Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Cygb protein expression in human GBM cells . Cygb expression was assessed by Western blot analyses after exposure to hypoxia (0.6% O 2 ) for 0, 6, 24 and 48 h (n = 4). The integrated intensities of Cygb and α- tubulin (control) bands were determined and expressed in arbitrary units (AU), and representative blots are shown. (A) M006x: (B) M006xLo; (C) M010B; (D) M059J; (E) U87T. * p < 0.05; ** p < 0.01; *** p < 0.001 (ANOVA).

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing, Western Blot, Control

Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Cygb mRNA expression in human GBM cells . Cygb mRNA expression was assessed by qRT-PCR after exposure to hypoxia (0.6% O 2 ) for 0, 6, 24 and 48 h. Data were expressed as fold increase relative to aerobic control (n = 4). (A) M006x: (B) M006xLo; (C) M010B; (D) M059J; (E) U87T. * p < 0.05; ** p < 0.01; *** p < 0.001 (ANOVA).

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Ngb, Cygb and CA IX expression in tissue microarrays of human solid tumors and adjacent normal tissues

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing

Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Expression of Ngb, Cygb and CA IX in human normal tissues . Tissue microarrays containing cores obtained from various human normal tissues were stained with to antibodies to Ngb, Cygb or CA IX. Positive staining was visualized by the chromogenic reaction of HRP with DAB. Photomicrographs were obtained at 20× magnification, and the scale bar indicates 50 μM. (A) stomach (fundus); (B) small bowel; (C) gallbladder.

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing, Staining

Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Expression of Ngb, Cygb and CA IX in human tumors . Tissue microarrays containing cores obtained from various human tumors were stained with to antibodies to Ngb, Cygb or CA IX. Positive staining was visualized by the chromogenic reaction of HRP with DAB. Photomicrographs were obtained at 20× magnification, and the scale bar indicates 50 μM. (A) ovarian carcinoma; (B) hepatocellular carcinoma; (C) breast infiltrating duct carcinoma.

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing, Staining

Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Expression of Ngb and Cygb in human astrocytomas . Tissue microarrays containing cores obtained from various human brain tumors were stained with to antibodies to Ngb or Cygb. Positive staining was visualized by the chromogenic reaction of HRP with DAB. Photomicrographs were obtained at 20× magnification, and the scale bar indicates 50 μM. Sections of representative examples of Grades I-IV astrocytoma are shown.

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing, Staining

Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Expression of Cygb and Ngb in human primary brain tumors . Tissue microarrays containing cores obtained from various human brain tumors were stained with to antibodies to Ngb or Cygb. Positive staining was visualized by the chromogenic reaction of HRP with DAB. Photomicrographs were obtained at 20× magnification, and the scale bar indicates 50 μM. (A) oligodendroglioma; (B) ependymoblastoma; (C) ganglioglioma.

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing, Staining

Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Ngb, Cygb and CA IX expression in tissue microarrays of human primary brain tumors

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing

Journal: Cancer Cell International

Article Title: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues

doi: 10.1186/1475-2867-10-33

Figure Lengend Snippet: Assessment of Cygb, Ngb and CA IX expression in tissue microarrays of human glioblastoma multiforme tumors

Article Snippet: Quantitative real-time PCR (qRT-PCR) analysis was performed with a 7900 HT Fast Real-Time PCR System (Applied Biosystems) using TaqMan fast universal PCR master mix and a validated TaqMan Gene Expression assay (Applied Biosystems) for the human CYGB gene (assay ID: Hs00370478_ml).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Cytoglobin Promotes Cardiac Progenitor Cell Survival against Oxidative Stress via the Upregulation of the NFκB/iNOS Signal Pathway and Nitric Oxide Production

