Figures & data
Table 1. Characteristics of 60 Non-small Cell Lung Cancer Patients.
Figure 1. Expression of cypA and HIF-1α in NSCLC carcinoma specimens. (A) Increased and decreased expression levels of HIF-1α and cypA on NSCLC specimen sections. (B) Relationship between cypA expression and HIF-1α expression. Spearman’s correlation = 0.346, p <0.0001.
Figure 2. Hypoxia treatment induced cypA expression in pulmonary carcinoma cell lines. H1299 and A549 cell lines were cultured under hypoxic or normoxic conditions. (A, B) RT-qPCR revealed upregulated HIF-1α and cypA mRNA levels in H1299 and A549 cells in response to anoxia in comparison with those under normoxia. (C, D) WB showed elevated protein expression levels of cypA and HIF-1α in H1299 and A549 cells in response to anoxia in comparison with those under normoxia. * p <0.05.
Figure 3. Artificial regulation of cypA expression in pulmonary carcinoma cells under hypoxic conditions. H1299 and A549 cells were transfected with an ITCH (WT/R55A mutant) overexpression vector or empty vector, and then subjected to hypoxic or normoxic conditions. (A, B) Dysregulated mRNA expression of cypA in H1299 and A549 cells in response to anoxia. (C, D) WB detected cypA protein expression levels in anoxic H1299 and A549 cells transfected with different plasmids. * p <0.05, ** p <0.01.
Figure 4. Effect of cypA on colony formation ability, ROS generation, and apoptosis of hypoxia-conditioned LC cells. H1299 and A549 cells were transfected with an ITCH (WT/R55A mutant) overexpression vector or empty vector, and then subjected to hypoxic or normoxic conditions. (A, B) A colony formation assay was conducted to examine the cell growth rate of H1299 and A549 cells. (C, D) DCFH-DA staining was used to measure ROS production in H1299 and A549 cell lines. (E, F) Apoptosis was measured by FC with Annexin V-FITC/PI staining. * p <0.05, ** p <0.01.
Figure 5. CypA interacts with and degrades TXNIP. (A) A549 and H1299 cells were co-transfected with cypA WT/R55A mutant. Whole-cell lysis products treated for the indicated period with 100 μg/mL CHX were probed for cypA and TXNIP using WB. (B) A549 and H1299 cells were co-transfected with HA-TXNIP plasmids and flag-cypA. Split products were immunoprecipitated by WB as well as anti-flag antibody using the indicated antibodies. (C) A549 and H1299 cells were transfected with cypA (WT/R55A mutant) overexpression vector or empty vector, and then subjected to hypoxic or normoxic conditions. WB detected TXNIP protein expression levels in hypoxic H1299 and A549 cells transfected with different plasmids.
Figure 6. Influence of TXNIP overexpression on colony-forming ability, ROS generation, and apoptosis of cypA-overexpressed and hypoxia-conditioned LC cells. H1299 and A549 cells were co-transfected with cypA and TXNIP overexpression vectors for 24 h and then subjected to anoxic conditions. (a, b) WB detected cypA and TXNIP protein levels in hypoxic A549 and H1299 cells transfected with different plasmids. (c, d) The colony-forming assay examined the cell growth rates of H1299 and A549 cells. (e, f) DCFH-DA staining assay was used to examine ROS generation in H1299 and A549 cells. (g, h) Apoptosis was tested by FC with Annexin V-FITC/PI staining. * p < 0.05, ** p < 0.01.
Figure 7. Effect of verapamil on colony formation ability, ROS generation, and apoptosis of cypA-silenced hypoxia-conditioned LC cells. H1299 and A549 cells were transfected with siTXNIP for24 h, treated with 1 μM verapamil for 12 h, and then subjected to hypoxic conditions. (A, B) WB detected cypA and TXNIP protein levels in hypoxic H1299 and A549 cells transfected with different plasmids. (C, D) The colony formation assay examined the cell growth rates of H1299 and A549 cells. (E, F) DCFH-DA staining assay was used to measure ROS production in H1299 and A549 cells. (G, H) Apoptosis was tested by FC with Annexin V-FITC/PI staining. * p <0.05, ** p <0.01.
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