Journal: Journal of translational medicine

Article Title: Cytosine-phosphate-guanine oligodeoxynucleotides alleviate radiation-induced kidney injury in cervical cancer by inhibiting DNA damage and oxidative stress through blockade of PARP1/XRCC1 axis.

doi: 10.1186/s12967-023-04548-y

Figure Lengend Snippet: Fig. 1 Protective effects of CpG-ODNs on RKI in relation to DNA damage and oxidative stress levels. A CpG-ODNs related sequence information; B Levels of blood urea nitrogen (BUN) and creatinine in mouse serum detected by automated biochemical analyzer; C Histopathological changes in mouse kidney tissues observed by HE staining (scale bar = 25 µm); D Levels of cellular apoptosis in mouse kidney tissues detected by TUNEL staining (scale bar = 25 µm), with brownish-gray indicating apoptotic cells; E Levels of DNA damage index (8-OHdG) in mouse serum detected by ELISA; F Levels of oxidative stress-related indexes in mouse serum detected by ELISA; G Levels of malondialdehyde (MDA) in mouse serum detected. * indicates P < 0.05 compared to the Normal group, # indicates P < 0.05 compared to the RKI group; each group consisted of 8 mice

Article Snippet: The levels of DNA damage index (8-OHdG) and each oxidative stress related factor in serum and cells were detected using mouse 8-OHdG (CSB-E10527m, Cusabio), human catalase (CAT) (JL14741, Shanghai Jianglai Biotechnology, Shanghai, China), mouse CAT (CSB-E14190m, Cusabio, Houston, TX), human superoxide dismutase (SOD) (CSB-E17064h, Cusabio), mouse SOD (JL12237, Shanghai Jianglai Biotechnology), human glutathione (GSH) (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), mouse GSH (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), mouse GSH (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), human glutathione peroxidase (GPx) (JL10355, Shanghai Jianglai Biotechnology), mouse GPx (JL49904, Shanghai Jianglai Biotechnology) kits [39, 40].

Techniques: Sequencing, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of translational medicine

Article Title: Cytosine-phosphate-guanine oligodeoxynucleotides alleviate radiation-induced kidney injury in cervical cancer by inhibiting DNA damage and oxidative stress through blockade of PARP1/XRCC1 axis.

doi: 10.1186/s12967-023-04548-y

Figure Lengend Snippet: Fig. 5 Effect of CpG-ODNs on DNA damage and oxidative stress in HK-2 cells by regulating PARP1/XRCC1 axis. A CCK-8 assay for detection of viability of radiation-treated HK-2 cells in response to CpG-ODN2006 alone or combined with oe-PARP1 or oe-XRCC1. B Caspase activity in radiation-treated HK-2 cells in response to CpG-ODN2006 alone or combined with oe-PARP1 or oe-XRCC1 as determined with enzymatic method. C Immunofluorescence assay to detect the γH2AX-positive cell ratio of radiation-treated HK-2 cells in response to CpG-ODN2006 alone or combined with oe-PARP1 or oe-XRCC1. Blue indicated DAPI fluorescence labeled nucleus, and green indicated labeled γH2AX. D ELISA detection of levels of oxidative stress-related factors (SOD, CAT, GSH, GPx) in radiation-treated HK-2 cells in response to CpG-ODN2006 alone or combined with oe-PARP1 or oe-XRCC1. E ELISA detection of MDA levels in radiation-treated HK-2 cells in response to CpG-ODN2006 alone or combined with oe-PARP1 or oe-XRCC1. * P < 0.05 versus HK-2 cells treated with saline + oe-NC. # P < 0.05 versus HK-2 cells treated with CpG-ODN2006 + oe-NC. All cell experiments were repeated three times

Article Snippet: The levels of DNA damage index (8-OHdG) and each oxidative stress related factor in serum and cells were detected using mouse 8-OHdG (CSB-E10527m, Cusabio), human catalase (CAT) (JL14741, Shanghai Jianglai Biotechnology, Shanghai, China), mouse CAT (CSB-E14190m, Cusabio, Houston, TX), human superoxide dismutase (SOD) (CSB-E17064h, Cusabio), mouse SOD (JL12237, Shanghai Jianglai Biotechnology), human glutathione (GSH) (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), mouse GSH (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), mouse GSH (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), human glutathione peroxidase (GPx) (JL10355, Shanghai Jianglai Biotechnology), mouse GPx (JL49904, Shanghai Jianglai Biotechnology) kits [39, 40].

