PMC:5608921 / 24633-32298 JSONTXT

{"project":"2_test","denotations":[{"id":"28846079-11063126-79081959","span":{"begin":401,"end":403},"obj":"11063126"},{"id":"28846079-18548112-79081960","span":{"begin":2087,"end":2089},"obj":"18548112"},{"id":"28846079-11063126-79081961","span":{"begin":6518,"end":6520},"obj":"11063126"}],"text":"Materials and Methods\n\nAnimal handling and treatments\nThe genetic background of Tg(K6b-E6/E7)-M8 transgenic mice (always hemizygous for the transgene) was that of CD1 outbred or Fvb/N inbred strains. These and the corresponding WT mice were maintained and treated in accordance with the regulations of the local Bioethical Committee. Genotype was determined by E7-specific PCR, as previously reported.26 Bilateral ovariectomy was performed at the moment of ear perforation, whereas castration in males was performed 2 weeks before ear punching. Pellets embedded with 0.05 mg ß-estradiol (E2) 17-acetate (90-day release; NE-271; Innovative Research of America, Sarasota, FL, USA) were implanted in the dorsal-anterior area of mice at the day of ear perforation for long-term ear regeneration experiments; whereas, pellets embedded with 0.25 mg E2 17-acetate (90-day release; NE-271) were implanted for estrous cycle determination (19 days) or for cancer experiments (up to 3 pellets for 9 months). For short-term regeneration studies, E2 (0.7 μg/100 μl dissolved in corn oil) or the vehicle were injected subcutaneously every day up to for 14 days; hormone was not injected on the day of ear perforation or of BrdU injection. For ER inhibition, female mice were intraperitoneally injected with raloxifene (Eli Lilly, Indianapolis, IN, USA; 10 mg/ml in phosphate-buffered saline (PBS)); for regeneration experiments, 1.5 mg of raloxifene was delivered daily for 14 days or 5 days/week for a month and, during the estrous cycle, the same amount was delivered daily through 19 days or once at proestrus. BrdU (50 μg/g weight of mice) was intraperitoneally injected (1 dose) and mice killed 2 h later.\n\nHole closure assay\nExcisional 2 mm punches were made with a metal ear puncher on the center of ears of 6 weeks and 3-month-old mice. The hole diameter was measured 4 weeks after punching under a dissecting microscope. Formation of new hair follicles and cartilage was determined by visualizing them in samples stained with fast green and safranin after 24 h or 3 months of ear perforation.21 Ear regeneration efficiency was classified into four categories (gray scale in bar graphs) based on the following estimation:\nPercent of regeneration (Pr)=100−(100·non-regenerated area/area excised)\n\nwhere r1=measured diameter (mm)/2, and r2 =2 mm/2=1 mm\nTherefore,\n\nAnalysis of estrous cycle\nMice for analysis of estrous cycles were CD1-based 9 weeks old, whereas those for promotion of cervical–uterine carcinomas were Fvb/N-based 1-month-old. The estrous cycle phase was determined by analysis of cell composition and morphology of vaginal smears, taken every day at the same hour, stained with hematoxylin–eosin. The number of estrous cycles was determined by counting the number of proestrus–estrus (P–E) sequence along 19 days.46 Samples for molecular analysis were obtained along the estrus cycle or at specific estrus phase in the case of E2-treated mice.\n\nHistochemistry and immunodetection procedures\nEars and/or cervix were dissected from mice treated as described above, fixed in 4% paraformaldehyde and kept in 30% sucrose at 4 °C until sectioning. Frozen sections (10 μm) were used for all determinations. Ki67 and incorporated BrdU were the antigens used to determine the number of proliferating cells in at least six sections per sample initially subjected to a permeabilizing solution (1% Tritón X-100, 3% H2O2 in TBS) at room temperature for 15 min, then incubated with 1 N HCl for 20–30 min and neutralized with 0.1 M boric acid pH 8.5; for BrdU detection, we additionally incubated with 0.001% trypsin. All tissue sections were subjected to heat-induced