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Fig. 1. PKD2 was highly expressed in LUAD and associated with ferroptosis. A PKD2 mRNA expression in TCGA tumors vs normal tissues (if available) by TIMER 2.0. B PKD2 mRNA expression in 8 pairs of LUAD and paracarcinoma tissues by qRT-PCR. C Representative immunohistochemical images of PKD2 in alveolus, mesenchyme, LUAD tissues. Left: 100X, scale bar, 200 μm; Right: 400X, scale bar, 50 μm. D IHC score of PKD2 in normal, paracarcinoma and LUAD tissues. E Comparison of tumor volume between PKD2 low- and high-score groups. F GSEA enrichment analyses. G Comparison of sensitivity of sorafenib between PKD2 high and low expression groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图1 . PKD2 在 LUAD 中高表达并与铁死亡相关。通过 TIMER 2.0 比较TCGA肿瘤与正常组织(如果有)中的 PKD2 mRNA 表达。 B通过 qRT-PCR 检测 8 对 LUAD 和癌旁组织中 PKD2 mRNA 的表达。 C肺泡、间充质、LUAD 组织中 PKD2 的代表性免疫组织化学图像。左:100X,比例尺,200 μm;右:400X,比例尺,50 μm。 D正常组织、癌旁组织和 LUAD 组织中 PKD2 的 IHC 评分。 E PKD2低分组和高分组之间肿瘤体积的比较。 F GSEA 富集分析。 G PKD2高、低表达组索拉非尼敏感性比较。 * P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。
Table 1. Demographics and clinicopathologic characteristics of LUAD patients from IHC microarray.
表 1 . IHC 微阵列中 LUAD 患者的人口统计学和临床病理特征。
Variables 变量 | LUAD | |||
---|---|---|---|---|
Total (n = 61) 总计(n = 61) | High score (n = 40) 高分 (n = 40) | Low score (n = 21) 低分(n = 21) | P value P值 | |
Age 年龄 | ||||
<60 years <60年 | 34 (55.7) | 23 (37.7) | 11 (18.0) | 0.702 |
≥60 years ≥60岁 | 27 (44.3) | 17 (27.9) | 10 (16.4) | |
Sex 性别 | ||||
Male 男性 | 36 (59.0) | 22 (36.1) | 14 (23.0) | 0.379 |
Female 女性 | 25 (41.0) | 18 (29.5) | 7 (11.5) | |
Histological subtype 组织学亚型 | ||||
Mural 壁画 | 2 (3.3) | 2 (3.3) | 0 (0.0) | 0.960 |
Papillary | 20 (32.8) | 12 (19.7) | 8 (13.1) | |
Solid | 10 (16.4) | 7 (11.5) | 3 (4.9) | |
Acinar-papillary | 3 (4.9) | 2 (3.3) | 1 (1.6) | |
Acinar-solid | 5 (8.2) | 3 (4.9) | 2 (3.3) | |
Acinar | 18 (29.5) | 12 (19.7) | 6 (9.8) | |
Mucous | 3 (4.9) | 2 (3.3) | 1 (1.6) | |
Grade | ||||
I | 15 (24.6) | 12 (19.7) | 3 (4.9) | 0.186 |
II | 31 (50.8) | 17 (27.9) | 14 (23.0) | |
III | 15 (24.6) | 11 (18.0) | 4 (6.6) | |
T stage | ||||
T1 | 11 (18.0) | 6 (9.8) | 5 (8.2) | 0.620 |
T2 | 23 (37.7) | 14 (23.0) | 9 (14.8) | |
T3 | 22 (36.1) | 16 (26.2) | 6 (9.8) | |
T4 | 5 (8.2) | 4 (6.6) | 1 (1.6) | |
N stage | ||||
N0 | 27 (44.3) | 17 (27.9) | 10 (16.4) | 0.660 |
N1 | 8 (13.1) | 4 (6.6) | 4 (6.6) | |
N2 | 22 (36.1) | 16 (26.2) | 6 (9.8) | |
N3 | 4 (6.6) | 3 (4.9) | 1 (1.6) | |
M stage M级 | ||||
M0 | 60 (98.4) | 40 (65.6) | 20 (32.8) | 0.164 |
M1 | 1 (1.6) | 0 (0.0) | 1 (1.6) | |
Tumor volume (cm3) 肿瘤体积(cm 3 ) | ||||
≤50 | 22 (36.1) | 10 (16.4) | 12 (19.7) | 0.044 |
>50 | 29 (47.5) | 22 (36.1) | 7 (11.5) | |
NA | 10 (16.4) | 8 (13.1) | 2 (3.3) |
