Journal: Biomedicines

Article Title: Expression of Spermine Oxidase Is Associated with Colorectal Carcinogenesis and Prognosis of Patients

doi: 10.3390/biomedicines10030626

Figure Lengend Snippet: Comparison of clinicopathological factors and SMOX expression in patients with CRC.

Article Snippet: Specific SMOX siRNA (specific target sequence, 5′-GUUGAGGAAUUCAGCGAUU-3’) was purchased from Bioneer (Daejeon, South Korea).

Techniques: Comparison, Expressing

Journal: Biomedicines

Article Title: Expression of Spermine Oxidase Is Associated with Colorectal Carcinogenesis and Prognosis of Patients

doi: 10.3390/biomedicines10030626

Figure Lengend Snippet: Expression of SMOX in colorectal cancer (CRC) cell lines and the effects of SMOX siRNA (small interfering RNA). SMOX expression and inhibition were confirmed by RT-PCR and immunoblot. ( A ) Screening by RT-PCR of SMOX in CRC cell lines and confirmation of SMOX inhibition compared with control cells. ( B ) Relative RNA expression of SMOX. The expression of SMOX siRNA-transfected cell lines was significantly lower than that in the control cell lines ( *** p < 0.001). ( C ) Expression levels of SMOX and β-actin were determined by Western blotting in CRC cell lines. ( D ) Relative protein levels of SMOX. SMOX siRNA-transfected cell expression levels were lower than those in the control (*** p < 0.001).

Article Snippet: Specific SMOX siRNA (specific target sequence, 5′-GUUGAGGAAUUCAGCGAUU-3’) was purchased from Bioneer (Daejeon, South Korea).

Techniques: Expressing, Small Interfering RNA, Inhibition, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, RNA Expression, Transfection

Journal: Biomedicines

Article Title: Expression of Spermine Oxidase Is Associated with Colorectal Carcinogenesis and Prognosis of Patients

doi: 10.3390/biomedicines10030626

Figure Lengend Snippet: The comparison of the cell proliferation rate between control cells and siRNA-transfected cells in the CRC cell lines. ( A ) HCT116, ( B ) HT29, ( C ) SW480, and ( D ) SW620 (** p < 0.01, *** p < 0.001).

Article Snippet: Specific SMOX siRNA (specific target sequence, 5′-GUUGAGGAAUUCAGCGAUU-3’) was purchased from Bioneer (Daejeon, South Korea).

Techniques: Comparison, Control, Transfection

Journal: Biomedicines

Article Title: Expression of Spermine Oxidase Is Associated with Colorectal Carcinogenesis and Prognosis of Patients

doi: 10.3390/biomedicines10030626

Figure Lengend Snippet: The results of the transwell assay regarding metastasis. Reduced expression of SMOX inhibits the migration and invasion abilities of CRC cells. The result of the chamber migration assay and invasion assay. Cells were visualized in violet color. ( A ) Migration of the siRNA treatment group was reduced in comparison with the control group. The images of the cells were on the bottom membrane (Original magnification ×40). ( B ) siRNA-treated cells were less numerous than control cells. The images of the cells were on the bottom membrane (Original magnification ×40). ( C ) The graph indicates the percentage of cells and the results of the chamber migration assay (HCT116, 35%; HT29, 46%; SW480, 34%; and SW620, 22%) (* p < 0.05, *** p < 0.001). ( D ) The graph expresses the percentage of cells in the chamber invasion assay (HCT116, 31%; HT29, 41%; SW480, 35%; and SW620, 18%) (** p < 0.01, *** p < 0.001).

Article Snippet: Specific SMOX siRNA (specific target sequence, 5′-GUUGAGGAAUUCAGCGAUU-3’) was purchased from Bioneer (Daejeon, South Korea).

Techniques: Transwell Assay, Expressing, Migration, Invasion Assay, Comparison, Control, Membrane

Journal: Biomedicines

Article Title: Expression of Spermine Oxidase Is Associated with Colorectal Carcinogenesis and Prognosis of Patients

doi: 10.3390/biomedicines10030626

Figure Lengend Snippet: The result of thee semisolid agar colony-forming assay. Anchorage-independent growth in soft agar was examined with control cells and cells that displayed downregulation of SMOX expression. The numbers of colonies were measured after 14 days. Colony formation in the siRNA-treated group was reduced compared to that in the control group (HCT116, 31%; HT29, 38%; SW480, 33%; and SW620, 21%). Colonies were visualized in violet color. ( A ) Comparison of the growth of colonies with control cells and siRNA-treated cells (Original magnification ×40). ( B ) The graph indicates the number of colonies and the percentage difference between the control cells and siRNA-treated cells (* p < 0.05, *** p < 0.001).

Article Snippet: Specific SMOX siRNA (specific target sequence, 5′-GUUGAGGAAUUCAGCGAUU-3’) was purchased from Bioneer (Daejeon, South Korea).

