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圖 1. Cadherin-11 對於細胞擴展作用之影響
轉染表現cadherin-11 載體或控制載體的 L 細胞 及 C4-2B4 細胞，在 cad11-Fc 或 Fc 塗層培養板上的表現。
(A) 表現有 cadherin-11 的 L-cell/cad11 細胞在 cad11-Fc 塗層培養板上呈現明顯擴展 的片狀/絲狀偽足扁平型態，且隨著 cad11-Fc 濃度存在劑量正相關性。
(B) 表現有 cadherin-11 的 C4-2B4 前列腺癌細胞 (C4-2B4/cad11) 在 cad11-Fc 塗層 培養板上具有相同的片狀/絲狀偽足扁平型態表現。
(C) 在自身已表現有 cadherin-11 的 PC3-mm2 細胞，在 Cad11-Fc 塗層培養板上同 樣表現扁平、偽足狀細胞擴展，在Fc 的塗層培養板上，則皆維持細胞的原型 型狀。
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圖 2. Cadherin-11 在各種成骨細胞株上的表現及對細胞吸附作用之影響
(A) 以西方墨點法並利用辨識 cadherin-11 細胞內區段的單殊抗體 5B2H5，檢視各 種成骨細胞株cadherin-11 的表達量，除了未分化的 C2C12 細胞，均有一定量 的表現。
(B) 以免疫細胞染色法檢視，利用 goat anti-cadherin-11 多株抗體 (R&D Systems) 及DAPI 染色 (藍色) ，Cadherin-11 (紅色) 表現於 MC3T3-E1 及 UMR106 細 胞間接合處。圖面標示Bar 代表 10 m 大小。
(C) MC3T3-E1 及 UMR 細胞株在細胞吸附實驗，可吸附於 cadherin-11-Fc 預先處理 過的培養盤，但未能吸附於BSA 或 cadherin-12-Fc, 且其作用會因為加入 EDTA 而被抑制。
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圖 3. Cadherin-11 促使前列腺癌細胞吸附於成骨細胞並改變細胞的形狀表現 (A) 在 C4-2B4/vector 與 MC3T3-E1 以 2:1 比例混合後，於細胞培養皿中，
C4-2B4/vector 細胞與 MC3T3-E1 細胞各自吸附至培養皿上，且 C4-2B4/vector 細胞多數維持圓形形狀。
(B) 在 C4-2B4/cad-11 與 MC3T3-E1 以 2:1 比例混合後，C4-2B4/cad-11 多數吸附於 MC3T3-E1 細胞上，且呈現扁平形狀。
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圖 4. Cadherin-11 介導前列腺癌細胞與成骨細胞間的吸附作用
(A) 以 GFP 螢光標記的 C4-2B4 前列腺癌細胞培養於長滿 MC3T3-E1 成骨細胞培 養皿，C4-2B4/cad11 細胞呈現擴展的偽足形狀，並嵌入 MC3T3-E1 細胞層中。
C4-2B4/vector 則呈現圓形形狀於 MC3T3-E1 細胞層上。
(B) 以共軛顯微鏡 Z 軸不同階層取得影像，在 a 層較小細胞核的 C4-2B4/cad11 呈 現偽足形狀，並藉由抗人類cadherin-11 單株抗體 4B6 染色，可觀察到
C4-2B4/cad11 嵌入 MC3T3-E1 細胞層(b 和 c 層，呈現較大且有小點的細胞核) 中。
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圖 5. Cadherin-11 促使前列腺癌細胞嵌入成骨細胞層
(A) 在 C4-2B4/cad-11 與 MC3T3-E1 共同培養實驗，於共軛顯微鏡下由上而下漸層 觀察。
(B) C4-2B4/cad-11(紅色免疫染色) 嵌入 MC3T3-E1 細胞層，且呈現扁平偽足形狀。
(C) C4-2B4/vector 與 MC3T3-E1 共同培養，並無任何被染色訊息。
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圖 6. Cadherin-11 對於細胞移行 (migration) 及侵襲 (invasion)作用之影響 (A) C4-2B4/cadherin-11 與 C4-2B4/vector 的細胞移行作用。左上角的圖為移行至
insert 另一側細胞之照片，右上角為量化的資料。左下角則為 PC3-mm2-shcad11 及PC3-mm2-shcontrol 以西方墨點 (Western blot) 法確認是否表現有
cadherin-11，並以 actin 作為檢測量之對照。右下角圖面則為 PC3-mm2-shcad11 及PC3-mm2-shcontrol 移行作用的量化資料。
(B) C4-2B4/cad11 相較於 C4-2B4/vector cells 及 PC3-mm2/shcontrol 相較於 PC3-mm2/shcad11 的細胞侵襲作用量化資料 。
圖面Bar 代表 10 m。資料為 3 組取平均值± SD。 *, P<0.05.