epitope retrieval with ImmunoDNA Retriever Citrate (Bio SB Inc, Santa Barbara, CA, USA) at 60 °C for 30 min and treated with 0.3% H2O2 in methanol for 30 min. Tissues for keratin 5 (K5) and 6b (K6b) and E7 oncogene detection were permeabilized at this stage with 0.1% Triton X-100 in PBS for 30 min. After washing with PBS, samples were treated for 30–60 min with a blocking solution (10% mouse serum in PBS for K5, K6b and E7, and 10% donkey serum, 0.1% Triton X-100 in TBS for Ki67 and BrdU). Slides were then incubated in a humid chamber for 30–60 min at 25 °C and overnight at 4 °C with the specific antibodies (Supplementary Table S1), washed twice with PBS and incubated with the corresponding secondary antibody (Supplementary Table S1) for 2 h. Regardless of the secondary antibody used for histochemical determinations, after washing with PBS, tissue sections were incubated with the ABC Elite (Avidin/Biotin) System (1:200 dilution; Vector Laboratories, Burlingame, CA, USA) at 25 °C for 30 min. The horseradish peroxidase activity was developed using H2O2 and 3,3-diaminobenzidine. Dying cells were detected with In Situ Cell Detection Kit (Roche, Mannheim, Germany). Finally, tissue sections were counterstained with DAPI or hematoxylin, mounted with ProLon Gold (ThermoFisher Scientific, Waltham, MA, USA) and photographed using an invert microscope (confocal LSM 510, Apotome Axio Observer Z1, Zeiss, Jena, Germany; confocal FV1000 or BX51, Olympus, Tokyo, Japan). To count Ki67+ and BrdU+ cells, a section of tissue was taken and positive nuclei counted along a defined linear extension (at least 400 μm) of epithelium.\n\nReverse transcription quantitative PCR (RT-qPCR) procedure\nRNA was obtained from cervix and ears of WT or Tg(K6b-E/6E7) mice using the Hybrid-R kit (GeneAll, Seoul, South Korea). For estrous cycle analysis, 9-week-old CD1 mice were selected at each estrous cycle phase and the whole cervix dissected. For ear regeneration, 3–4 perforations were done in one ear of 3-month-old CD1 female mice and, 7 days later, both injured and non-injured ears were collected. First-strand cDNA was synthetized using HyperScript Reverse Transcriptase (GeneAll, Seoul, South Korea) and random primers (Invitrogen, Carlsbad, CA, USA). The real-time quantitative PCR was performed using KAPA SYBR FAST mix (KAPA Biosystems, Wilmington, MA, USA) in the presence of specific primers (Supplementary Table S2) and the Rotor-Gene Q (Qiagen, Wilmington, MA, USA). Gene expression was evaluated using a ΔΔCt method. The housekeeping gene Rplp0 was used to normalize gene expression levels.\n\nStatistic analysis\nAnimals were sorted only by genotype or treatment, and although exclusion or inclusion of an animal was not predetermined, some mice of the Tg(K6b-E6/E7) died during the experimental treatment (the majority died before 2 months of age26); it is important to mention, however, that the cause of death was never associated with the presence of tumors. Specific blinding or randomization method was not applied. The size of each experimental group was limited according with reproducibility and extent of difference; generally, small groups (3–4 independent individuals) were only considered for determination with an evident qualitative difference between groups. Fisher's exact test was used for the analysis of ear regeneration data; since there is a low correlation between a mouse and the regeneration efficiency of its ears, each ear was taken as an independent regeneration event. For other experiments, the t-Student test was performed when the distribution was normal, or the Mann–Whitney Rank-Sum Test when the data distribution was not normal. Since the intact and perforated ear sample for gene expression analysis derived from the same mouse, the paired t-Student test was used in this case. The results are shown as the mean±s.d. and the variance analysis was performed when two data sets were analyzed. We considered significant differences when the P-value was ≤0.05. "}