ANE 30-100K 所誘導的自體吞噬在兩種不同的上皮癌細胞中為 Atg5

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附圖與表

圖 1. 人 類 口 腔 鱗 狀 上 皮 癌 細 胞 株 OECM-1 和 食 道 上 皮 癌 細 胞 株 CE81T/VGH 經 RNAi 之技術抑制 Atg5 的表現

(a)OECM-1 的母株細胞、感染含空質體的慢病毒之純化系細胞株(Virus control B5, VC-B5)與感染含 Atg5 RNAi 構築的慢病毒之純化系的細胞株 (si-Atg5 B5 與 si-Atg5 B7)，以西方墨點法偵測 Atg5 與 β-actin 的表現情形。

(b)以同樣的方法在 CE81T/VGH 中取得 VC-A3, VC-A4, si-Atg5 A3 與 si-Atg5 A5，其中以 si-Atg5 A5 的抑制效果最佳。

(a)

(b)

圖 2.抑制 Atg5 的表現對 ANE 30-100K 的細胞毒性之影響

圖 1(a)與圖 1(b)中的細胞以不同濃度的 ANE 30-100K 處理 24 小時後，

以 XTT 分析的方法測定細胞的存活率。結果顯示與 OECM-1 以及 VC-B5 細胞相較之下，OECM-1 的 si-Atg5 B5 與 B7 純化系細胞對 ANE 30-100K 皆具有顯著的耐受性。si-Atg5 B5 vs OECM-1: *p<0.05, **p<0.01，si-Atg5 B7 vs OECM-1: ^#p<0.05, ^##p<0.01；而 CE81T/VGH 則只有 si-Atg5 A5 純化系細 胞對 ANE 30-100K 具有顯著的耐受性，si-Atg5 A3 則不明顯。si-Atg5 A5 vs

(a)

(b)

CE81T: *p<0.05, **p<0.01。

圖 3.抑制 Atg5 的表現對 ANE 30-100K 所誘導的 LC3-II 堆積之影響

(a)OECM-1, VC-B5 與 si-Atg5 B7 細胞與(b)CE81T/VGH, VC-A3, VC-A4, si-Atg5 A3 與 si-Atg5 A5 細胞，經 ANE 30-100K(分別為 8.3 與 10 mg/ml)處 理 24 小時後，進行西方墨點法分析 LC3-II 與β-actin 的表現情形。結果發現 OECM-1 的 si-Atg5 B7 細胞中 LC3-II/actin 的比值低於 OECM-1 與 VC-B5；

而 CE81T/VGH 的 si-Atg5 A3 與 si-Atg5 A5 細胞中 LC3-II/actin 的比值同樣 低於 CE81T 與 VC-A3 與 VC-A4。

(a)

(b)

(a)

(b)

圖 4. 抑制 Atg5 的表現對 ANE 30-100K 所誘導的酸性泡產生之影響 (a)OECM-1, VC-B5 與 si-Atg5 B7 細胞與(b)CE81T/VGH, VC-A3, VC-A4, si-Atg5 A3 與 si-Atg5 A5 細胞，經 ANE 30-100K(分別為 8.3 與 10 µg/ml)處 理 24 小時後，進行 acridine orange 的染色並在螢光顯微鏡下觀察酸性泡產 生的情形。Bar=20 µM。照片下方的圖為在一個顯微鏡的視野下隨機計算 100 顆細胞中含有酸性泡細胞的數目，並且計算了 3 個視野。結果顯示在所 有 Atg5 表現遭到抑制的 OECM-1 與 CE81T/VGH 純化系細胞中，酸性泡的 產生均有顯著減少的情形。*p<0.05, **p<0.01。