Pan-Inhibition of Protein Disulfide Isomerase Caused Cell Death through Disrupting Cellular Proteostasis in Pancreatic Ductal Adenocarcinoma Cells
蛋白二硫键异构酶的泛抑制通过破坏胰腺导管腺癌细胞中的细胞蛋白质稳态导致细胞死亡

2.1. The PDI Inhibitor, E64FC26, Inhibited Proliferation of PDAC Cells
2.1. PDI 抑制剂 E64FC26 抑制 PDAC 细胞增殖

We first investigated messenger (m)RNA expressions of PDI family members using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) database [25] in normal human pancreatic and PDAC tissues. As shown in Figure 1, most PDI family members were upregulated except for PDIA2 and ERP27 in human PDAC tissues. Compared to normal pancreatic tissues, the top three members with the highest fold changes in expression in human PDAC tissues were AGR2 (fold change 6.392), AGR3 (fold change 4.689), and PDIA3 (fold change 1.961). These results suggested that PDI family members might play important roles in PDAC cell proliferation.
我们首先使用基因表达分析交互式分析 2 （GEPIA2） 数据库 [ 25 ] 在正常人胰腺和 PDAC 组织中研究了 PDI 家族成员的信使 （m）RNA 表达。如图所示 Figure 1 ，除人 PDAC 组织中 PDIA2 和 ERP27 外，大多数 PDI 家族成员均上调。与正常胰腺组织相比，人 PDAC 组织中表达倍数变化最高的前三个成员是 AGR2 （倍数变化 6.392） 、 AGR3 （倍数变化 4.689） 和 PDIA3 （倍数变化 1.961）。这些结果表明 PDI 家族成员可能在 PDAC 细胞增殖中发挥重要作用。

To examine whether inhibition of PDI activity could decrease the proliferation of PDAC cells, we used E64FC26, a novel PDI inhibitor, to treat human pancreatic cancer cell lines AsPC-1 and BxPC-3 cells for 24 and 48 h and determined cell viability by an MTT assay. As shown in Figure 2, E64FC26 significantly decreased the viability of both cell lines in dose-dependent manners. The 50% inhibitory concentrations (IC₅₀) with 24 h of treatment were 6.13 ± 0.08 and 0.93 ± 0.33 µM in AsPC-1 and BxPC-3 cells, respectively; and IC₅₀ values after 48 h of treatment were 3.41 ± 0.11 and 0.87 ± 0.16 µM, respectively. Prolonged drug treatment for 48 h was more effective than 24 h treatment in AsPC-1 cells, but there was no significant difference in BxPC-3 cells. These results suggest that the E64FC26 PDI inhibitor significantly inhibited proliferation of PDAC cells.
为了检查抑制 PDI 活性是否可以减少 PDAC 细胞的增殖，我们使用新型 PDI 抑制剂 E64FC26 处理人胰腺癌细胞系 AsPC-1 和 BxPC-3 细胞 24 和 48 小时，并通过 MTT 测定测定细胞活力。如 Figure 2 所示，E64FC26 以剂量依赖性方式显著降低两种细胞系的活力。AsPC-1 和 BxPC-3 细胞处理 24 h 的 50% 抑制浓度 （IC₅₀） 分别为 6.13 ± 0.08 和 0.93 ± 0.33 μM;处理 48 小时后的 IC₅₀ 值分别为 3.41 ± 0.11 和 0.87 ± 0.16 μM。在 AsPC-1 细胞中，延长药物治疗 48 h 比处理 24 h 更有效，但在 BxPC-3 细胞中无显著差异。这些结果表明，E64FC26 PDI 抑制剂显着抑制了 PDAC 细胞的增殖。