Techniques: Control, Expressing, Comparison

Journal: Biomedicines

Article Title: Expression of Spermine Oxidase Is Associated with Colorectal Carcinogenesis and Prognosis of Patients

doi: 10.3390/biomedicines10030626

Figure Lengend Snippet: The result of IHC for SMOX expression in CRC ( A – D ). In total, 350 specimens were used, which were scored by multiplying the staining frequency and staining intensity. Low/High expression of SMOX was discovered in 182/168 CRC tissues. The Kaplan–Meier survival analysis was performed for all the sampled CRC patients, based on numerous variables and their expression of SMOX. SMOX proteins and nuclei were visualized by brown and violet, respectively. ( A ) Normal tissue without expression of SMOX (Original magnification ×200). ( B ) Cancer tissue without expression of SMOX (Original magnification ×200). ( C ) Cancer tissue with low expression of SMOX (Original magnification ×200). ( D ) Cancer tissue with high expression of SMOX (Original magnification ×200). ( E ) SMOX expression and overall survival of colon cancer patients were related. Following the expression of SMOX, patients were divided into two groups and the survival rate was determined using a Kaplan-Meier analysis ( p = 0.001).

Article Snippet: Specific SMOX siRNA (specific target sequence, 5′-GUUGAGGAAUUCAGCGAUU-3’) was purchased from Bioneer (Daejeon, South Korea).

Techniques: Expressing, Staining

Journal: Biomedicines

Article Title: Expression of Spermine Oxidase Is Associated with Colorectal Carcinogenesis and Prognosis of Patients

doi: 10.3390/biomedicines10030626

Figure Lengend Snippet: Cox regression analysis of the clinicopathological factors in CRC.

Article Snippet: Specific SMOX siRNA (specific target sequence, 5′-GUUGAGGAAUUCAGCGAUU-3’) was purchased from Bioneer (Daejeon, South Korea).

Techniques: Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Regulation of Spermine Oxidase through Hypoxia-Inducible Factor-1α Signaling in Retinal Glial Cells under Hypoxic Conditions

doi: 10.1167/iovs.61.6.52

Figure Lengend Snippet: Expression and production of SMOX in TR-MUL5 cells with silencing of Hif1a or Hif2a under hypoxic condition (1% O 2 ). TR-MUL5 cells were transfected with control siRNA (Ctrl-siRNA), siRNAs for Hif-1α ( Hif1a siRNA-1 and -2), or siRNAs for Hif2a ( Hif2a siRNA-1 and -2) and cultivated with or without hypoxic stimulation. ( A ) Real-time qPCR analysis for Smox mRNA expression 6 hours after hypoxic stimulation. ( B ) SMOX protein levels in TR-MUL5 cells measured by ELISA 24 hours after hypoxia stimulation. ( C ) Cell viability assay. n = 3 in each group. ** P < 0.01, n.s., not significant.

Article Snippet: Levels of SMOX protein in the cell lysate were analyzed using ELISA kits for rat SMOX (MyBioSource, San Diego, CA, USA) following the manufacturer's protocol.

Techniques: Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Viability Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Regulation of Spermine Oxidase through Hypoxia-Inducible Factor-1α Signaling in Retinal Glial Cells under Hypoxic Conditions

doi: 10.1167/iovs.61.6.52

Figure Lengend Snippet: Identification of HRE sites in the Smox promoter. ( A ) Six potential HRE sites located in the rat Smox promoter in the sequence analysis. ( B ) Luciferase assay for the transcriptional activity of the Smox promoter in TR-MUL5 cells under either normoxic or hypoxic conditions for 24 hours (20% O 2 or 1% O 2 ). ( C ) Effects of Hif1a silencing on Smox expression in TR-MUL5 cells under hypoxic condition (1% O 2 ) for 24 hours. ( D ) Mutant luciferase constructs designed for each of the six HRE sites via site-direct mutagenesis using the –1067 Luc reporter plasmid as a backbone. ( E ) Effects of Smox promoter mutations on the Smox transcriptional activity in TR-MUL5 cells stimulated with hypoxia for 24 hours. n = 6 in each group. ** P < 0.01.

Article Snippet: Levels of SMOX protein in the cell lysate were analyzed using ELISA kits for rat SMOX (MyBioSource, San Diego, CA, USA) following the manufacturer's protocol.

Techniques: Sequencing, Luciferase, Activity Assay, Expressing, Mutagenesis, Construct, Plasmid Preparation

Journal: PLoS Genetics

Article Title: Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

doi: 10.1371/journal.pgen.1003941

Figure Lengend Snippet: (A) Tissue-specific gene expression pattern of daw . (B) Tissue-specific distribution of transcription factor Smox using 7-day-old Oregon R females. (C–E) Lifespan analysis of Activin signaling using muscle-specific Gal4 driver (MHC-Gal4). Lifespan was extended by inactivating Activin genes ( daw , Smox and babo ) in muscle (Log-rank test, p <0.0001). (F–H) Lifespan analysis of Activin signaling using adult fat body-specific Gal4 driver (S106-GS-Gal4). Fat body-specific inactivation of Activin genes ( daw and Smox ) shortens lifespan (Log-rank test, p <0.0001). See for survival analysis. (I, J) mRNA expression of daw and phosphorylation of Smox are down-regulated by chico mutation and rescued by mutation of dFOXO. Muscle and fat body were dissected from 7-day-old female wildtype, chico −/− and chico;foxo double mutants. Band intensity was quantified using Bio-Rad Image Lab software. The average band intensity from four independent experiments is shown. Asterisk indicates significant difference between treatment and control ( p <0.05).