doi: 10.1038/s41598-017-11342-6

Figure Lengend Snippet: The expression of CYGB is associated with the cell survival ability against oxidative stress in hCPCs. ( a ) Representative images and quantitative data of Western blot showed that the expression of CYGB is observed in both hCPCs and human right atrial appendage (hRAA). However, the expression of myoglobin is not detectable in hCPCs compared to its abundant expression in hRAA. The total protein loading is 5 µg for hRAA and 20 µg for hCPCs. Full-length blots are presented in Supplemental Fig. . ( b ) Western blot confirmed that CYGB was overexpressed in hCPCs. Full-length blots are presented in Supplemental Fig. . ( c ) CPCs were infected with lentivirus expressing CYGB or vector for 24 h, challenged with 2 mM H 2 O 2 for 3 h, and then evaluated by LDH assay. ( d ) Western blot confirmed that CYGB was knocked-down in hCPCs with shRNAs for clone #1&4. Full-length blots are presented in Supplemental Fig. . ( e ) Human CPCs were infected with lentivirus expressing shRNA against CYGB, or scramble shRNA for 24 h, challenged with 2 mM H 2 O 2 for 3 h, then evaluated by LDH assay. Data presented in this figure were mean values on a ratio of H 2 O 2 -induced LDH release to total LDH in cells with standard error (means ± SEM). *Indicates p < 0.05 vs. control; **indicates p < 0.01 vs. control; n = 4 independent experiments.

Article Snippet: ORF expression clone for CYGB (purified plasmid), empty control vector for pReceiver-Lv202, shRNA scrambled control clone for psi-LVRH1GP, and shRNA clone set against human CYGB were purchased from GeneCopoeia.

Techniques: Expressing, Western Blot, Infection, Plasmid Preparation, Lactate Dehydrogenase Assay, shRNA, Control

Journal: Scientific Reports

Article Title: Cytoglobin Promotes Cardiac Progenitor Cell Survival against Oxidative Stress via the Upregulation of the NFκB/iNOS Signal Pathway and Nitric Oxide Production

doi: 10.1038/s41598-017-11342-6

Figure Lengend Snippet: Overexpression of CYGB displays the anti-oxidant effect. ( a ) Representative images for DHE staining and quantitative analysis for hCPCs infected with CYGB- or vector-lentivirus for 48 hours. ( b ) Representative merged histogram and quantitative analysis for ROS measurement for hCPCs overexpressing CYGB vs. vector control by the FACS analysis with the ROS Detection Assay Kit (Deep Red Fluorescence). ( c ) Representative merged histogram and quantitative analysis for ROS measurement for hCPCs expressing shRNA against CYGB vs. scramble shRNA. The open blue traces are unstained cells as negative controls in panel b and c. ( d ) Examination of anti-oxidant gene expression at the mRNA level after CYGB was overexpressed. ( e ) Representative images and quantitative data of Western blot showing anti-oxidant protein expression level after CYGB was overexpressed. Full-length blots are presented in Supplemental Fig. . *Indicates p < 0.05 vs. control; **indicates p < 0.01 vs. control; n = 3 independent experiments.

Article Snippet: ORF expression clone for CYGB (purified plasmid), empty control vector for pReceiver-Lv202, shRNA scrambled control clone for psi-LVRH1GP, and shRNA clone set against human CYGB were purchased from GeneCopoeia.

Techniques: Over Expression, Staining, Infection, Plasmid Preparation, Control, Detection Assay, Fluorescence, Expressing, shRNA, Gene Expression, Western Blot

Journal: Scientific Reports

Article Title: Cytoglobin Promotes Cardiac Progenitor Cell Survival against Oxidative Stress via the Upregulation of the NFκB/iNOS Signal Pathway and Nitric Oxide Production

doi: 10.1038/s41598-017-11342-6

Figure Lengend Snippet: Disruption of NFκB expression abolished the cytoprotective effect of overexpressing CYGB. ( a ) Representative FACS analysis with annexin V/PI staining showing H 2 O 2 -induced apoptosis in hCPCs stably expressing scrambled or NFκB-p65 shRNA following with or without CYGB overexpression. ( b ) Quantitative data analysis for panel a. ( c ) Representative images and quantitative data of Western blot showing NFκB-related protein expression levels after NFκB was knocked-down. Full-length blots are presented in Supplemental Fig. . ( d ) Representative images and quantitative data of Western blot showing anti-apoptotic and anti-oxidant protein expression levels after NFκB was knocked down. Full-length blots are presented in Supplemental Fig. . *Indicates p < 0.05 vs. control; **indicates p < 0.01 vs. control; n = 3 independent experiments.