2.2. The PDI Inhibitor, E64FC26, Induced ER Stress and the UPR
2.2. PDI 抑制剂 E64FC26 诱导的 ER 应激和 UPR

PDI inhibitors are known to be able to impede the correct disulfide bond formation of nascent proteins and cause protein misfolding [26]. Accumulation of misfolded proteins can increase ER stress and the UPR. To explore whether E64FC26 could induce ER stress and the UPR in PDAC cells, we detected protein expressions of ER stress- and UPR-related signaling pathways. Grp78, a major ER stress-inducible chaperone, was time-dependently upregulated by E64FC26 treatment in both cell lines (Figure 3A). E64FC26 also significantly increased Grp78 expressions in dose-dependent manners in both cell lines (Figure 3B). E64FC26-induced ER stress led to significant PERK phosphorylation, and activated PERK then directly phosphorylated eIF2α in both cell lines. E64FC26 did not change the PDI protein expression in either cell line (Figure 3B). These results suggest that the PDI inhibitor, E64FC26, significantly increased ER stress and the UPR in PDAC cells.
已知 PDI 抑制剂能够阻碍新生蛋白质的正确二硫键形成并导致蛋白质错误折叠 [ 26 ]。错误折叠蛋白的积累会增加 ER 应激和 UPR。为了探讨 E64FC26 是否可以诱导 PDAC 细胞中的 ER 应激和 UPR，我们检测了 ER 应激和 UPR 相关信号通路的蛋白表达。Grp78 是一种主要的 ER 应激诱导伴侣，在两种细胞系中，E64FC26处理均呈时间依赖性上调 （ Figure 3 A）。E64FC26 还以剂量依赖性方式显着增加了两种细胞系中 Grp78 的表达 （ Figure 3 B）。E64FC26诱导的 ER 应激导致显著的 PERK 磷酸化，激活的 PERK 随后在两种细胞系中直接磷酸化 eIF2α。E64FC26 没有改变两种细胞系 （ Figure 3 B） 中的 PDI 蛋白表达。这些结果表明，PDI 抑制剂 E64FC26 显著增加了 PDAC 细胞的 ER 应激和 UPR。

2.3. The PDI Inhibitor, E64FC26, Caused Cell Death Partially via Apoptosis
2.3. PDI 抑制剂 E64FC26 部分通过细胞凋亡导致细胞死亡

It is known that persistent ER stress and the UPR can eventually trigger cell apoptosis [27]. To examine whether the PDI inhibitor, E64FC26, can induce apoptosis in PDAC cells, we detected expressions of cleaved caspases by Western blotting and caspase activities. E64FC26 at 10 and 1.5 µM in AsPC-1 and BxPC-3 cells, respectively, induced very limited expressions of cleaved PARP and cleaved caspase-3 (Figure 4A), but cleaved caspase-8 and caspase-9 were absent from Western blots. To further confirm whether E64FC26 can induce cell apoptosis in PDAC cells, we treated cells with a higher concentration of E64FC26 and directly detected caspase activity with a commercially available caspase activity assay kit. After increasing the E64FC26 concentration to 15 and 2 µM, respectively, in AsPC-1 and BxPC-3 cells, E64FC26 significantly induced caspase-3, -8, and -9 activities using a caspase activity assay kit (Figure 4B). These results suggest that the PDI inhibitor, E64FC26, partially induced cell death through apoptosis in PDAC cells.
众所周知，持续的 ER 应激和 UPR 最终会触发细胞凋亡 [ 27 ]。为了检查 PDI 抑制剂 E64FC26 是否可以诱导 PDAC 细胞凋亡，我们通过 Western blotting 和 caspase 活性检测了裂解的 caspase 的表达。在 AsPC-1 和 BxPC-3 细胞中分别以 10 μM 和 1.5 μM E64FC26诱导非常有限的裂解 PARP 和裂解的 caspase-3 （ Figure 4 A） 的表达，但裂解的 caspase-8 和 caspase-9 在蛋白质印迹中不存在。为了进一步确认 E64FC26 是否可以诱导 PDAC 细胞凋亡，我们用较高浓度的 E64FC26 处理细胞，并用市售的 caspase 活性测定试剂盒直接检测 caspase 活性。在 AsPC-1 和 BxPC-3 细胞中将 E64FC26 浓度分别增加到 15 和 2 μM 后，E64FC26 使用 caspase 活性测定试剂盒 （ Figure 4 B） 显著诱导 caspase-3 、 -8 和 -9 活性。这些结果表明，PDI 抑制剂 E64FC26 通过 PDAC 细胞凋亡部分诱导细胞死亡。