Fig. 2. Abrogation of PKD2 promoted erastin-induced ferroptosis of LUAD cells. A The ROS content in A549 cells with silencing PKD2 with or without erastin treatment (20 μM, 24h). B The ROS content in PC9 cells with PKD2 overexpression with or without erastin treatment (20 μM, 24h). C, D Erastin-induced ROS content detected by flow cytometry in A549 and PC9 cells with or without PKD2 knockdown treated by erastin treatment (20 μM, 24h). E, F Erastin-induced ROS content detected by flow cytometry in A549 and PC9 cells with or without PKD2 inhibitor CRT0066101. G, H Relative MDA content in A549 and PC9 cells with erastin (20 μM, 24h) treatment when PKD2 was knocked down. I, J Percentage of PI staining positive cells in A549 and PC9 cells with or without PKD2 knockdown treated by erastin (50 μM, 24h). K, L Relative total iron content in A549 and PC9 cells with PKD2 knockdown stimulated by erastin (20 μM, 24h). M, N Relative total iron content in A549 and PC9 cells with PKD2 overexpression stimulated by erastin (20 μM, 24h). All statistical data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图2 . PKD2 的废除促进了erastin 诱导的LUAD 细胞铁死亡。 A沉默 PKD2 的 A549 细胞中 ROS 含量,无论是否经过erastin 处理(20 μM,24 小时)。 B PKD2 过表达的 PC9 细胞中 ROS 含量,无论是否经过erastin 处理(20 μM,24 小时)。 C、D通过流式细胞术检测经过erastin处理(20μM,24小时)的PKD2敲低或未敲除的A549和PC9细胞中Erastin诱导的ROS含量。 E、F通过流式细胞术检测有或没有 PKD2 抑制剂 CRT0066101 的 A549 和 PC9 细胞中 Erastin 诱导的 ROS 含量。 G、H当 PKD2 被敲低时,用erastin(20 μM,24 小时)处理的 A549 和 PC9 细胞中的相对 MDA 含量。 I、J经过erastin(50 μM,24 小时)处理或未敲除PKD2 的A549 和PC9 细胞中PI 染色阳性细胞的百分比。 K、L经erastin(20 μM,24 小时)刺激PKD2 敲低的A549 和PC9 细胞中的相对总铁含量。 M、N经erastin(20 μM,24 小时)刺激PKD2 过表达的A549 和PC9 细胞中的相对总铁含量。所有统计数据均以平均值±标准差表示; n = 3。* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。
Fig. 3. PKD2 inhibited autophagic degradation of ferritin. A Immunoblots of ferroptosis regulator in PC9 cells with PKD2 knockdown or overexpression with erastin (20 μM, 24h) treatment. B, C Immunoblots of FTL protein in A549 and PC9 cells with PKD2 knockdown or overexpression treated by erastin (20 μM, 24h). D, E mRNA expression of NCOA4, FTH1, FTL in A549 cells with PKD2 knockdown or overexpression. F–I Immunoblots of FTH1 and NCOA4 in A549 and PC9 cells with PKD2 knockdown or overexpression treated by erastin (20 μM, 24h) with or without bafilomycin A1 (100 nM, 4h). J Representative immunohistochemical images of PKD2, FTH1 in LUAD tissue from two cases. 100X, scale bar, 200 μm; 400X, scale bar, 50 μm. H Representative confocal images show the colocalization of FTL and LAMP2 in experimental cells. The coefficient of colocalization was calculated by Image J. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图3 . PKD2 抑制铁蛋白的自噬降解。 A PKD2 敲低或用erastin(20 μM,24 小时)处理过表达的 PC9 细胞中铁死亡调节因子的免疫印迹。 B、C经过erastin(20 μM,24 小时)处理的PKD2 敲低或过表达的A549 和PC9 细胞中FTL 蛋白的免疫印迹。 D、E PKD2 敲低或过表达的 A549 细胞中 NCOA4、FTH1、FTL 的 mRNA 表达。 PKD2 敲低或过度表达的 A549 和 PC9 细胞中 FTH1 和 NCOA4 的F–I免疫印迹,经erastin(20 μM,24 小时)、加或不加巴弗洛霉素 A1(100 nM,4 小时)处理。 J两例 LUAD 组织中 PKD2、FTH1 的代表性免疫组织化学图像。 100X,比例尺,200μm; 400X,比例尺,50 μm。 H代表性共焦图像显示实验细胞中 FTL 和 LAMP2 的共定位。通过Image J计算共定位系数。数据以平均值±SD表示; n = 3。* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。