Techniques: CCK-8 Assay, Activity Assay, Immunofluorescence, Fluorescence, Labeling, Enzyme-linked Immunosorbent Assay, Saline

Journal: Journal of translational medicine

Article Title: Cytosine-phosphate-guanine oligodeoxynucleotides alleviate radiation-induced kidney injury in cervical cancer by inhibiting DNA damage and oxidative stress through blockade of PARP1/XRCC1 axis.

doi: 10.1186/s12967-023-04548-y

Figure Lengend Snippet: Fig. 7 Schematic diagram of the molecular mechanism by which CpG-ODNs regulate PARP1/XRCC1 axis to affect DNA damage and oxidative stress response in RKI in cervical cancer. CpG-ODNs may attenuate RKI in cervical cancer by blocking PARP1/XRCC1 axis activation and inhibiting DNA damage and oxidative stress in renal tubular epithelial cells

Article Snippet: The levels of DNA damage index (8-OHdG) and each oxidative stress related factor in serum and cells were detected using mouse 8-OHdG (CSB-E10527m, Cusabio), human catalase (CAT) (JL14741, Shanghai Jianglai Biotechnology, Shanghai, China), mouse CAT (CSB-E14190m, Cusabio, Houston, TX), human superoxide dismutase (SOD) (CSB-E17064h, Cusabio), mouse SOD (JL12237, Shanghai Jianglai Biotechnology), human glutathione (GSH) (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), mouse GSH (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), mouse GSH (ml063305, Shanghai Meilian Biotechnology Co., Ltd.), human glutathione peroxidase (GPx) (JL10355, Shanghai Jianglai Biotechnology), mouse GPx (JL49904, Shanghai Jianglai Biotechnology) kits [39, 40].

Techniques: Blocking Assay, Activation Assay

Journal: Thrombosis and Haemostasis

Article Title: NF-κB-Mediated Upregulation of Tissue Factor Contributes to the Procoagulant Phenotype of Smooth Muscle Cells from Abdominal Aorta Aneurysm in Human

doi: 10.1055/a-2665-2510

Figure Lengend Snippet: Involvement of the PAR-2/NF-κB axis in increased tissue factor expression in AAA. ( A ) Fluorescent detection of cellular oxidant levels using CM-H 2 DCFDA ( n = 9). ( B ) Levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) in cell supernatants ( n = 9–10). ( C ) Seahorse respiratory measurements, namely basal respiration, ATP-linked respiration, proton-linked respiration, maximum respiratory capacity, and reserve capacity in AAA-SMCs compared with HA-SMCs ( n = 9–12). ( D ) TNF-α mRNA levels in SMCs cultured from HA and AAA aortas ( n = 8). ( E ) Representative images of Western blot to assess levels of PAR-2, phosphorylated and total IKK, and NF-κB in SMCs. ( F–H ) Quantification of PAR-2, p-IKK/IKK, and p-NF-κB/NF-κB band intensities ( n = 5–6). ( I ) Immunofluorescence staining of α-SMA (red) and p-NF-κB (green) in aortic tissues. Nuclei were counterstained using DAPI. ( J ) Immunofluorescence staining of p-NF-κB in SMCs. ( K, L ) TF activity at the surface of SMCs incubated for 4 hours with the PAR-2 antagonist GB83 or the inhibitor of NF-κB activation BAY 11–7082 (BAY). Values are normalized by TF activity in the absence of GB83 or BAY ( n = 5). Data represent means ± SD. Groups were statistically analyzed by the Mann–Whitney test.

Article Snippet: Extracellular 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) was measured in cell culture supernatants using the HT 8-oxo-dG ELISA Kit II assay (R&D Systems).

Techniques: Expressing, Cell Culture, Western Blot, Immunofluorescence, Staining, Activity Assay, Incubation, Activation Assay, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus

doi: 10.1084/jem.20151876

Figure Lengend Snippet: MtDNA oxidation is required for pDC activation. (A, top) pDCs were incubated with supernatants from neutrophils treated with or without IFN ± αRNP. IFN-α levels were quantified after 18 h culture. (bottom) Extruded mtDNA or TFAM quantification (mtDNA copy number) in the supernatants from neutrophils treated with or without IFN ± αRNP. (B) Dot blot analysis, using anti-8OHdG or anti-dsDNA (loading control) antibodies, of the DNA isolated from the supernatants of neutrophils treated with or without IFN ± αRNP. Bars represent the quantification of 8OHdG intensity. (C) Neutrophils were stimulated with IFN/αRNP in the absence or in the presence of the mtROS scavenger MitoTempo (MT). Extruded mtDNA was assessed for its interferogenic effect on pDCs (top) and for its oxidation status (bottom). (D, top) Neutrophils were treated with IFN/αRNP in the presence of IRS661 (TLR7 inhibitor) or DVX42 (TLR8 inhibitor) and the corresponding supernatants were assessed for their interferogenic effect on pDCs. (bottom) The oxidation status of extruded mtDNA was assessed by dot blot. (E) IFN-α production by pDCs stimulated with LL-37 added to genomic DNA, Non-Ox, or Ox mtDNA, or with supernatants from IFN/αRNP-treated neutrophils. (F) IFN-α production by pDCs stimulated with Ox mtDNA with our without the TLR9 inhibitor ODN-TTAGGG. (G, top) DNA from IFN/αRNP activated neutrophil supernatants were immunoprecipitated with anti-dsDNA antibody. The IP was analyzed by Western blot with anti-LL37 or anti-HMGB1 antibodies. (bottom) IFN-α production by pDCs stimulated with interferogenic neutrophil supernatants in the presence or absence of the HMGB1 inhibitor BoxA. (H) IFN-α production was assessed after pDC incubation with interferogenic neutrophil supernatants in the presence or absence of anti-TFAM antibodies or recombinant RAGE-Fc chimera. (I) Neutrophils were treated as shown and mitochondrial depolarization was assessed with MitoTracker DeepRed; the amount of extruded mtDNA was assessed by Real-Time PCR and its oxidation status by 8OHdG dot blot. Bars represent mean ± SD from at least three independent experiments with n = 3–8. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Antibody against 8OHdG (J-1; rabbit polyclonal IgG2b) and all chemicals were purchased from Santa Cruz Biotechnology, Inc. Antibodies against LL-37, TFAM (Clone 18G102B2E11), Mitofilin (Clone 2E4AD5), γH2A.X, TOMM20 (Clone 4F3), Pyruvate Dehydrogenase E2/E3bp (PDH), VDAC/Porin, Glyceraldehyde 3 - phosphate dehydrogenase (GADPH), dsDNA, LAMP1 (Clone H4A3), and Rab7 were purchased from Abcam.

