Prognostic Significance of CD200 Protein Expression and its Correlation with COX-2 in Cutaneous Melanoma

Supplementary Figure 1. Expression of CD200 and COX-2 in melanoma tissue. Immunohistochemical staining for (A) CD200 (X 100) and (B) COX-2 (X 40) in melanoma tissue. Paraffin-embedded sections were immunostained with anti-CD200 (R&D systems, Minneapolis, MN, USA; 1:5000) and anti-COX-2 (Neomarkers, CA, USA; 1:50). Double immunofluorescence staining in melanoma tissue of (C) melan-A and CD200 (melan-A, red; CD200, green), (D) melan-A and COX-2 (melan A, red; COX-2, green), and (E) CD200 and COX-2 (CD200, green; COX-2, red). Double-positive cells are indicated by the yellow signals. (F) Correlation between CD200 and COX-2 protein expression in cutaneous melanoma.

Supplementary Figure 2. Progression-free survivals depending on (A) CD200 (B) COX-2 expression in melanoma patients. COX-2, cyclooxygenase-2

Supplementary Figure 3. Effects of COX-2 inhibitor on the expression of CD200 in melanoma cell line. (A, B) Western blotting analysis to evaluate the effect of COX-2 inhibitor on the level of CD200 in Human BRAFV600E A375 melanoma cell. A375 cells were treated with the selective COX-2 inhibitor, celecoxib at different doses. CD200 was downregulated by celecoxib at dose from 60 μM. *p < 0.05, **p < 0.01.

补充图1 黑色素瘤组织中CD200与环氧合酶-2（cyclooxygenase-2，简称COX-2）的表达。对黑色素瘤组织进行免疫组织化学染色：(A) CD200（放大倍数×100），(B) COX-2（放大倍数×40）。石蜡包埋组织切片分别以抗CD200抗体（R&D Systems公司，美国明尼苏达州明尼阿波利斯市；稀释比例1:5000）与抗COX-2抗体（Neomarkers公司，美国加利福尼亚州；稀释比例1:50）进行免疫染色。黑色素瘤组织的双重免疫荧光染色结果如下：(C) 黑素-A（melan-A）与CD200（黑素-A呈红色荧光，CD200呈绿色荧光），(D) 黑素-A与COX-2（黑素-A呈红色荧光，COX-2呈绿色荧光），(E) CD200与COX-2（CD200呈绿色荧光，COX-2呈红色荧光），双重阳性细胞以黄色信号标注。(F) 皮肤黑色素瘤中CD200与COX-2蛋白表达的相关性分析。

补充图2 黑色素瘤患者的无进展生存期分别依据(A) CD200、(B) COX-2的表达水平进行分组的结果。注：COX-2即环氧合酶-2（cyclooxygenase-2）。

补充图3 COX-2抑制剂对黑色素瘤细胞系中CD200表达的影响。(A、B) 通过蛋白质印迹法（Western blotting）分析选择性COX-2抑制剂塞来昔布不同剂量处理后人BRAFV600E型A375黑色素瘤细胞内CD200的表达水平。实验结果显示，当塞来昔布剂量达到60 μM及以上时，CD200的表达被显著下调。*p < 0.05，**p < 0.01。