TOX stimulated MF cell proliferation and invasion through AKT phosphorylation.

<p>(A) Western blot assays showed that LY294002 can inhibited AKT phosphorylation. The expression of p-AKT was normalized to GAPDH. (B) Western blot assays showed that overexpression of TOX stimulated AKT phosphorylation. The expression of p-AKT was normalized to GAPDH. (C) The proliferative role of TOX overexpression was largely blocked by LY294002, an AKT inhibitor, in MyLa cells were transfected with TOX vector, control or not transfected or TOX vector and LY294002. (D) The invasive role of TOX overexpression was largely blocked by LY294002, an AKT inhibitor, in MyLa cells were transfected with TOX vector, control or not transfected or TOX vector and LY294002. ** p<0.01, *p<0.05, and ***p<0.001.</p

TOX expression in MF and comparison group.

<p>All p values were compared to MF.</p><p>MF mycosis fungoides, BID Benign inflammatory diseases,NSNormal skin, Pos no. positive numbers</p><p>TOX expression in MF and comparison group.</p

Primer sequences for TOX, CD4 and GAPDH.

<p>Primer sequences for TOX, CD4 and GAPDH.</p

Representative immunohistochemical staining of TOX in different stages of MF lesions.

<p>(A) Patch stage MF. Only a few infiltrating MF cells were positive for TOX while the majority of lymphocytes stained negative. (B) Plaque stage MF. Atypical infiltrating lymphocytes in the dermis were positive for TOX. (C) Tumor stage MF. The majority of infiltrating lymphocyteswere stained positive.</p

Characteristics of subjects with mycosis fungoides (n = 35).

<p>Characteristics of subjects with mycosis fungoides (n = 35).</p

TOX accelerates the MyLa cell migration and invasion.

<p>(A) Migration assays of the Myla cells after treatment with TOX vector, control or not transfected; the relative ratio of migratory cells per field is shown on the right.(B) Invasion analysis of the Myla cells after treatment with TOX vector, control or not transfected; the relative ratio of invasive cells per field is shown on the right, ***p<0.001.</p

Representative immunohistochemical staining for TOX and CD4 in a patch stage MF lesion.

<p>Bright focal nuclei staining on lymphoid cells were considered to be positive. The arrow indicates the epidermotropic MF cells in a Pautrier’s microabscess and papillary dermal MF cells staining positive with TOX and CD4.</p

The TOX expression in MF lesion by Western blot analysis.

<p>(A) CD4 protein expression in MF、BID and NS. (B) TOX protein expression in MF、BID and NS.The signal in each lane was quantified using Image J software and the ratio of TOX and CD4 to GAPDH were determined. Error bars represent SD. MF mycosis fungoides, BID Benign inflammatory diseases, NS Normal skin</p

TOX accelerates the MyLa cell proliferation.

<p>(A) Western blot analysis showed that TOX vector could enhance the expression of TOX. The expression of TOX was normalized to GAPDH. (B)CCK8 assays have showed that overexpression of TOX increased the Myla cell proliferation compared with either the control vector-transfected cells or the untreated cells. (C)The effect of TOX on cell cycle progression of the Myla cell after 48-h incubation was detected by flow cytometry analysis. (D) TOX promoted Ki-67 mRNA expression. The Myla cells were transfected with TOX vector, controlornot transfected. Ki-67were detected by real-time PCR.(E)TOXpromotedKi-67protein expression. The Myla cells were transfected with TOX vector, controlornot transfected. Ki-67was detected by western blot.The expression of ki-67 was normalized to GAPDH. Values are presented as mean±SD. As compared with control, ** p<0.01, *p<0.05, and ***p<0.001</p

The TOX expression in MF lesion by real-time RT PCR.

<p>(A) TOX mRNAs in MF、BID and NS. (B) CD4 mRNAs in MF、BID and NS. RT-PCR analysis was performed in triplicate and the expression levels of TOX and CD4 mRNAs were normalized to GAPDH mRNAs. Error bars represent as SD.</p