Fig. 4. Augmenting PKD2 blocked the fusion of autophagosome and lysosome in LUAD. A GO enrichment analyses, including BP (biological process), CC (cellular component), MF (molecular function). B The correlation analysis of PKD2 expression and ssGSEA score of negative regulation of autophagy. C, D The alteration of autophagy-related genes in mRNA level in A549 and PC9 cells when PKD2 was knocked down. E, F The alteration of other autophagy-related genes in mRNA level in A549 and PC9 cells when PKD2 was overexpressed. G-M Immunoblots of LC3 and P62 proteins in A549 or PC9 cells with PKD2 knockdown or overexpression treated by rapamycin (5 μM, 4h), EBSS (4h), serum-free medium (SFM) starvation (6h) with or without bafilomycin A1 (100 nM, 4h). N, O Immunoblots of autophagy-related genes in A549 and PC9 cells with PKD2 knockdown treated by SFM starvation (6h). P A549 cells with PKD2 knockdown were infected by StubRFP-sensGFP-LC3 adenovirus and starved for 6 h. Representative confocal images show GFP puncta, RFP puncta and merged puncta in experimental cells. The data were presented as the mean ± SD; n = 3. Q Representative confocal images show the colocalization of LC3B and LAMP2 in experimental cells. The coefficient of colocalization was calculated by Image J. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图4 .增强 PKD2 可阻断 LUAD 中自噬体和溶酶体的融合。 GO富集分析,包括BP(生物过程)、CC(细胞成分)、MF(分子功能)。 B PKD2表达与自噬负调控ssGSEA评分的相关性分析。 C、D当 PKD2 被敲低时,A549 和 PC9 细胞中自噬相关基因 mRNA 水平的变化。 E、F PKD2过表达时,A549和PC9细胞中其他自噬相关基因mRNA水平的变化。 PKD2 敲低或过表达的 A549 或 PC9 细胞中 LC3 和 P62 蛋白的GM免疫印迹,经雷帕霉素(5 μM,4 小时)、EBSS(4 小时)、无血清培养基 (SFM) 饥饿(6 小时)、加或不加巴弗洛霉素 A1(100)处理纳米,4 小时)。通过 SFM 饥饿(6 小时)处理 PKD2 敲低的 A549 和 PC9 细胞中自噬相关基因的N、O免疫印迹。 PKD2敲低的PA549细胞被StubRFP-sensGFP-LC3腺病毒感染并饥饿6小时。代表性共焦图像显示实验细胞中的 GFP 点、RFP 点和合并点。数据以平均值±标准差表示; n = 3。Q代表性共焦图像显示实验细胞中 LC3B 和 LAMP2 的共定位。通过Image J计算共定位系数。数据以平均值±SD表示; n = 3。* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。
Fig. 5. PKD2 blocked autophagosome-lysosome fusion in a TFEB-independent way. A Immunoblots of TFEB in A549 cells with PKD2 overexpression. B Extraction of cytoplasmic and nuclear protein was to detect subcellular location of TFEB in HEK cells with rapamycin (5 μM, 4h) treatment when PKD2 was overexpressed. C, D Representative immunofluorescence images show PKD2 (green), TFEB (red), nucleus (blue) and merged images in A549 cells with or without rapamycin (5 μM, 4h) treatment when PKD2 was overexpressed or knocked down.