Article Snippet: Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156 (Covance, Dedham, MA, USA) and affinity purified (Thermo/Pierce, Waltham, MA, USA) (specificity documented in ).

Techniques: Gene Expression, Expressing, Phospho-proteomics, Mutagenesis, Software, Control

Journal: PLoS Genetics

Article Title: Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

doi: 10.1371/journal.pgen.1003941

Figure Lengend Snippet: (A–C) Decline of flight with age is delayed in daw , Smox and babo RNAi flies. 40 females were scored for each genotype at each time point. Flying ability was measured at one week, three weeks and five weeks. (D) Poly-Ubiquitin-positive protein aggregates are reduced at old age in daw , Smox and babo RNAi flies. Aggregates were visualized with Poly-Ubiquitin FK2 antibody at one week, three weeks and five weeks. Scale bar: 20 µm. (E) RNAi for daw , Smox and babo preserves the decline of lysotracker-positive organelles (lysosomes). Scale bar: 20 µm. (F) Quantification of the cumulative area of protein aggregates for (n = 20). (G) Quantification of the number of lysotracker-positive stain for (n = 10). Asterisk indicates significant difference between treatment and control ( p <0.05).

Article Snippet: Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156 (Covance, Dedham, MA, USA) and affinity purified (Thermo/Pierce, Waltham, MA, USA) (specificity documented in ).

Techniques: Ubiquitin Proteomics, Staining, Control

Journal: PLoS Genetics

Article Title: Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

doi: 10.1371/journal.pgen.1003941

Figure Lengend Snippet: (A) Autophagosomes indicated using an Atg8a -Cherry reporter in Activin RNAi flies or babo over-expressing ( babo-Act ) flies. 3-day old females. (B) Quantification of autophagosomes for (n = 20). (C) mRNA expression of autophagy genes ( Atg1 , Atg5 , Atg6 and Atg8a ) in aging muscle, at 10 days, 25 days and 45 days. (D) Phosphorylation of Smox in muscle increases with age. The average band intensity from three independent experiments was quantified using Image Lab software. (E) Inactivation of daw in muscle up-regulates autophagy gene expression. (F) RNAi against Smox in muscle up-regulates autophagy gene expression. (G) Constitutive activation of babo ( babo-Act ) in muscle inhibits autophagy gene expression. Asterisk indicates significant difference between treatment and control ( p <0.05).

Article Snippet: Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156 (Covance, Dedham, MA, USA) and affinity purified (Thermo/Pierce, Waltham, MA, USA) (specificity documented in ).

Techniques: Expressing, Phospho-proteomics, Software, Gene Expression, Activation Assay, Control

Journal: PLoS Genetics

Article Title: Activin Signaling Targeted by Insulin/dFOXO Regulates Aging and Muscle Proteostasis in Drosophila

doi: 10.1371/journal.pgen.1003941

Figure Lengend Snippet: (A) Schematic of Atg8a genomic region (Smad box and ChIP-PCR target regions (P1–P3) are shown). Gray bar represents UTR and orange bar represents exon. (B) ChIP-PCR shows Smox binds to the promoter of Atg8a with binding enrichment calculated as the fold change of ChIP DNA vs. input DNA. The binding to the coding region of Actin gene (Act5C) was used as a negative control. (C) Smox binds to the promoter of Atg8a , but not Atg1 and Atg6 . The primers targeting the promoter regions containing putative Smad box in Atg8a , Atg1 and Atg6 were used in ChIP-PCR. (D) The binding of Smox to Atg8a promoter is abolished by chico mutation. Asterisk indicates significant difference between treatment and control ( p <0.05). (E). EMSA analysis reveals that recombinant Smox protein (MH1-DNA binding domain) binds to Smad binding element (AGAC AGAC) located in Atg8a promoter. Biotin-labeled Atg8a oligonucleotide probe ( 5′- CATATT AGAC AGAC ACCATT -3′ ) and its mutated forms are labeled with biotin. Halo-tagged Smox-MH1 DNA binding domain (amino acids 1–140) are expressed in E. coli and purified before used in EMSA analysis. (F) Recombinant Smox protein can also bind to mammalian SBE ( GTAT GTCT AGAC TGAA ).

Article Snippet: Smox polyclonal antibody was generated against the peptide sequence (DSIVDYPLDNHTHQ) corresponding to amino acids 143–156 (Covance, Dedham, MA, USA) and affinity purified (Thermo/Pierce, Waltham, MA, USA) (specificity documented in ).

Techniques: Binding Assay, Negative Control, Mutagenesis, Control, Recombinant, Labeling, Purification