Nitric oxide synthase 1 inhibits the progression of esophageal cancer through interacting with nitric oxide synthase 1 adaptor protein

Figure 1 Expression of nitric oxide synthase 1 in different tissues and Kaplan-Meier analysis. A: Expression of nitric oxide synthase 1 (NOS1) in different tumor tissues and normal tissues; B: Expression of NOS1 in esophageal cancer tissues (left) and normal esophageal tissues (right); C: Relationship between the expression of NOS1 and overall survival of patients with esophageal cancer. ^bP < 0.01. ^cP < 0.001. ACC: Adrenocortical cancer; BLCA: Bladder cancer; BRCA: Breast cancer; CESC: Cervical cancer; CHOL: Bile duct cancer; COAD: Colon cancer; DLBC: Large B-cell lymphoma; ESCA: Esophageal cancer; GBM: Glioblastoma; HNSC: Head and neck cancer; HNSC-HPV: Head and neck cancer-human papillomavirus; KICH: Kidney chromophobe; KIRC: Kidney clear cell carcinoma; KIRP: Kidney papillary cell carcinoma; LAML: Acute myeloid leukemia; LGG: Lower grade glioma; LIHC: Liver cancer; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; MESO: Mesothelioma; OV: Ovarian cancer; PAAD: Pancreatic cancer; PCPG: Pheochromocytoma and paraganglioma; PRAD: Prostate cancer; READ: Rectal cancer; SARC: Sarcoma; SKCM: Melanoma; STAD: Stomach cancer; TGCT: Testicular cancer; THCA: Thyroid cancer; THYM: Thymoma; UCEC: Endometrioid cancer; UCS: Uterine carcinosarcoma; UVM: Ocular melanomas; TPM: Transcripts per million; NOS1: Nitric oxide synthase 1; HR: Hazard ratio.

Figure 2 Effect of nitric oxide synthase 1 on esophageal cancer cell function. A: Quantitative real-time polymerase chain reaction to detect the expression of nitric oxide synthase 1 (NOS1) in Het-1A, KYSE-70, TE-9, TE-15, OE-19 cells; B: Western Blotting to detect the expression of NOS1 in Het-1A, KYSE-70, TE-9, TE-15, OE-19 cells.; C: Cell counting kit-8 to detect cell proliferation and viability; D: Flow cytometry to analyze the effect of NOS1 on cell apoptosis; E: Effect of NOS1 on cell invasion ability; F: Effect of NOS1 on cell migration ability; G: Effect of NOS1 on the cell cycle. Values are mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. n = 3 experiments. NOS1: Nitric oxide synthase 1; oe-NOS1: Overexpression of nitric oxide synthase 1; OD: Optical density.

Figure 3 Effect of nitric oxide synthase 1 on tumor growth in mice. A: Expression of nitric oxide synthase 1 in tumor tissues detected by immunohistochemistry; B: Representative images of tumors; C: Tumor weight; D: Daily tumor volume; E: Total tumor volume; F: Body weight of mice; G: Tumor tissues stained with hematoxylin-eosin. Values are mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. n = 3 experiments. NOS1: Nitric oxide synthase 1; oe-NOS1: Overexpression of nitric oxide synthase 1.

Figure 4 Western blotting to detect the effect of nitric oxide synthase 1 on the levels of autophagy, apoptosis, pyroptosis, and ferroptosis in esophageal cancer cells. A: Autophagy; B: Apoptosis; C: Pyroptosis; D: Ferroptosis. Values are mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. n = 3 experiments. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NOS1: Nitric oxide synthase 1; oe-NOS1: Overexpression of nitric oxide synthase 1.

Figure 5 Construction of nitric oxide synthase 1-mediated producer price index network in esophageal cancer. CALM3: Calmodulin 3; CALML3: Calmodulin-like 3; CALML4: Calmodulin-like 4; CALML5: Calmodulin-like 5; CALML6: Calmodulin-like 6; DLG4: Discs large MAGUK scaffold protein 4; GRIN2B: Glutamate receptor, ionotropic, N-methyl D-aspartate 2B; NOS1: Nitric oxide synthase 1; NOS1AP: Nitric oxide synthase 1 adaptor protein; RASD1: Ras-related dexamethasone-induced 1; SNTA1: Syntrophin alpha-1.

Figure 6 Nitric oxide synthase 1 and nitric oxide synthase 1 adaptor protein interaction. A: Immunoprecipitation analysis of the interaction between nitric oxide synthase 1 (NOS1) and nitric oxide synthase 1 adaptor protein (NOS1AP); B and C: Effect of NOS1 expression level on NOS1AP expression level; Quantitative real-time polymerase chain reaction (B); Western blotting (C); D and E: Western Blotting to detect the changes of NOS1 and NOS1AP expression in TE-15 and OE-19 cell lines after inhibition of autophagy; TE-15 cell line (D); OE-19 cell line (E). Values are mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. n = 3 experiments. NOS1: Nitric oxide synthase 1; NOS1AP: Nitric oxide synthase 1 adaptor protein; IB: Immunoblotting; IP: Immunoprecipitation; oe-NOS1: Overexpression of nitric oxide synthase 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; si-NOS1AP: Silencing of nitric oxide synthase 1 adaptor protein.

Figure 7 Nitric oxide synthase 1 and nitric oxide synthase 1 adaptor protein affect the level of cell autophagy and apoptosis. A and B: Western blotting detected the changes in the expression of autophagy-related proteins LC3B and P62 in TE-15 and OE-19 cell lines; C: Flow cytometry analyzed the level of apoptosis; D: TUNEL staining analyzed the level of apoptosis; E: 5-Ethynyl-2’-deoxyuridine assay analyzed the cell proliferation ability; F and G: Immunohistochemistry analysis of P62, LC3B expression in mouse tumor tissues. Values are mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. n = 3 experiments. EDU: 5-Ethynyl-2’-deoxyuridine; oe-NOS1: Overexpression of nitric oxide synthase 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; si-NOS1AP: Silencing of nitric oxide synthase 1 adaptor protein.

Figure 8 Effect of nitric oxide synthase 1 and nitric oxide synthase 1 adaptor protein on the expression of nitric oxide, nitrite, and phosphatidylinositol 3-kinase/protein kinase B pathway-related proteins. Enzyme-linked immunosorbent assay to detect nitric oxide and nitrite in different samples. A: TE-15 cell line; B: OE-19 cell line; C and D: Mouse serum; E and F: Tumor tissues; G: Western Blotting was performed to detect the expression of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway-related proteins E-cadherin, phosphorylated (p)-PI3K/PI3K, p-AKT/AKT. Values are mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. n = 3 experiments. oe-NOS1: Overexpression of nitric oxide synthase 1; NO: Nitric oxide; si-NOS1AP: Silencing of nitric oxide synthase 1 adaptor protein; PI3K: Phosphatidylinositol 3-kinase; AKT: Protein kinase B.