Journal: Journal of cellular biochemistry

Article Title: Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand-mediated triple negative breast cancer cell death.

doi: 10.1002/jcb.27887

Figure Lengend Snippet: FIGURE 3 5F 203 induces CYGB‐dependent lysosomal membrane permeabilization and cathepsin B release. A and B, T47D, and MDA‐MB‐468 cells after treatment with 0.1% DMSO (CTL) or 5F 203 (1 μM for T47D cells and 100 nM for MDA‐MB‐468 cells) for 24 hours followed by analysis using acridine orange or the Magic Red assays to measure LMP and cathepsin B release, respectively. C and D, shCYGB and shGFP MDA‐MB‐468 cells evaluated for LMP and cathepsin B release, respectively, after 5F 203 treatment. Scale bar = 50 μm. CTL, control; CYGB, cytoglobin; DMSO, dimethyl sulfoxide; LMP, lysosomal membrane permeabilization

Article Snippet: The CYGB complementary DNA (cDNA) clones were purchased from OriGene Technologies, Inc (Rockville, MD).

Techniques: Membrane, Control

Journal: Journal of cellular biochemistry

Article Title: Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand-mediated triple negative breast cancer cell death.

doi: 10.1002/jcb.27887

Figure Lengend Snippet: FIGURE 4 5F 203 confers anticancer activity, induces CYGB and proapoptotic protein expression, and produces caspase‐3 cleavage in vitro. A, Colony formation analysis in MDA‐MB‐468 cells untransfected, transfected with empty vector or transfected to overexpress CYGB. Lower panel: quantification of colony numbers. B, Assessment of migration in untransfected, shGFP, and shCYGB MDA‐MB‐468 cells after 5F 203 treatment. Lower panel: wound closure, normalized to control. C and D, Determination of CYGB, BAK‐1, LTA, and GADD45A protein expression as well as caspase‐3 cleavage in untransfected, shGFP, and shCYGB MDA‐MB‐468 cells after treatment with 5F 203 (1 μM, 72 hours). Scale bar = 100 μm. Bars, SEM. Statistically significant where indicated as designated *P < 0.05, **P < 0.01, or ***P < 0.001. BAK‐1, Bcl‐2 homologous antagonist killer; CYGB, cytoglobin; GADD45A, growth arrest and DNA‐damage‐inducible protein alpha; LTA, lymphotoxin alpha; SEM, standard error of the mean

Article Snippet: The CYGB complementary DNA (cDNA) clones were purchased from OriGene Technologies, Inc (Rockville, MD).

Techniques: Activity Assay, Expressing, In Vitro, Transfection, Plasmid Preparation, Migration, Control

Journal: Journal of cellular biochemistry

Article Title: Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand-mediated triple negative breast cancer cell death.

doi: 10.1002/jcb.27887

Figure Lengend Snippet: FIGURE 5 5F 203 confers anticancer activity, induces CYGB and proapoptotic protein expression, and produces caspase‐3 cleavage in vivo. A and B, Effect of Phortress on tumor growth (as measured in tumor volume, N = 11 and tumor mass, N = 9) for three cycles of three doses every other day (Q2D × 3) followed by a 10‐day rest period as determined in materials and methods. C, and D, CYGB, BAK‐1, and GADD45A protein expression as well as caspase‐3 cleavage determination in tumor xenografts. N = 6. Bars, SEM. Statistically significant where indicated as designated *P < 0.05, **P < 0.01, or ***P < 0.001. BAK‐1, Bcl‐2 homologous antagonist killer; CYGB, cytoglobin; GADD45A, growth arrest and DNA‐damage‐inducible protein alpha; SEM, standard error of the mean

Article Snippet: The CYGB complementary DNA (cDNA) clones were purchased from OriGene Technologies, Inc (Rockville, MD).

Techniques: Activity Assay, Expressing, In Vivo

Journal: Biochimica et biophysica acta

Article Title: Peroxidase activation of cytoglobin by anionic phospholipids: Mechanisms and consequences.

doi: 10.1016/j.bbalip.2016.02.022

Figure Lengend Snippet: Fig. 7. Formation of disulfide bond between Cys38 and Cys83 induces the bending of the E helix and an increase of the distance between E and F helices. The figure shows the overlay of the hexa-coordinated crystal structure of the C38S/C83S mutant of human Cygb (purple, PDB ID: 1UT0) with a model of the penta-coordinated, ferric wild-type structure (cyan). The yellow dashed line indicates the distance between the alpha carbons of Ala88 (E helix) and Val105 (F helix). Molecular dynamics simulations indicate that the formation of the disulfide bond causes an increase in this interhelical distance (8.04 ± 0.64 Å vs 7.2 Å, respectively), allowing for the binding of hydrophobic chains in the hydrophobic core of Cygb.

Article Snippet: In summary, the region encoding the human Cygb protein was cloned from the Origene (Rockville, MD) cDNA clone SC321813 (NM_134268) and inserted into the NcoI/HindIII restriction sites of the pET28a plasmid (Novagen) generating the pET28–HsaCygb plasmid.

Techniques: Mutagenesis, Binding Assay

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: 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