图5 。 PKD2 以不依赖 TFEB 的方式阻断自噬体-溶酶体融合。 A PKD2 过表达的 A549 细胞中 TFEB 的免疫印迹。 B提取细胞质和核蛋白,检测 PKD2 过表达时用雷帕霉素(5 μM,4h)处理的 HEK 细胞中 TFEB 的亚细胞定位。 C、D代表性免疫荧光图像显示当 PKD2 过度表达或敲低时,用或不用雷帕霉素(5 μM,4 小时)处理的 A549 细胞中的 PKD2(绿色)、TFEB(红色)、细胞核(蓝色)和合并图像。
Fig. 6. PKD2 knockdown-mediated ferroptosis could be impaired by bafilomycin A1 stimulation or regulating FTH1. A The ROS content in PKD2-depleted PC9 cells with or without erastin, bafilomycin A1 treatment. B The data of the ROS content in PC9 cells were presented as the mean ± SD; n = 3. C The ROS content in PKD2-depleted A549 cells with or without erastin, bafilomycin A1 treatment was detected by flow cytometry. D, E Relative MDA content in A549 and PC9 cells with erastin (20 μM, 24h) and bafilomycin A1 (100 nM, 4h) treatment when PKD2 was knocked down. The data were presented as the mean ± SD; n = 3. F The ROS content in PKD2 overexpressed PC9 cells treated by erastin with or without FTH1 knockdown was detected by flow cytometry. G Percentage of PI staining positive cells in PKD2 overexpressed A549 cells with or without FTH1 knockdown. All statistical data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 7. PKD2 promoted proliferation, migration, invasion of LUAD cells in vitro. A, B Effects of silencing PKD2 on the proliferative ability of A549 and PC9 cells by SRB staining assay. C, D Effects of PKD2 overexpression on the proliferative ability of A549 and PC9 cells by SRB staining assay. E, F The EdU assay detected the effects of silencing PKD2 on the proliferative ability of A549 and PC9 cells. G Effects of PKD2 knockdown on the colony-formation ability of A549 and PC9 cells. H Effects of PKD2 overexpression on the colony-formation ability of A549 and PC9 cells. I, J Wound healing assay validated that the role of silencing PKD2 on migrative ability of A549 and PC9 cells. K, L Effects of silencing or overexpressed PKD2 on migrative and invasive ability of PC9 cells by Transwell assay. All statistical data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图7 . PKD2在体外促进LUAD细胞的增殖、迁移、侵袭。 A、B通过SRB染色测定沉默PKD2对A549和PC9细胞增殖能力的影响。 C、D通过SRB染色测定PKD2过表达对A549和PC9细胞增殖能力的影响。 E、F EdU 测定检测了沉默 PKD2 对 A549 和 PC9 细胞增殖能力的影响。 G PKD2 敲低对 A549 和 PC9 细胞集落形成能力的影响。 H PKD2 过表达对 A549 和 PC9 细胞集落形成能力的影响。 I、J伤口愈合实验验证了沉默 PKD2 对 A549 和 PC9 细胞迁移能力的作用。 K、L通过 Transwell 实验观察沉默或过表达 PKD2 对 PC9 细胞迁移和侵袭能力的影响。所有统计数据均以平均值±标准差表示; n = 3。* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。
Fig. 8. PKD2 knockdown suppressed LUAD progression in vivo. A A549 cells, infected with lentivirus containing shPKD2 or shNT, at a concentration of 2 × 106 mixed with 50% Matrigel were implanted subcutaneously in 4-week-old male nude mice. Mice were sacrificed at 42 days after injection with no tumor volume reaching 2000 mm3. B Tumor volumes were recorded every 6 days. C Tumor samples separated surgically from mice. D Tumor weight of samples separated surgically from mice was compared between shPKD2 and shNT groups.
图8 . PKD2 敲低可抑制体内 LUAD 进展。将含有shPKD2或shNT的慢病毒感染的A549细胞以2×10 6的浓度与50% Matrigel混合,植入4周龄雄性裸鼠皮下。注射后42天处死小鼠,肿瘤体积未达到2000mm3。 B每6天记录一次肿瘤体积。 C通过手术从小鼠中分离出肿瘤样本。 D比较 shPKD2 组和 shNT 组之间通过手术从小鼠分离的样品的肿瘤重量。
Fig. 9. Targeting PKD2 could enhance sensitivity of LUAD cells to carboplatin. A, B The cell viability of A549 and PC9 cells with PKD2 knockdown with or without carboplatin treatment. The data were presented as the mean ± SD; n = 3. C, D The cell death of A549 and PC9 cells with PKD2 knockdown with or without carboplatin treatment. The data were presented as the mean ± SD; n = 3. E, F IC50 experiment was conducted to detect cell viability of A549 and PC9 cells with carboplatin treatment when PKD2 was knocked down or not. G, H The ROS content in A549 and PC9 cells in the indicated treatment was detected by flow cytometry. The data were presented as the mean ± SD; n = 3. I, J Relative MDA content in A549 and PC9 cells in the indicated treatment was detected. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
图9 .靶向 PKD2 可以增强 LUAD 细胞对卡铂的敏感性。 A、B PKD2 敲低的 A549 和 PC9 细胞在有或没有卡铂处理的情况下的细胞活力。数据以平均值±标准差表示; n = 3。C 、D PKD2 敲低的 A549 和 PC9 细胞在有或没有卡铂治疗的情况下的细胞死亡。数据以平均值±标准差表示; n = 3。E 、F IC50实验检测卡铂处理的A549和PC9细胞在PKD2敲低与否时的细胞活力。 G、H通过流式细胞术检测指定处理中的 A549 和 PC9 细胞中的 ROS 含量。数据以平均值±标准差表示; n = 3. I、J检测指定处理中 A549 和 PC9 细胞中的相对 MDA 含量。数据以平均值±标准差表示; n = 3。* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。
Fig. 10. Proposed model of PKD2-mediated ferroptosis suppression by blocking autophagosome-lysosome fusion to suppress ferritinophagy and targeting PKD2 could enhance efficacy of carboplatin on LUAD.