2.4. PDI Inhibitor E64FC26-Induced Cell Death Might Be Associated with Ferroptosis
2.4. PDI 抑制剂 E64FC26 诱导的细胞死亡可能与铁死亡有关

Ferroptosis is a kind of non-apoptotic cell death that depends on iron and lipid peroxidation (LPO) [28]. It was reported that upregulation of ER stress contributes to ferroptosis [29]. To investigate whether E64FC26 caused cell death through ferroptosis, we used the iron-chelating agent, deferoxamine (DFO), and the ROS scavengers, ferrostatin-1 and NAC, to reverse E64FC26-caused cell death. As shown in Figure 5A, pretreatment with 5 and 10 µM of DFO significantly reversed 5 and 10 µM of E64FC26-induced cell death, respectively, in AsPC-1 cells with both 24 and 48 h of treatment. Although DFO alone caused partial cell death, 1 and 5 µM of DFO also reversed 3 µM E64FC26-induced cell death in BxPC-3 cells at both 24 and 48 h of treatment. At both 24 and 48 h, 1 and 5 µM of ferrostatin-1 reversed the 5 µM E64FC26-induced death of AsPC-1 cells, and 2 and 3 µM of E64FC26-induced cell death in BxPC-3 cells (Figure 5B). In addition, 10 mM of NAC prevented cell death under 10 µM E64FC26 treatment for both 24 and 48 h in AsPC-1 cells, and 5 and 10 mM of NAC increased cell survival under 2 and 3 µM of E64FC26 treatment at both 24 and 48 h in BxPC-3 cells (Figure 5C). Interestingly, we detected no significant increase in ROS by the cytosolic ROS sensor, 2’-7’-dichlorodihydrofluorescein diacetate (DCF-DA), in E64FC26-treated cells. These results suggest that E64FC26-caused cell death might be partially mediated through ferroptosis in PDAC cells.
铁死亡是一种非凋亡性细胞死亡，依赖于铁和脂质过氧化 （LPO） [ 28 ]。据报道，ER 应激的上调会导致铁死亡 [ 29 ]。为了研究 E64FC26 是否通过铁死亡导致细胞死亡，我们使用铁螯合剂去铁胺 （DFO） 和 ROS 清除剂铁司他汀-1 和 NAC 来逆转 E64FC26 引起的细胞死亡。如 Figure 5 A 所示，在处理 24 小时和 48 小时的 AsPC-1 细胞中，用 5 μM 和 10 μM DFO 预处理分别显着逆转了 5 μM 和 10 μM E64FC26诱导的细胞死亡。尽管单独的 DFO 导致部分细胞死亡，但在处理的 24 小时和 48 小时，1 μM 和 5 μM 的 DFO 也逆转了 BxPC-3 细胞中 3 μM E64FC26诱导的细胞死亡。在 24 和 48 小时，1 μM 和 5 μM 的 ferrostatin-1 逆转了 5 μM E64FC26诱导的 AsPC-1 细胞死亡，以及 BxPC-3 细胞中 2 μM 和 3 μM E64FC26诱导的细胞死亡 （ Figure 5 B）。此外，在 AsPC-1 细胞中，10 mM 的 NAC 在 10 μM E64FC26处理下 24 和 48 小时可防止细胞死亡，并且在 2 μM 和 3 μM E64FC26处理下，5 和 10 mM 的 NAC 在 BxPC-3 细胞中 24 和 48 小时均能增加细胞存活率 （ Figure 5 C）。有趣的是，我们在 E64FC26 处理的细胞中检测到胞质 ROS 传感器 2'-7'-二氯二氢荧光素二乙酸酯 （DCF-DA） 的 ROS 没有显着增加。这些结果表明，E64FC26 引起的细胞死亡可能是通过 PDAC 细胞中的铁死亡部分介导的。