Article Snippet: ORF expression clone for CYGB (purified plasmid), empty control vector for pReceiver-Lv202, shRNA scrambled control clone for psi-LVRH1GP, and shRNA clone set against human CYGB were purchased from GeneCopoeia.

Techniques: Disruption, Expressing, Staining, Stable Transfection, shRNA, Over Expression, Western Blot, Control

Journal: Scientific Reports

Article Title: Cytoglobin Promotes Cardiac Progenitor Cell Survival against Oxidative Stress via the Upregulation of the NFκB/iNOS Signal Pathway and Nitric Oxide Production

doi: 10.1038/s41598-017-11342-6

Figure Lengend Snippet: Knocking down iNOS diminishes the cytoprotective effect of overexpressing CYGB. ( a ) Representative FACS analysis with annexin V/PI staining showing H 2 O 2 -induced apoptosis in hCPCs stably expressing scrambled or iNOS shRNA following with or without CYGB overexpression. ( b ) Quantitative data analysis for panel a. ( c ) Representative images and quantitative data of Western blot showing the expression of anti-apoptotic and anti-oxidant proteins after iNOS was knocked down. Full-length blots are presented in Supplemental Fig. . ( d ) Descriptive diagram of proposed molecular mechanism for CYGB regulated hCPC survival. *Indicates p < 0.05 vs. control; **indicates p < 0.01 vs. control; n = 3 independent experiments.

Article Snippet: ORF expression clone for CYGB (purified plasmid), empty control vector for pReceiver-Lv202, shRNA scrambled control clone for psi-LVRH1GP, and shRNA clone set against human CYGB were purchased from GeneCopoeia.

Techniques: Staining, Stable Transfection, Expressing, shRNA, Over Expression, Western Blot, Control

Journal: Molecular and cellular biochemistry

Article Title: Cytoglobin augments ferroptosis through autophagic degradation of ferritin in colorectal cancer cells.

doi: 10.1007/s11010-024-05148-0

Figure Lengend Snippet: Fig. 5 CYGB combined TFR to regulate ferroptosis. (A) Western blotting analysis of FLAG-CYGB-overexpression in HCT116 and SW620. (B) The immunoprecipitation and immunoblotting were per- formed using FLAG-CYGB and TFR antibodies in HCT116. (C) Co-localization of TFR and FLAG-CYGB in HCT116 cells based on immunofluorescence staining

Article Snippet: The separated proteins were separated in a 10% gel through sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes, blocked in 0.3% Tris-buffered saline with Tween 20 solution containing 5% nonfat dry milk, and further incubated with the primary antibodies LC3B (1:1000 dilution, #3868; Cell Signaling), β-Actin (1:20000 dilution, AC026; ABclonal), CYGB (1:1000 dilution, 60228-1-Ig; Proteintech), DDDDKTag (1:1000 dilution, HT201; TransGen), NCOA4 (1:1000 dilution, A5695; ABclonal), TFR (1:1000 dilution, #13113; Cell Signaling), FTH (1:1000 dilution, A19544; ABclonal), FTL (1:2000 dilution, A11241; ABclonal), PCBP1 (1:1000 dilution, A22141; ABclonal) and ACSL4 (1:1000 dilution, A20414; ABclonal).

Techniques: Western Blot, Over Expression, Immunoprecipitation, Immunofluorescence, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cytoglobin promotes sensitivity to ferroptosis by regulating p53‐YAP1 axis in colon cancer cells

doi: 10.1111/jcmm.16400

Figure Lengend Snippet: CYGB promoted YAP1 expression and was involved in ferroptosis signalling pathway. A. Demonstration of significant changes in the expression of genes based on RNA‐seq in the CYGB‐overexpressing HCT116 cells compared to MOCK cells. B. The most twenty significantly enriched KEGG pathways based on CYGB overexpression. C. Immunoblotting analysis of YAP1, P‐YAP1 and ACSL4 in the MOCK‐ and CYGB‐overexpressing cells. GAPDH was used as a loading control. D. The comparison of obviously changed genes between RNA‐seq in present study and the colon cancer data from TCGA in Figure . E. The most twenty significantly enriched KEGG pathways based on overlapped genes in D. F, G. The list of obviously changed ferroptosis‐related genes between comparison of CYGB‐low and CYGB‐high expression in colon cancer from TCGA data (F) and RNA‐seq in present study (G). H, I. Gene set enrichment analysis (GSEA) indicates that high expression of CYGB is associated with the hippo (H) and ferroptosis signalling pathway (I) in the TCGA database