图10 。提出的通过阻断自噬体-溶酶体融合来抑制铁蛋白自噬和靶向 PKD2 的 PKD2 介导的铁死亡抑制模型可以增强卡铂对 LUAD 的疗效。
Multimedia component 1.
Supplementary Fig. 1. PKD2 overexpression and knockdown systems were established by using siRNA, shRNA and plasmid in A549 and PC9 cell lines. A, B Immunoblots of A549 and PC9 cells with PKD2 knockdown by PKD siRNAs. C Immunoblots of A549 cells with PKD2 knockdown by PKD shRNA. D, E Immunoblots of A549 cells with PKD2 overexpression by PKD plasmid.
Supplementary Fig. 2. PKD2 blocked autophagic degradation of ferritin in LUAD. A, B The mRNA expression of FTL, FTH1, NCOA4 in PC9 cells with PKD2 knockdown or overexpression by qRT-PCR. C, D Immunoblots of NCOA4, FTL in A549 and PC9 cells with PKD2 knockdown with erastin (20 μM, 24h) and chloroquine (40 μM, 6h) treatment. E Representative immunohistochemical images of FTH1 in alveolus, mesenchyme, LUAD tissues. Left: 100X, scale bar, 200 μm; Right: 400X, scale bar, 50 μm. F Representative confocal images show the colocalization of FTL and LAMP2 in experimental cells. The coefficient of colocalization was calculated by Image J. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplementary Fig. 3. Regulation of PKD2 on autophagy of LUAD. A GSEA enrichment analysis. B, C P62 mRNA expression of A549 and PC9 cells with silencing PKD2 by qRT-PCR. D, E P62 mRNA expression of A549 and PC9 cells with PKD2 overexpression by qRT-PCR. F, G LC3 mRNA expression of A549 cells with PKD2 knockdown and PC9 cells with PKD2 overexpression. H, I Immunoblots of LC3 and P62 proteins in PC9 cells with PKD2 knockdown or overexpression treated by rapamycin (5 μM, 4h) with or without bafilomycin A1 (100 nM, 4h). J A549 cells with PKD2 overexpression were infected by StubRFP-sensGFP-LC3 adenovirus and starved for 6 h. Representative confocal images show GFP puncta, RFP puncta and merged puncta in experimental cells. The data were presented as the mean ± SD; n = 3. K Representative confocal images show the colocalization of LC3B and LAMP2 in experimental cells. The coefficient of colocalization was calculated by Image J. The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplementary Fig. 4. PKD2 promote proliferation, migration, invasion of LUAD cells in vitro. A Representative immunohistochemical images of PKD2, Ki67 in LUAD tissue from two cases. 100X, scale bar, 200 μm; 400X, scale bar, 50 μm. B, C The EdU assay was used to detect proliferative ability of A549 and PC9 cells with PKD2 overexpression. D, E Wound healing assay was used to detect migrative ability of A549 and PC9 cells with PKD2 overexpression. F, G The role of PKD2 on A549 cell migration and invasion was validated by Transwell assay. All statistical data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplementary Fig. 5. Targeting PKD2 could enhance sensitivity of LUAD cells to carboplatin. A, B The EdU assay detected proliferation of A549 and PC9 cells with PKD2 knockdown when cells were treated by carboplatin (20 μM, 24h). C The effects of silencing PKD2 or pharmacological inhibition of PKD2 on carboplatin (20 μM, 24h)-induced apoptosis of A549 cell. D, E The data were presented as the mean ± SD; n = 3. F The effects of silencing PKD2 or pharmacological inhibition of PKD2 on carboplatin (20 μM, 24h)-induced apoptosis of PC9 cells. G, H The data were presented as the mean ± SD; n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
多媒体组件 1 .
