging kit (C10339, Invitrogen). In Edu proliferation assay we have administered Edu in two different pregnant females or two different litters for each stage. For each stage analyzed, we have used three pups (nine sections from each pup). A total of 27 sections each of thickness 12 µm (covering around 108 µm of the cochlea length in each sample) were analyzed. Statistical analysis was done by using a 2-tailed student t test. A p-value ≤0.05 was considered as statistically significant.

To test for repair/DNA synthesis, cells were incubated with 10μM EdU (5-ethynyl-2’-deoxyuridine) 1-20min before laser exposure. A 10mM EdU stock was prepared according to protocol (Invitrogen catalogue #C10339). Cells that require prolonged mitosis were concurrently incubated with colcemid and EdU between 1–20 minutes prior to irradiation. Cells were fixed at time points ranging from 10–120 minutes after irradiation with 4% paraformaldehyde in PBS for 5-10minutes, and followed by blocking buffer containing 10% fetal bovine serum and 0.2% Saponin in PBS for 30minutes

To examine cell proliferation within aggregates by EdU labeling, aggregates were generated in ultra-low adherence 24-well plates as described above. 3 days after aggregation was initiated, 100 μl of growth medium containing EdU was added to aggregates to result in a 150 μM EdU concentration. Aggregates were incubated with EdU for 4 hr and then fixed and resuspended in 300 μl PEM buffer as described above. Aggregates were then processed for imaging following the manufacturer’s protocol (Thermo Fisher C10339) using the 24-well plate membrane insert method described above to transfer aggregates between solutions. For all steps, wells with 1 ml of solution were used for washing or staining cells in membrane inserts, and wells with 100 μl of solution were used to drain wells in preparation for transfer into the next solution.

EdU and other reagents were prepared according to the manufacturer’s instructions (ThermoFisher C10339). In vivo EdU incorporation assays were carried out according to previous publications⁴⁵ . To analyse EdU incorporation in cultured neonatal CMs, cells in 2-well chamber slides were labelled with 10 µM EdU for 12 h. After two washes with pre-warmed PBS, cells were fixed with 4% PFA for 10 min at room temperature and EdU incorporation was visualized using the Click-iT EdU kit (Invitrogen), following the manufacturer’s protocol.

EdU assay was performed to measure OSCC cell proliferation. Cells were seeded in the 96-well plate at 1 × 10⁴ cells/well and treated with or without IR (2 Gy). The cells were incubated with 10 μM of EdU (C10339, Thermo Fisher) at 37 ˚C for 2 h, then fixed in 4% paraformaldehyde and permeabilized in 0.5% Triton X-100 for 30 min. After blocking in 5% BSA for 1 h, 100 μl of staining solution was added to each well and incubated in the dark for 1 h. Next, nuclei were counterstained with DAPI (P36935, Thermo Fisher Scientific). Images were analyzed using the confocal fluorescence microscope system (Nikon C1si; NIKON Instruments Co.).

EdU and other reagents were prepared according to the manufacturer’s instructions (ThermoFisher C10339). In vivo EdU incorporation assays were carried out according to previous publications⁴⁵ . To analyse EdU incorporation in cultured neonatal CMs, cells in 2-well chamber slides were labelled with 10 µM EdU for 12 h. After two washes with pre-warmed PBS, cells were fixed with 4% PFA for 10 min at room temperature and EdU incorporation was visualized using the Click-iT EdU kit (Invitrogen), following the manufacturer’s protocol.