2.5. The PDI Inhibitor, E64FC26, Caused Autophagic Cell Death by Blocking Autolysosome Formation
2.5. PDI 抑制剂 E64FC26 通过阻断自噬溶酶体的形成导致自噬细胞死亡

Previous studies demonstrated that PDAC cells have a high basal level of autophagy, which enables them to continue cell proliferation in vivo and in vitro and enhances resistance to chemotherapy and radiation therapy [30,31]. LC3B-II is commonly used as an indicator of mammalian autophagosome formation. To further examine whether the PDI inhibitor, E64FC26, can induce cell death through autophagy, we detected LC3B-II expression by Western blotting and LC3B puncta formation in PDAC cells. As shown in Figure 6A, E64FC26 treatment resulted in LC3B-II accumulation in a dose-dependent manner in both cell lines. Moreover, E64FC26 increased LC3B puncta formation as revealed by IF staining (Figure 6B,C). Expressions of components of autophagy initiation, including VPS34 and Atg7, did not change upon E64FC26 treatment. The final process of autophagy is the fusion of autophagosomes and lysosomes into autolysosomes, which allows degradation of their contents, including SQSTM1/p62. Interestingly, E64FC26 did not induce SQSTM1/p62 degradation but significantly increased SQSTM1/p62 expressions in a dose-dependent manner in both cell lines (Figure 6A). These results suggest that E64FC26 induced incomplete autophagy by increasing the LC3B-II level but failed to degrade SQSTM1/p62.
先前的研究表明，PDAC 细胞具有高水平的基础自噬，这使它们能够在体内和体外继续细胞增殖，并增强对化疗和放疗的抵抗力 [ 30 ， 31 ]。LC3B-II 通常用作哺乳动物自噬体形成的指标。为了进一步研究 PDI 抑制剂 E64FC26 是否可以通过自噬诱导细胞死亡，我们通过 Western blotting 检测 LC3B-II 在 PDAC 细胞中的表达和 LC3B 点状形成。如 Figure 6 A 所示，E64FC26处理导致 LC3B-II 在两种细胞系中以剂量依赖性方式积累。此外，E64FC26 增加了 IF 染色 （ Figure 6 B，C） 显示的 LC3B 点形成。自噬起始成分的表达，包括 VPS34 和 Atg7，在 E64FC26 处理后没有变化。自噬的最后一个过程是自噬体和溶酶体融合成自噬溶酶体，这允许降解其内容物，包括 SQSTM1/p62。有趣的是，E64FC26 没有诱导 SQSTM1/p62 降解，但在两种细胞系中以剂量依赖性方式显着增加 SQSTM1/p62 表达 （ Figure 6 A）。这些结果表明，E64FC26通过增加 LC3B-II 水平诱导不完全自噬，但未能降解 SQSTM1/p62。