Article Snippet: Primary antibodies against CYGB (#60228, Proteintech), YAP1 (#14074, Cell Signaling Technology), phospho‐YAP1 (Ser127) (#13008, Cell Signaling Technology), p53 (#2524, Cell Signaling Technology), phospho‐p53 (Ser15) (#9284, Cell Signaling Technology), SLC7A11 (#12691, Cell Signaling Technology), ACSL4 (#ab155282, Abcam) and GAPDH (#AP0063, Bioworld Antibodies) were used.

Techniques: Expressing, RNA Sequencing, Over Expression, Western Blot, Control, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cytoglobin promotes sensitivity to ferroptosis by regulating p53‐YAP1 axis in colon cancer cells

doi: 10.1111/jcmm.16400

Figure Lengend Snippet: CYGB promoted ferroptosis sensitivity is YAP1 dependent. A. Immunoblotting analysis of YAP1 and ACSL4 in HCT116 cells treated with siRNAs targeting YAP1 for 72 hours. B, C. Cellular (B) and lipid (C) ROS detection with DCFDA and C11‐BODIPY, respectively. D. Cell death analysis with propidium iodide (PI) staining by flow cytometry after the application of RSL3, with or without YAP1 siRNAs. Data represent the mean ± SD of three biological replicates. * P < 0.05, ** P < 0.05. E, F. Positive correlations between CYGB and YAP1 (E); and between CYGB and ACSL4 (F) mRNA expression in colon cancer from TCGA

Article Snippet: Primary antibodies against CYGB (#60228, Proteintech), YAP1 (#14074, Cell Signaling Technology), phospho‐YAP1 (Ser127) (#13008, Cell Signaling Technology), p53 (#2524, Cell Signaling Technology), phospho‐p53 (Ser15) (#9284, Cell Signaling Technology), SLC7A11 (#12691, Cell Signaling Technology), ACSL4 (#ab155282, Abcam) and GAPDH (#AP0063, Bioworld Antibodies) were used.

Techniques: Western Blot, Staining, Flow Cytometry, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cytoglobin promotes sensitivity to ferroptosis by regulating p53‐YAP1 axis in colon cancer cells

doi: 10.1111/jcmm.16400

Figure Lengend Snippet: YAP1 was a downstream target of p53. A. Immunoblotting analysis of p53, P‐p53 and SLC7A11 in the MOCK‐ and CYGB‐overexpressing cells. B. Immunoblotting analysis of p53, YAP1 and SLC7A11 in the HCT116 cells treated with siRNAs targeting p53 for 72 hours. C. Transcriptional level of p53, SLC7A11 and YAP1 in the HCT116 cells treated with siRNAs targeting p53 for 72 hours. D. Effects on the lipid peroxidation after p53 knock‐down in the MOCK‐ and CYGB‐overexpressing HCT116 were assessed with C11‐BODIPY by flow cytometry. Data represent the mean ± SD of three biological replicates. * P < 0.05, ** P < 0.05. E. Correlations between CYGB and P53 mRNA expression in colon cancer from TCGA. F. A proposed working model of CYGB to promote sensitivity of ferroptosis. YAP1 was the downstream of p53 which was activated by CYGB to promote lipid peroxidation and ferroptosis

Article Snippet: Primary antibodies against CYGB (#60228, Proteintech), YAP1 (#14074, Cell Signaling Technology), phospho‐YAP1 (Ser127) (#13008, Cell Signaling Technology), p53 (#2524, Cell Signaling Technology), phospho‐p53 (Ser15) (#9284, Cell Signaling Technology), SLC7A11 (#12691, Cell Signaling Technology), ACSL4 (#ab155282, Abcam) and GAPDH (#AP0063, Bioworld Antibodies) were used.

Techniques: Western Blot, Knockdown, Flow Cytometry, Expressing