补充图 1 。利用siRNA、shRNA和质粒在A549和PC9细胞系中建立PKD2过表达和敲低系统。 A、B 通过 PKD siRNA 敲低 PKD2 的 A549 和 PC9 细胞的免疫印迹。 C通过 PKD shRNA 敲低 PKD2 的 A549 细胞的免疫印迹。 D、E PKD 质粒过度表达 PKD2 的 A549 细胞的免疫印迹。
补充图 2 。 PKD2 阻断 LUAD 中铁蛋白的自噬降解。 A、B 通过 qRT-PCR 检测 PKD2 敲低或过表达的 PC9 细胞中 FTL、FTH1、NCOA4 的 mRNA 表达。
C 、 D使用erastin(20 μM,24h)和氯喹(40 μM,6h)处理敲低PKD2的A549和PC9细胞中NCOA4、FTL的免疫印迹。 E肺泡、间充质、LUAD 组织中 FTH1 的代表性免疫组织化学图像。左:100X,比例尺,200 μm;右:400X,比例尺,50 μm。 F代表性共焦图像显示实验细胞中 FTL 和 LAMP2 的共定位。通过Image J计算共定位系数。数据以平均值±SD表示; n = 3。* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。补充图 3 。 PKD2 对 LUAD 自噬的调节。 GSEA 富集分析。 B、C通过 qRT-PCR 观察沉默 PKD2 的 A549 和 PC9 细胞的 P62 mRNA 表达。 D、E通过 qRT-PCR 检测 PKD2 过表达的 A549 和 PC9 细胞的 P62 mRNA 表达。 F,G PKD2敲低的A549细胞和PKD2过表达的PC9细胞的LC3 mRNA表达。 PKD2 敲低或过表达的 PC9 细胞中 LC3 和 P62 蛋白的H、I免疫印迹,经雷帕霉素(5 μM,4 小时)、加或不加巴弗洛霉素 A1(100 nM,4 小时)处理。
PKD2过表达的J A549细胞被StubRFP-sensGFP-LC3腺病毒感染并饥饿6小时。代表性共焦图像显示实验细胞中的 GFP 点、RFP 点和合并点。数据以平均值±标准差表示; n = 3. K代表性共焦图像显示实验细胞中 LC3B 和 LAMP2 的共定位。通过Image J计算共定位系数。数据以平均值±SD表示; n = 3。* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。补充图 4 。 PKD2在体外促进LUAD细胞增殖、迁移、侵袭。 A 两例 LUAD 组织中 PKD2、Ki67 的代表性免疫组织化学图像。 100X,比例尺,200μm; 400X,比例尺,50 μm。 B、C EdU测定用于检测PKD2过表达的A549和PC9细胞的增殖能力。 D、E伤口愈合实验检测 PKD2 过表达的 A549 和 PC9 细胞的迁移能力。 F、G通过 Transwell 实验验证了 PKD2 对 A549 细胞迁移和侵袭的作用。所有统计数据均以平均值±标准差表示; n = 3。*P < 0。
05,**P < 0.01,***P < 0.001,****P < 0.0001。补充图 5 。靶向 PKD2 可以增强 LUAD 细胞对卡铂的敏感性。 A、B EdU 测定检测到用卡铂(20 μM,24 小时)处理细胞时,PKD2 敲低的 A549 和 PC9 细胞的增殖。 C沉默PKD2或药理抑制PKD2对卡铂(20 μM,24h)诱导的A549细胞凋亡的影响。 D、E数据表示为平均值±SD; n = 3. F沉默 PKD2 或药理抑制 PKD2 对卡铂(20 μM,24 小时)诱导的 PC9 细胞凋亡的影响。 G、H数据表示为平均值±SD; n = 3。* P < 0.05,** P < 0.01,*** P < 0.001,**** P < 0.0001。
For example, GCH1 knockdown induced ferroptosis and activated ferritinophagy in colorectal cancer [17], while d-Borneol enhanced cisplatin sensitivity in NSCLC by promoting ferritinophagy [18]. Recent studies have reported that PKD2 deletion in LUAD exacerbates ferritinophagy and weakens resistance to chemotherapeutic drugs [51]. In our study, NCOA4 was downregulated in LUAD clinical samples.