To investigate why SQSTM1/p62 was not degraded with E64FC26 treatment, we first transfected the autophagy reporter plasmid FUW mCherry-GFP-LC3 into PDAC cells to express the mCherry-GFP-LC3 fusion protein. The mCherry protein is stable at a neutral pH and in an acidic condition, while the GFP protein is only stable at neutral pH but is acid-labile. Therefore, the mCherry-GFP-LC3 fusion protein displays both green and red fluorescence in the neutral environment of the autophagosome lumen, whereas it only exhibits red fluorescence in the acidic condition of the autolysosome lumen. Compared to control cells, E64FC26 treatment significantly increased yellow fluorescent spots in both cell lines (Figure 7A), indicating that both mCherry and GFP were expressed in autophagosomes of these cells. These results suggest that E64FC26 treatment might block autolysosome formation through inhibiting the fusion of autophagosomes and lysosomes in PDAC cells. To further confirm whether E64FC26 blocks the fusion of autophagosomes and lysosomes, we used AO dye to stain acidic vesicles of cells. As shown in Figure 7B, control cells displayed a lot of reddish-orange fluorescence dots, indicating the formation of mature lysosomes and acidic autolysosomes. However, cells treated with E64FC26 exhibited few reddish-orange fluorescence dots, indicating that E64FC26 blocked autolysosome formation or induced lysosome defects. CTSL is a lysosomal protease that is activated by lysosomal cleavage of pre-CTSL to form mature CTSL. To examine whether E64FC26 impaired lysosome function, we detected expressions of pre-CTSL and mature CTSL by Western blotting in PDAC cells. As shown in Figure 7C, E64FC26 treatment dose-dependently caused the accumulation of pre-CTSL, but decreased mature CTSL in both cell lines. These results suggest that E64FC26 might block autolysosome formation through inducing lysosome defects in PDAC cells.
为了研究 SQSTM1/p62 在 E64FC26 处理后未降解的原因，我们首先将自噬报告基因质粒 FUW mCherry-GFP-LC3 转染到 PDAC 细胞中以表达 mCherry-GFP-LC3 融合蛋白。mCherry 蛋白在中性 pH 值和酸性条件下稳定，而 GFP 蛋白仅在中性 pH 值下稳定，但耐酸性。因此，mCherry-GFP-LC3 融合蛋白在自噬体管腔的中性环境中同时显示绿色和红色荧光，而它仅在自噬溶酶体管腔的酸性条件下显示红色荧光。与对照细胞相比，E64FC26处理显着增加了两种细胞系 （ Figure 7 A） 中的黄色荧光斑点，表明 mCherry 和 GFP 在这些细胞的自噬体中均表达。这些结果表明，E64FC26治疗可能通过抑制 PDAC 细胞中自噬体和溶酶体的融合来阻止自噬溶酶体的形成。为了进一步确认 E64FC26 是否阻断自噬体和溶酶体的融合，我们使用 AO 染料对细胞的酸性囊泡进行染色。如 Figure 7 B 所示，对照细胞显示大量红橙色荧光点，表明成熟溶酶体和酸性自溶酶体的形成。然而，用 E64FC26 处理的细胞表现出很少的红橙色荧光点，表明E64FC26阻断了自噬溶酶体的形成或诱导了溶酶体缺陷。CTSL 是一种溶酶体蛋白酶，可被 pre-CTSL 的溶酶体裂解激活以形成成熟的 CTSL。为了检查E64FC26溶酶体功能是否受损，我们通过 Western blotting 检测 PDAC 细胞中 pre-CTSL 和成熟 CTSL 的表达。 如 Figure 7 C 所示，E64FC26 处理剂量依赖性地导致 pre-CTSL 的积累，但在两种细胞系中成熟 CTSL 降低。这些结果表明，E64FC26 可能通过诱导 PDAC 细胞中的溶酶体缺陷来阻断自体溶酶体的形成。

