Fig 1: Validation of microarray data using qRT-PCR.The bar chart shows the gene expression patterns (presented as fold change) of selected significantly regulated genes calculated using qRT–PCR and microarray analysis. The down-regulated genes selected were CYP24A1, ANXA3 and AREG, while the up-regulated genes were LEAP2, FGA, FGG, SERPINE1, IFNGR1, MVK, DHCR24, ALDH6A1 and ADH6. All qRT-PCR data were normalized to that of GAPDH, a housekeeping gene.
Fig 2: ELISA analyses of selected proteins.(A) Enzyme-Linked Immunosorbent Assay (ELISA) analyses of the human IFNGR1, LEAP2, SERPINE1, ANXA3, KNG1, MX1, FGG, MVK, ALDH6A1 and ADH6 antibodies level in the untreated and leaf-treated HepG2 cells. ELISA analyses were done according to manufacturer’s protocols (Cloud-clone, Houston, Texas, USA; Cusabio Biotech, Wuhan, China). Bars not sharing the same superscript letter indicate significant difference at p < 0.05 (B) Up regulation of IFNGR1 (a) and SERPINE1 (b) after treatment with IC20 concentration of the leaf extract for 24 h. Protein levels were measured with specific antibodies by western blot analysis; β-actin was the loading control. Untreated cells were used as control. The experiments were repeated in triplicates and the representative blot was shown. Bars not sharing the same superscript letter indicate significant difference at p < 0.05.
Fig 3: IPA graphical representation.(A) IPA graphical representation of the molecular relationships between the significantly regulated genes to the predicted canonical pathways in response to the leaf treatment in HepG2 cells. The network is displayed graphically as nodes (genes) and edges (the biological relationships between the nodes). Nodes in red indicate up-regulated genes while those in green represent down-regulated genes. Various shapes of the nodes represent functional class of the proteins. Edges are displayed with various labels that describe the nature of the relationship between the nodes. Name of genes with their corresponding abbreviations are as follows: ADH6, Alcohol dehydrogenase 6; ALDH6A1, Aldehyde dehydrogenase 6 family, member A1; ALDH9A1, Aldehyde dehydrogenase 9 family, member A1; ANXA3, Annexin A3; CYP24A1, Cytochrome P450, family 24, subfamily A, polypeptide 1; DHCR24, 24-dehydrocholesterol reductase; EPHX1, Epoxide hydrolase 1; FGA, Fibrinogen alpha chain; FGG, Fibrinogen gamma chain; GSTM4, Glutathione S-transferase mu 4; IFNGR1, Interferon gamma receptor 1; KNG1, Kininogen 1; LEAP2, Liver-expressed antimicrobial peptide; LSS, Lanosterol synthase; MVK, Mevalonate kinase; MX1, Myxovirus resistance 1; SERPINC1, Serpin peptidase inhibitor, clade C (antithrombin), member 1; SERPIND1, Serpin peptidase inhibitor, clade D (heparin cofactor), member 1, SERPINE1, Serpin peptidase inhibitor, clade E (Nexin, Plasminogen activator inhibitor, type 1), member 1; TM7SF2, Transmembrane 7 superfamily member 2. (B) IPA graphical representation showing the effect of significantly regulated genes, ALDH6A1, ALDH6 and GSTM4 in the detoxification process of 4-HNE and MDA. This figure demonstrates that the three genes are involved in the xenobiotic metabolism signaling pathway as on one of the top canonical pathway generated by IPA. The process is displayed graphically as nodes (genes) and edges (the biological relationships between the nodes). Nodes in red indicate up-regulated genes. Various shapes of the nodes represent functional class of the proteins. Edges are displayed with various labels that describe the nature of the relationship between the nodes. (C) IPA graphical representation of the molecular relationships between KNG1, SERPINE1, SERPINC1, SERPIND1 and Fibrinogen that are involved in “Coagulation System”, the top predicted canonical pathway in HepG2 cells affected by the methanol leaf extract of T. indica. The network is displayed graphically as nodes (genes) and edges (the biological relationships between the nodes). Nodes in red indicate up-regulated genes. Various shapes of the nodes represent functional class of the proteins. Edges are displayed with various labels that describe the nature of the relationship between the nodes. Name of genes/proteins with their corresponding abbreviations are as follows: A2M, Alpha-2-macroglobulin; BDK, Bradykinin; BDKR, Bradykinin receptor; F2, Coagulation factor II (thrombin); F2a, Coagulation factor IIa (thrombin); F2R, Coagulation factor II receptor; F3, Coagulation factor III (thromboplastin, tissue factor); F5a, Coagulation factor V (proaccelerin, labile factor); F7, Coagulation factor VII (serum prothrombin conversion accelerator); F7a, Coagulation factor VIIa (serum prothrombin conversion accelerator); F8, Coagulation factor VIII (procoagulant component); F8a, Coagulation factor VIIIa (procoagulant component); F9, Coagulation factor IX; F9a, Coagulation factor IXa; F10, Coagulation factor X; F10a, Coagulation factor Xa; F11, Coagulation factor XI; F11a, Coagulation factor XIa; F12, Coagulation factor XII (Hageman factor); F12a, Coagulation factor XIIa (Hageman factor); F13, Coagulation factor XIII; F13a, Coagulation factor XIIIa; KLKB1a, Kallikrein B plasma 1a; PLG, Plasminogen; SERPINA1, Serpin peptidase inhibitor, clade A, member 1; SERPINA5, Serpin peptidase inhibitor, clade A, member 5; SERPINF2, Serpin peptidase inhibitor, clade F, member 2; TFPI, Tissue factor pathway inhibitor; THBD, Thrombomodulin; TPA, Tissue plasminogen activator; UPA, Urokinase plasminogen activator; UPAR, Urokinase plasminogen activator receptor; vWF, von Willebrand factor.
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