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圖 7. Cadherin-11 對於前列腺癌細胞的增殖、存活、
docetaxel 治療敏感性，以及非附著依賴性生長 的影響
(A) Cadherin-11 的表現，不影響 C4-2B4 細胞在培養皿 或MC3T3-E1 細胞層上的生長。
(B) (C)在 serum starvation 與 docetaxel 的存活測試，
cadherin-11 的表現，不影響 C4-2B4 細胞的生長。
(D) Cadherin-11 的表現，不影響 C4-2B4 細胞非附著依 賴性生長。
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圖 8.表現完整 cadherin-11 及其突變序列之 C4-2B4 細胞鑑別分析
(A) Cadherin-11 mutant 簡要圖示。E1-E5: extracellular cadherin repeats 1-5.
TM: transmembrane domain. JMD: juxtamembrane cytoplasmic domain.
CBS: -catenin-binding sequence.
(B) 以免疫沉澱法及西方墨點法分析 C4-2B4/vector, C4-2B4/cad11, C4-2B4/JMD, C4-2B4/CBS 之 cadherin-11 及 p120-catenin 或 -catenin 的表現。
C4-2B4/JMD, C4-2B4/CBS 有較小的分子量。以抗體免疫沉澱法分析，
C4-2B4/cad11 同時結合了 p120-catenin 及-catenin 。C4-2B4/JMD 及 C4-2B4/CBS 則分別失去了 p120-catenin 及 -catenin 的結合能力
(C) 以 anti-human cadherin-11 4B6 單株抗體進行免疫染色及 FACS 分析，確認各細 胞表面均正常表現cadherin-11。
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圖 9. Cadherin-11 細胞內區域對細胞擴展 (spreading) 作用之影響
(A) C4-2B4/cad11, C4-2B4/JMD 及 C4-2B4/CBS 在覆蓋有 cadherin-11-Fc 培養 板上均有良好的細胞擴展作用。
(B) 將 C4-2B4 細胞培養在長滿 MC3T3-E1 成骨細胞培養皿上，C4-2B4/cad11, C4-2B4/JMD 及 C4-2B4/CBS 均呈現細胞擴展及偽足狀外型，C4-2B4/vector 則仍呈現圓形形狀。
C4-2B4/cad11, C4-2B4/JMD 及 C4-2B4/CBS 有相同的 cadherin-11 細胞外區 域表現，其細胞擴展及與成骨細胞間的互動也有一致的呈現。
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圖 10. Cadherin-11 細胞內區域對細胞移行 (migration) 及侵襲 (invasion) 作用之 影響
(A) 以 Fc 或 cad11-Fc coating 的 Falcon HTS FluoroBlok Inserts，檢測 C4-2B4/vector, C4-2B4/cad11, C4-2B4/JMD 及 C4-2B4/CBS 細胞的移行作用，
C4-2B4/JMD 及 C4-2B4/CBS 之作用明顯不如 C4-2B4/cad11，左側為顯微 鏡下螢光染色照片，右側為量化資料。
(B) 以 Biocoat Matrigel invasion chambers 檢測 C4-2B4/vector, C4-2B4/cad11, C4-2B4/JMD 及 C4-2B4/CBS 細胞的侵襲作用，C4-2B4/JMD 及
C4-2B4/CBS 之作用明顯不如 C4-2B4/cad11，且與 C4-2B4/vector 相似，左 側為顯微鏡下DAPI 染色照片，右側為量化資料。 (照片中 Bar 代表 10 m)
A
B
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圖 11. Cadherin-11 的表現對 C4-2B4 細胞侵襲 (invasion) 作用相關基因表現之影 響
以Ilumina 基因分析平台進行 C4-2B4/vector, C4-2B4/cad11, C4-2B4/JMD 及 C4-2B4/CBS 細胞的基因微陣列分析，紅色代表基因表現量增加，綠色代表基因 表現量下降。
56
圖 12. Cadherin-11 的表現對 C4-2B4 細胞移行 (migration) 作用相關基因表現之影 響
以Ilumina 基因分析平台進行 C4-2B4/vector, C4-2B4/cad11, C4-2B4/JMD 及 C4-2B4/CBS 細胞的基因微陣列分析，紅色代表基因表現量增加，綠色代表基因 表現量下降。
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圖 13.Cadherin-11 及其多株抗體對細胞聚集 (aggregation) 作用之影響
(A) Cadherin-11 介導的細胞黏附作用促使 L-cad11 細胞隨著時間發生細胞聚集作 用，故可觀察到單細胞數目逐漸減少，但L-vector 細胞則無明顯之細胞聚集作 用。
(B) Anti-cadherin-11 多株抗體 (20 g/mL)可抑制 L-cad11 細胞之聚集作用。
(C) Anti-cadherin-11 多株抗體抑制 L-cad11 細胞之聚集作用具有劑量相關性。
*, P<0.05.
58
圖 14. Anti-cadherin-11 單株抗體對於細胞聚集作用之影響
將L-cad11 細胞懸浮於含 10% FBS 的 DMEM 細胞培養液，加入 20 g/mL 單株抗體，持續以搖臂式混合1 小時，並計數單一細胞比例。1A5 及 2C7 單株抗體有較明顯之細胞聚集抑制作用，並達約80%。