4.1. Chemicals and Antibodies
4.1. 化学品和抗体

Deferoxamine (DFO) and E64FC26 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Ferrostatin-1 was purchased from Cayman Chemical (Ann Arbor, MI, USA), and N-acetylcysteine (NAC) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary rabbit polyclonal anti-PKR-like ER kinase (PERK), rabbit polyclonal anti-eukaryotic initiation factor 2α (eIF2-α), and mouse polyclonal anti-cathepsin L (CTSL) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); rabbit polyclonal anti-phosphorylated (p)-PERK, rabbit polyclonal anti-p-eIF2-α, rabbit polyclonal anti-autophagy related 7 (Atg7), rabbit polyclonal anti-light chain 3 (LC3), rabbit polyclonal anti-cleavage caspase-3, rabbit polyclonal anti-p-mammalian target of rapamycin (mTOR), and rabbit polyclonal anti-mTOR were obtained from Cell Signaling Technology (Danvers, MA, USA); rabbit polyclonal anti-glucose-regulated protein 78 (GRP78), mouse polyclonal anti-α-tubulin, rabbit polyclonal anti-vacuolar sorting protein 34 (VPS34), mouse polyclonal anti-sequestosome 1 (SQSTM1)/p62, and rabbit polyclonal anti-GAPDH were purchased from Genetex (Irvine, CA, USA); and rabbit polyclonal anti-PDI was obtained from Abcam (Waltham, MA, USA).
去铁胺 （DFO） 和 E64FC26 购自 MedChemExpress（美国新泽西州蒙茅斯章克申）。Ferrostatin-1 购自 Cayman Chemical（美国密歇根州安娜堡），N-乙酰半胱氨酸 （NAC） 购自 Sigma-Aldrich（美国密苏里州圣路易斯）。原代兔多克隆抗 PKR 样 ER 激酶 （PERK）、兔多克隆抗真核起始因子 2α （eIF2-α） 和小鼠多克隆抗组织蛋白酶 L （CTSL） 购自 Santa Cruz Biotechnology（美国加利福尼亚州圣克鲁斯）;兔多克隆抗磷酸化 （p）-PERK、兔多克隆抗 p-eIF2-α、兔多克隆抗自噬相关 7 （Atg7）、兔多克隆抗轻链 3 （LC3）、兔多克隆抗裂解 caspase-3、兔多克隆抗 p-哺乳动物雷帕霉素靶标 （mTOR） 和兔多克隆抗 mTOR 从 Cell Signaling Technology（美国马萨诸塞州丹佛斯）获得;兔多克隆抗葡萄糖调节蛋白 78 （GRP78） 、小鼠多克隆抗 α-微管蛋白、兔多克隆抗液泡分选蛋白 34 （VPS34）、小鼠多克隆抗螺隔线体 1 （SQSTM1）/p62 和兔多克隆抗 GAPDH 购自 Genetex （Irvine， CA， USA）;兔多克隆抗 PDI 购自 Abcam （Waltham， MA， USA）。

4.2. Cell Culture  4.2. 细胞培养

The human AsPC-1 and BxPC-3 PDAC cell lines were kindly provided by Prof. Shiow-Lin Pan (Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical University, Taipei, Taiwan). Both AsPC-1 and BxPC-3 cells were cultured in Roswell Park Memorial Institute (RPMI) medium (Gibco; ThermoFisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (FBS) and a 1% penicillin/streptomycin solution, and maintained in a humidified incubator at 37 °C with 5% CO₂.
人 AsPC-1 和 BxPC-3 PDAC 细胞系由 Shiow-Lin Pan 教授（台北医学大学癌症生物学和药物发现研究所，台北，台湾）友情提供。AsPC-1 和 BxPC-3 细胞均在罗斯威尔公园纪念研究所 （RPMI） 培养基 （Gibco;ThermoFisher Scientific，美国马萨诸塞州沃尔瑟姆）加入 10% 胎牛血清 （FBS） 和 1% 青霉素/链霉素溶液，并维持在 37 °C 和 5% CO₂ 的加湿培养箱中。

4.3. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay
4.3. 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物 （MTT） 测定

AsPC-1 and BxPC-3 cells were seeded in 96-well plates at 6 × 10³ and 9 × 10³ cells/well, respectively. At the end of each experiment, cells were changed to 50 µL of MTT medium and incubated for another 3~4 h. The MTT medium was removed, and 100 µL of DMSO was added to dissolve the MTT formazan, and finally the absorbance at an optical density (OD) of 570 nm was measured on an enzyme-linked immunosorbent assay (ELISA) plate reader [57].
将 AsPC-1 和 BxPC-3 细胞分别以 6 × 10³ 和 9 × 10³ 个细胞/孔接种在 96 孔板中。每次实验结束时，将细胞更换为 50 μL MTT 培养基，再孵育 3~4 h。去除 MTT 培养基，加入 100 μL DMSO 以溶解 MTT 甲臜，最后在酶联免疫吸附测定 （ELISA） 读板器上测量 570 nm 光密度 （OD） 处的吸光度 [ 57 ]。

