TP63 and SOX2 staining in cervical biopsies.

<p>Immunohistochemical staining of cervical biopsies. Bars  =  50µm.(A) normal cervix no primary control; (B) normal cervix stained with anti-SOX2; (C) Squamous cell cervical carcinoma no primary control; (D) representative TP63, and (E) representative SOX2, staining of tumour cells. For both TP63 and SOX2 staining was seen in the nucleus of positive cells (examples indicated by solid arrows); negative cells were a minority of tumour cells (examples indicated by unfilled arrows). (F) Image analysis results of % nuclear +ve tumour cells in biopsies. Parallel sections from 11 cases were stained with SOX2 and TP63. Tumour cells were evaluated for their nuclear expression of the transcription factors. There was no significant difference between the data for SOX2 and TP63 (Wilcoxon signed rank test).</p

Proteomic array detection of stem cell associated proteins in pooled aliquots from 9 HR-HPV+ve cervical samples from women with CIN3 (A) and 9 HPV –ve samples with normal cytology (B); (C & D) map and key for array spots; (E) average pixel density ranked by ratio of pooled CIN3:normal samples for each protein on the array; (F) sox2, TP63 and HCG mRNAs are upregulated in HPV+ve cervical samples with severe dyskaryosis compared to HPV–ve samples with normal morphology (* = p<0.05, ** p<0.01, Mann Whitney test).

<p>Proteomic array detection of stem cell associated proteins in pooled aliquots from 9 HR-HPV+ve cervical samples from women with CIN3 (A) and 9 HPV –ve samples with normal cytology (B); (C & D) map and key for array spots; (E) average pixel density ranked by ratio of pooled CIN3:normal samples for each protein on the array; (F) sox2, TP63 and HCG mRNAs are upregulated in HPV+ve cervical samples with severe dyskaryosis compared to HPV–ve samples with normal morphology (* = p<0.05, ** p<0.01, Mann Whitney test).</p

Samples with increased TRA-1-60+ve cells from patients with CIN3 have enhanced expression of stem cell proteins.

<p>A) Flow cytometric analysis of samples used for proteomic microarray. Dotted lines show that the three samples with a significant increase in TRA-1-60 +ve cells have similar levels of cycling cells to the other three CIN3 samples. *  = p<.05. B) Image-J pixel density analysis of stem cell proteomic microarray. SOX2, TP63 and HCG are further increased in samples with increased TRA-1-60+ve cells. C) Inverted image of the scan of the array from TRA1-60+ve samples; D) Inverted image of the scan of the array from TRA1-60 –ve samples.</p

Flow cytometric detection of cycling cells in cervical samples.

<p>(A) LBC samples stratified by HPV status and histology; samples from women with CIN3 are significantly different from samples with normal cytology and from CIN1 and CIN2, 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus CIN3 group); (B) LBC samples stratified by HPV status and cytology results only; samples with severe dyskaryosis are significantly different from all normal samples 1 way ANOVA (Kruskall Wallis test with Dunn's post-test versus severe disease group). * p = <0.05, ** p = <0.01, *** p = <0.001.</p

Disease status, age range and cytology results for cervical LBC samples obtained from the Scottish national HPV Archive.

<p>Disease status, age range and cytology results for cervical LBC samples obtained from the Scottish national HPV Archive.</p

Sample Flow Cytometry analysis showing gating strategy.

<p>Debris (no DNA stain) and cell clumps (high SSC) are excluded from the analysis (A). Doublets excluded by plotting DRAQ5 area versus height (B). (C) and (D), Cells in HPV-ve normal samples do not stain with anti-TRA-1-60 but strong staining is seen in some samples from women with CIN3 (gates set against individual sample isotype controls). (E) and (F), representative cell cycle profiles from an HPV-ve normal sample (E) and a sample from a patient with CIN3 (F), showing an increased G2/M peak in (F).</p