Association between the characteristic phenotype of HPV–associated lesions and genotype of HPV E6/E2 DNA of mice.

<p>Association between the characteristic phenotype of HPV–associated lesions and genotype of HPV E6/E2 DNA of mice.</p

Primer sequences.

<p>Primer sequences.</p

Normalized relative expression of miR-155 in transgenic mice (HPV+) and wild-type mice (HPV-), in chest (a) and ear tissue (b).

<p>Normalized relative expression of miR-155 in transgenic mice (HPV+) and wild-type mice (HPV-), in chest (a) and ear tissue (b).</p

Normalized relative expression of miR-155 in ear and chest normal tissue (a).

<p>Normalized expression of miR-155 in different lesions of K14-HPV16 transgenic mice (CIS and hyperplasia) (b).</p

Wild-type (-/-) and K14-HPV16 transgenic (+/-) mice.

<p>Transgenic mice show a hunched position, partial thoracic and cephalic alopecia, together with extensive hyperkeratosis and auricular erythema.</p

Histology of wild-type and transgenic mice.

<p>a) Wild-type (-/-) female FVB/n mouse. Chest skin showing normal histology. H&E, 200x bar = 50μm b) K14-HPV16 transgenic (+/-) female FVB/n mouse. Chest skin showing epidermal hyperplasia and orthokeratotic hyperkeratosis. Note increased number of epidermal strata with conserved orderly squamous differentiation. c) K14-HPV16 transgenic (+/-) female FVB/n mouse. Ear skin showing in situ carcinoma. Note loss of epidermal stratification and progressive differentiation, presence of suprabasal mitotic figures and anisocytosis and abrupt parakeratotic keratinization with hyperkeratosis. The underlying stroma exhibits intense mixed inflammatory cell infiltration and neovascularization.</p

Mice genotyping.

<p>The presence of integrated HPV was assessed by amplification of HPV-E2 (b) and HPV-E6 (c) genes by polymerase chain reaction methodology (PCR) in-house. Samples 1 and 3 are HPV+; sample 2 is HPV-. Mouse-β-globin gene was used as endogenous control (a). M: molecular weight size marker: (a)100 bp, (b,c)50 bp; C-: negative control.</p

Overview of genotyping, histological and miR-155 profiling results.

<p>MiR-155 levels are significantly higher in normal chest skin compared with ear skin samples. Targeted expression of HPV-16 oncogenes to basal keratinocytes leads to multistep skin carcinogenesis–transgenic ear skin samples showed CIS while chest samples showed epidermal hyperplasia. Hyperplastic (chest) skin samples showed a significant miR-155 downregulation compared with matched wild-type samples. No differences were observed between wild-type and transgenic ear samples or between transgenic ear and chest samples.</p

Molecular features and neuroretinal function.

<p><b>(A)</b> Retinal VEGF was measured by qPCR at intervals (0,1,2,3,5 and 8 days) following exposure to hyperoxia (n = 4–7 per group. <b>(B)</b> GFAP expression measured by qPCR (n = 5–7 per group). <b>(C)</b> Representative images of GFAP immunostaining in retinal sections from mice after the conventional and the accelerated protocol. <b>(C)</b> and <b>(C’)</b> show the variability within the conventional OIR group. <b>(D to G)</b> Electroretinographic (ERG) a-wave and b-wave amplitudes in scotopic conditions were measured at P26-27 <b>(D, E)</b> and at P60 <b>(F, G)</b> in mice previously exposed to the conventional and accelerated OIR protocols, and in age-matched non-OIR controls (n = 4–13 per group). Data are expressed as means ± SEM.</p

Retinal vasculature regression and pre-retinal neovascularization following hyperoxia and 5 days in room air.

<p><b>(A)</b> The extent of oxygen-induced retinal vascular regression was consistently greater (in 3 independent experimental groups) in the accelerated protocol (85% O₂ from P8 to P11) than in the conventional protocol (75% O₂ from P7 to P12). No significant differences were evident between independent experimental groups within each protocol, and the variances were similar (Barlett's test, p = 0.6429 for retinal vascular regression and p = 0.1415 for pre-retinal neovascularization). <b>(B)</b> Representative images show the extent of retinal vascular regression (delineated in white) in isolectin B4-stained flat-mounted retinas of mice in the conventional and the accelerated OIR protocols. Five days following return to room air, the extents of both persistent retinal vascular regression <b>(C)</b> and pre-retinal neovascularization <b>(D)</b> were similar in retinas of mice in both protocols. <b>(E)</b> Representative images of flat-mounted retinas of mice from the conventional and accelerated protocols illustrate the area of persistent retinal vascular regression (delineated in white), and the area of pre-retinal neovascularization (delineated in yellow). Scale bars: 0.5 mm. n = 6–14 per group. Data are expressed as means ± SEM.</p