Figure 1.
Immunohistochemical analysis of CTIP2 in HNSCC.
(A) Expression in adjacent Normal epithelium is restricted to basal cell layers. (B) Expression in dysplasia is stronger than normal and extended to differentiated cell layers. (C–D) In well differentiated tumors, positive staining is restricted to the basal layers at the periphery of the keratinized horn pearl. (E–F) Homogeneous expression of CTIP2 was observed in tumor cells in moderately differentiated tumors. (G–H) Expression of stronger intensities (score 3) of CTIP2 was observed in poorly differentiated tumors. Arrows indicates the CTIP2 positive staining. Yellow dotted lines represents the margin of epidermis and dermis (A), and margin of undifferentiated tumor cell clusters (E) which is magnified in (F). The pictures in the insets (A, B, G and H) are magnified. (Original Magnification: 20×).
Figure 2.
Staining intensities of CTIP2 by immuno-histochemistry in HNSCC.
Staining intensities were 0–3 as described in materials and method sections. (A) Staining intensities of CTIP2 in normal epithelium (NE), dysplasia and HNSCC. Plotted are mean measurements (±S.E.M). *p<0.05. (B) Staining intensities in different histological grades of HNSCC. Plotted are mean measurements (±S.E.M). *p<0.05. (C–D) Co-staining of CTIP2 and BMI1 protein. The insets are magnified. Yellow arrows show the CTIP2 and Bmi1 double positive cells. Yellow arrowhead indicates single positive cells. (E) CTIP2 expression in HNSCC cell lines by western blot analysis. CTIP2 antibody recognizes the two isoforms (Long and the actual CTIP2). Green box represents the normal cell lines.
Table 1.
Staining intensities and percent positivity in normal epithelium and in HNSCC.
Figure 3.
Co-staining of CTIP2, cytokeratin 10 and Ki-67 using immunofluorescence.
(A) Co-staining of CTIP2 and differentiation marker cytokeratin 10 in dysplasia and in moderately differentiated tumor. CTIP2 in green, Cytokeratin 10 in red and DAPI in blue. Yellow dotted lines represents the margin of epidermis and dermis. (B) Co-staining of CTIP2 and a proliferation marker Ki-67 in poorly differentiated tumor. Insets are in yellow boxes and are magnified. Yellow arrows shows the CTIP2-Ki-67 double positive cells. (Original Magnification: 20×).
Figure 4.
Scheme of CTIP2 transcripts and the expression pattern of CTIP2L and CTIP2 in HNSCC.
(A) Two transcripts of human CTIP2 are shown. CTIP2 with all the 4 exons, is the long form of CTIP2 (CTIP2L), and, lacking exon 3 is the actual CTIP2. Primers for the long form were taken from Exon 2 for Forward and exon 3 for reverse, and for the shorter form, the forward from boundary of Exon 2 & 4 and the reverse from exon 4. Expression of the CTIP2 long (B) and CTIP2 (C), by qRT-PCR in HNSCC (n = 28) and 10 normal uvulas based on T size, Tumor staging, and differentiation status. Plotted are the Relative quantification levels normalized with RPLPO as S.E.M±. p values were not significant between the groups in (B) but a significant correlation of higher CTIP2 expression was observed with a poorer histological grade of the tumor, and a trend was noted in a relationship between expression and advanced T or clinical stage in (C) [P<0.05].
Table 2.
Correlation between CTIP2 mRNA expression levels and Clinicopathological characteristics in HNSCC.