Dnase 1 solution

Secretory-stage and maturation-stage enamel organ cells from mandibular incisors of 4-week old Wistar Hanover rats were collected as previously described (Lacruz et al., 2012b (link); Wen et al., 2014 (link)), and RNA extraction was performed using a QIAshredder, an RNeasy Protect Mini Kit, and DNase I solution from Qiagen (Valencia, CA, USA). Reverse transcription and real-time PCR were performed using the iScript cDNA Synthesis kit and SYBR Green Supermix from BioRad, respectively. Real-time PCR was performed on the CFX96 system (BioRad Laboratories, Hercules, CA, USA) in 10 μl volumes with a final primer concentration of 100 nm, for 40 cycles at 95°C for 10 s and 58°C for 45 s. Six independent real-time PCR analyses were conducted using samples from a total of 6 rats, 3 males, and 3 females, for each gene of interest (primers are listed in Table 2), and for both stages of amelogenesis. The male and female data were analyzed separately and no significant differences were noted between the sexes, so the data presented in the graph were generated from all 6 animals (n = 6). Rat enamel organ is preferred to mouse enamel organ for real-time PCR and western blot studies because separating secretory and maturation stage from adult mouse incisors is technically difficult and yields less RNA and protein per animal.

TUNEL assay was performed on paraffin-embedded testis sections using the DeadEnd Fluorometric TUNEL System according to manufacturer’s instructions (Promega, Madison, WI, USA). The positive control sections were pre-incubated with DNase I solution (Qiagen, Valencia, CA, USA), while negative control sections were incubated in incubation buffer without recombinant terminal deoxynucleotidyl transferase (rTdT enzyme). Slides were mounted with fluoromount mounting medium containing DAPI (SouthernBiotech, Birmingham, AL, USA) and visualized using a Nikon Eclipse fluorescent microscope (Nikon Corporation). 10 pictures were taken for each animal and TUNEL-positive cells were counted using FIJI software (Schindelin et al., 2012 (link)).

Nematodes (~50μL pellet) were harvested after 0 hours (L1 stage), 44 hours (day 1 adult) and 92 hours (day 3 adults) on OP50. RNA was isolated using TRIzol (Invitrogen) and purified by RNeasy Mini Kit (Qiagen) and DNase I solution (Qiagen), and cDNA was made by ProtoScript First Strand cDNA Synthesis Kit (New England Biolabs). A 10μL reaction consisted of 5ng of cDNA, 1X Power SYBR Green PCR Master Mix (Life Technologies), and 5pmol each of the forward and reverse primers. The qRT-PCR reactions were executed on ABI 7900HT Fast Realtime PCR platforms and the cycle threshold (CT) values were exported from the Sequence Detection System 2.3 software. qRT-PCR efficiency values were calculated from mean CT values and were then used to calculate the relative expression of each gene as described [27 (link)], normalized to the expression of the housekeeping gene, cdc-42.