Fig 1: The prognosis value of ALDOA-PLD1 for lung cancer patients. (A) Scores (0–3) indicate PLD1 protein levels in representative lung tumor tissues. (B) The expression level of the PLD1 protein in tumor tissue compared with the corresponding normal adjacent tissue. (C) Kaplan–Meier analysis of PLD1 protein expression at concurrently low or high levels as determined by IHC staining at the endpoint of overall survival probability and disease-free survival probability in lung cancer patients. (D) Kaplan–Meier analysis of PLD1 combined with ALDOA protein expression at concurrently low or high levels or others as determined by IHC staining at the endpoint of overall survival probability in lung cancer patients. (E) Multivariate analysis of ALDOA/PLD1/PLD2 and clinical parameters in a clinical cohort. The significance of the differences in (C, D) was analyzed using the Student’s t-test.
Fig 2: Events and activities related to PLD depend on ALDOA. (A) The production of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) were obtained from the available CCLE metabolomics results. Combined with previous research, we collected the survival fraction after radiation exposure in each CCLE cell. A549, H1299, H2122 (radioresistant group), and H1792, H23, H1975 (radiosensitivity group) lung cancer cells were divided into two groups to quantify their LPC and LPE concentrations. (B) The dependence of LPC or LPE products on glycolytic pathways from the available CCLE metabolites profile. These configuration files are calculated based on the RNA-seq expression of each candidate in the CCLE omics database. (C) Trends between glycolysis and phospholipase D metabolic pathways. This result shows that the performance of LPC/LPE depends on HK2, ALDOA, and PLD. The importance of TPI1 has decreased. Red color means positive dependence. Blue color means negative dependence for LPC/LPE products. (D) Two-way model of immunoprecipitation using ALDOA and PLD2 antibodies in CL1-0 cells with or without forced expression of an exogenous ALDOA gene. IgG served as the negative control. (E) The protein levels of ALDOA, PLD1, and PLD2 in A549 cells with or without shALDOA. Tubulin serves as an internal control. (F) The PLD2 protein level after treatment with MG-132 is time-dependent in A549 cells with vector control or ALDOA overexpression. Tubulin serves as an internal control. The significance of the difference was analyzed using the nonparametric Mann–Whitney U test.
Fig 3: Inhibited PLD1 expression induces apoptosis and attenuates proliferation, autophagy, and repair by alkylating agent treatment. (A) The protein levels of Ki-67, PCNA, PARP, BAX, LC3B, LAMP2, ?-H2AX, and RRM2 in A549 cells after alkylating agent exposure, with or without specific inhibitor treatment (PLD1-specific, VU0359595, 10 µM; and PLD2-specific, CAY10594, 50 µM). Tubulin serves as an internal control. (B) Quantitation of the activity of Caspase 3 in A549 cells after alkylating agent exposure with or without specific inhibitors treatment (PLD1-specific, VU0359595, 10 µM; and PLD2-specific, CAY10594, 50 µM). (C) Quantitation of colony-forming ability in A549 cells after alkylating agent exposure with or without specific inhibitors treatment (PLD1-specific, VU0359595, 10 µM; and PLD2-specific, CAY10594, 50 µM). (D) A measure of the growth curve in A549 cells after alkylating agent exposure with or without specific inhibitor treatment (PLD1-specific, VU0359595; and PLD2-specific, CAY10594). In this study, 10 µM of cisplatin, 100 µM of temozolomide, 10 µM of VU0359595, and 50 µM of CAY10594. The data from three independent experiments are presented in (B–D) as the means ± SEM. The significance of the difference was analyzed using the nonparametric Mann–Whitney U test. **p < 0.01, ***p < 0.001. ns, non significant.
Fig 4: Phospholipase D expression in lung cancer cells with alkylating agent events. (A) The cell models in the GSE116436 profile are classified according to NCI-60, and only lung cancer models are extracted and quantified for various drug response analyses. The heat map contains candidates whose high/low dose vs. control >1.5 fold-changes. (B) The Venn diagram shows the overlapping targets of GSE116436 during treatment of the alkylating agents in four lung cancer cells (H23, NCI-322M, HOP-62, and EKVX). (C) KEGG predicts the highest ranking of metabolic and canonical pathways based on selected features of the 44 candidates in panel (B). (D) The expression level of PLD1 in lung cancer cell lines was detected in the GSE116436 profile. Conditions include cisplatin 3 µM and 15 µM. The drug treatments were 2, 6, and 24 hours, respectively. (E) The expression level of PLD2 in lung cancer cell lines was detected in the GSE116436 profile. Conditions include cisplatin 3 µM and 15 µM. The drug treatments were 2, 6, and 24 hours, respectively.
Fig 5: ALDOA increases autophagy and invasion sensitivity after radiation exposure. (A) The survival fraction after radiation exposure in various lung cancer cells (CL1-0, CL1-5, H1299, and H1355). Radiation dose: 0~10 Gy. (B) The survival fraction after radiation exposure in CL1-0 cells with vector control or ALDOA overexpression. Radiation dose: 0~10 Gy. (C) Quantify the expression levels of PLD1 after radiation exposure in CL1-0 cells with vector control or ALDOA overexpression. Radiation dose: 8 Gy (D) The LC3-I/II protein level in CL1-0 cells with vector control or ALDOA overexpression. Tubulin serves as an internal control. Radiation dose: 8 Gy (E) Quantitation of the activity of caspase-3 in A549 cells after alkylating agent exposure in CL1-0 cells with vector control or ALDOA overexpression. Radiation dose: 8 Gy (F) The LC3-I/II and LAMP2 protein level after radiation exposure in a time-dependent manner in CL1-0 cells with vector control or ALDOA overexpression. Tubulin serves as an internal control. Radiation dose: 8 Gy. (G) The Ki-67, PCNA, PARP, ?-H2AX, and RRM2 protein level after radiation exposure in CL1-0 cells with vector control or ALDOA overexpression. Tubulin serves as an internal control. Radiation dose: 8 Gy. The data from three independent experiments are presented in (B–D) as the means ± SEM. The significance of the difference was analyzed using the nonparametric Mann–Whitney U test.
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