4.4. Western Blot Analysis
4.4. 蛋白质印迹分析

Cells were seeded at (8~9) × 10⁵ cells in 6 cm dishes. After treatment, cells were lysed in gold lysis buffer (137 mM of NaCl, 20 mM of Tris at pH 7.9, 10 mM of NaF, 1% Triton X-100, 10% glycerol, 5 mM of EDTA, 1 mM of EGTA, 1 mM of phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mM of sodium orthovanadate, 1 mM of sodium pyrophosphate, and 100 µM of β-glycerophosphate), and 10~30 µg of total cell lysates was used in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in the gel were then transferred to polyvinylidene difluoride (PVDF) membranes and visualized using enhanced chemiluminescence kits (Amersham, Arlington, IL, USA) in an ImageQuant^TM LAS4000 Imager system (GE Healthcare Life-Sciences, Taiwan Branch, Taipei, Taiwan) [58]. Relative band intensities of Western blots were quantified using ImageJ software (version 1.54d; National Institutes of Health, Bethesda, MD, USA).
将细胞接种在 （8~9） × 10⁵ 个细胞中，放入 6 cm 培养皿中。处理后，在金裂解缓冲液（137 mM NaCl、20 mM pH 值为 7.9 的 Tris、10 mM NaF、1% Triton X-100、10% 甘油、5 mM EDTA、1 mM EGTA、1 mM 苯甲基磺酰氟、10 μg/mL 抑肽酶、10 μg/mL 亮肽素、1 mM 原钒酸钠、1 mM 焦磷酸钠、 和 100 μM β-甘油磷酸酯），10~30 μg 总细胞裂解物用于十二烷基硫酸钠聚丙烯酰胺凝胶电泳 （SDS-PAGE）。然后将凝胶中的蛋白质转移到聚偏二氟乙烯 （PVDF） 膜上，并使用增强的化学发光试剂盒（美国伊利诺伊州阿灵顿阿默舍姆）在 ImageQuant^TM LAS4000成像系统（GE Healthcare Life-Sciences，台湾分公司，台北，台湾）中使用增强化学发光试剂盒（美国伊利诺伊州阿灵顿）进行可视化 [ 58 ]。使用 ImageJ 软件（版本 1.54d;美国国立卫生研究院，贝塞斯达，马里兰州，美国）。

4.5. Caspase Activity Assay
4.5. 半胱天冬酶活性测定

Cells were seeded at 7 × 10⁵ cells in 6 cm dishes for 1 day and treated with E64FC26 for another 24 h. Cells were lysed in cell lysis buffer, and 100 µg of total cell lysates was transferred into a 96-well plate. The reaction buffer and individual caspase substrates were added and incubated for 1 h at 37 °C, and then the absorbance at OD 405 nm was measured with an ELISA plate reader according the manufacturer’s instructions (Colorimetric Caspase Assay Kit; BioVision, Waltham, MA, USA) [59].
将细胞在 7 × 10⁵ 个细胞接种在 6 cm 培养皿中 1 天，并用 E64FC26 再处理 24 小时。在细胞裂解缓冲液中裂解细胞，并将 100 μg 总细胞裂解物转移至 96 孔板中。加入反应缓冲液和单个半胱天冬酶底物并在37°C下孵育1小时，然后根据制造商的说明用ELISA读板器测量OD 405 nm处的吸光度（比色半胱天冬酶测定试剂盒;BioVision，美国马萨诸塞州沃尔瑟姆）[ 59 ]。

4.6. Transient Transfection
4.6. 瞬时转染

Cells were plated at 4 × 10⁵ cells in 3.5 cm dishes for 1 day and then transfected with the FUW mCherry-GFP-LC3 plasmid (Addgene plasmid no. 110060) using the Lipofectamine^TM 3000 reagent (Life Technologies, Taiwan Brand, Taipei, Taiwan). After cells were treated with drugs, green fluorescent protein (GFP) and mCherry fluorescence levels were observed and photographed with a Confocal Spectral Microscope Imaging System (Leica TCS SP5, Singapore) [59].
将细胞在 4 × 10⁵ 个细胞中接种在 3.5 cm 培养皿中 1 天，然后使用 Lipofectamine^TM 3000 试剂（Life Technologies，台湾品牌，台北，台湾）转染 FUW mCherry-GFP-LC3 质粒（Addgene 质粒编号 110060）。用药物处理细胞后，观察绿色荧光蛋白 （GFP） 和 mCherry 荧光水平，并使用共聚焦光谱显微镜成像系统（Leica TCS SP5，新加坡）拍照 [ 59 ]。

4.7. Acridine Orange (AO) Staining
4.7. 吖啶橙 （AO） 染色

AsPC-1 and BxPC-3 cells (4 × 10⁵ cells) were cultured in 3.5 cm confocal dishes. Cells were treated with drugs and stained with 2 µg/mL of an AO (3,6-bis (dimethylamino) acridine hydrochloride) solution for 15 min in a cell culture incubator [60]. Fluorescence microscopic images were obtained with a Leica TCS SP5 confocal microscope (Leica Microsystems) using excitation at 458 nm and an emission filter at 480~560 or 590~660 nm.
AsPC-1 和 BxPC-3 细胞 （4 × 10⁵ 个细胞） 在 3.5 cm 共聚焦培养皿中培养。用药物处理细胞，并在细胞培养箱中用 2 μg/mL AO（3,6-双（二甲氨基）吖啶盐酸盐）溶液染色 15 分钟 [ 60 ]。使用 Leica TCS SP5 共聚焦显微镜 （Leica Microsystems） 在 458 nm 激发和 480~560 或 590~660 nm 发射滤光片下获得荧光显微图像。

4.8. Immunofluorescence (IF) Staining
4.8. 免疫荧光 （IF） 染色

A cover slide was plated in a 12-well plate, and 1 × 10⁵ cells were seeded for 1 day. After drug treatment, cells were subjected to IF staining. First, cells were fixed with 4% paraformaldehyde for 20 min, and permeabilized in a 0.5% Triton X-100 solution for 15 min. Then, cells were incubated in a blocking buffer (5% bovine serum albumin in phosphate-buffered saline) for 1 h, in a primary antibody solution (prepared in blocking buffer) overnight at 4 °C, and finally incubated with a CF^®488A-conjugated secondary antibody (Biotium, Fremont, CA, USA) for 1 h at room temperature. After IF staining, cell nuclei were stained with 1 mg/mL DAPI for 10 min, and cover slides with cells were mounted with 10 µL Fluromount-G (SouthernBiotech, Birmingham, AL, USA). Fluorescence microscopic images were obtained with a Leica TCS SP5 confocal microscope (Leica Microsystems) [59].
将盖玻片接种在 12 孔板中，将 1 × 10^{个 5} 个细胞接种 1 天。药物治疗后，对细胞进行 IF 染色。首先，用 4% 多聚甲醛固定细胞 20 分钟，并在 0.5% Triton X-100 溶液中透化 15 分钟。然后，将细胞在封闭缓冲液（5% 牛血清白蛋白在磷酸盐缓冲盐水中）中孵育 1 小时，在 4 °C 下在一抗溶液（在封闭缓冲液中制备）中孵育过夜，最后与 CF^®488A 偶联的二抗（Biotium，Fremont，CA，USA）在室温下孵育 1 小时。IF 染色后，用 1 mg/mL DAPI 对细胞核染色 10 分钟，并用 10 μL Fluromount-G（SouthernBiotech，美国阿拉巴马州伯明翰）封片细胞。使用徕卡 TCS SP5 共聚焦显微镜 （Leica Microsystems） [ ] 获得荧光显微图像 59 。

4.9. Statistical Analysis
4.9. 统计分析

Data are presented as the mean ± standard error (S.E.) for the indicated number of independently performed experiments. Statistical analyses were performed using a one-way Student’s t-test by GraphPad Prism 9 software, and differences were considered significant at p < 0.05.
数据表示为指定数量的独立执行实验的平均值±标准误差 （SE）。使用 GraphPad Prism 9 软件的单向学生 t 检验进行统计分析，差异被认为显著，p < 0.05。