Techniques: Activation Assay, Incubation, Dot Blot, Control, Isolation, Immunoprecipitation, Western Blot, Recombinant, Real-time Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus

doi: 10.1084/jem.20151876

Figure Lengend Snippet: Ox mtDNA is exported from mitochondria to lysosomes under steady-state conditions . (A) Cytoplasmic 8OHdG(+) staining in neutrophils and monocytes. Bar, 10 µm. (B) Live or apoptotic neutrophils were stained with anti-8OHdG antibody and the damaged nuclear DNA marker γH2A.X (top) or with TUNEL assay (bottom). Bar, 5 µm. (C and D) Cytosolic mtDNA copy number (C) and 8OHdG staining and quantification (D) in neutrophils treated with media, Rotenone or MitoTempo. The purity of the cytosolic fraction is also shown in (C). TCE = total cell extract. TOMM20 was used as mitochondrial marker. Bars, 10 µm. (E) Neutrophils were treated with the lysosomal inhibitor Bafilomycin A1 (BafA1) and stained with anti-8OHdG and anti-LAMP1 antibodies. Bars, 10 µm. (F) Supernatant (Sup) from the mitochondrial budding assay was subjected to Western blot analysis (top) or analyzed for mtDNA content (bottom) by agarose gel. The mitochondrial fraction (Mitos) was loaded as control. * indicates nonspecific band. Data in B–F are representative of three independent experiments, with n = 3. Bars represent mean ± SD from at least three independent experiments, with n = 3–5. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Antibody against 8OHdG (J-1; rabbit polyclonal IgG2b) and all chemicals were purchased from Santa Cruz Biotechnology, Inc. Antibodies against LL-37, TFAM (Clone 18G102B2E11), Mitofilin (Clone 2E4AD5), γH2A.X, TOMM20 (Clone 4F3), Pyruvate Dehydrogenase E2/E3bp (PDH), VDAC/Porin, Glyceraldehyde 3 - phosphate dehydrogenase (GADPH), dsDNA, LAMP1 (Clone H4A3), and Rab7 were purchased from Abcam.

Techniques: Staining, Marker, TUNEL Assay, Western Blot, Agarose Gel Electrophoresis, Control

Journal: The Journal of Experimental Medicine

Article Title: Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus

doi: 10.1084/jem.20151876

Figure Lengend Snippet: IFN/ α RNP activation of neutrophils induces intramitochondrial retention of Ox mtDNA. (A) Cytoplasmic 8OHdG(+) staining in neutrophils treated with or without IFN ± αRNP. 8OHdG quantification is also shown. Bars, 10 µm. (B) Neutrophils were treated with or without IFN ± αRNP and co-stained with anti-8OHdG and anti-TOMM20 antibodies. Bars, 10 µm. (C) Alternatively, mtDNA was extracted and analyzed by dot blot with anti-8OHdG and anti-dsDNA antibodies. TOMM20 was used as loading control. (D) Neutrophils were treated with or without IFN ± αRNP, and the production of mtROS or total cellular ROS was quantified by MitoSox or CellRox, respectively. Bars represent mean ± SD from at least three independent experiments, with n = 3–6. **, P < 0.01; ***, P < 0.001.

Article Snippet: Antibody against 8OHdG (J-1; rabbit polyclonal IgG2b) and all chemicals were purchased from Santa Cruz Biotechnology, Inc. Antibodies against LL-37, TFAM (Clone 18G102B2E11), Mitofilin (Clone 2E4AD5), γH2A.X, TOMM20 (Clone 4F3), Pyruvate Dehydrogenase E2/E3bp (PDH), VDAC/Porin, Glyceraldehyde 3 - phosphate dehydrogenase (GADPH), dsDNA, LAMP1 (Clone H4A3), and Rab7 were purchased from Abcam.

Techniques: Activation Assay, Staining, Dot Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus

doi: 10.1084/jem.20151876

Figure Lengend Snippet: IFN/ α RNP inhibit the dissociation of Ox mtDNA and TFAM. (A) Neutrophils were treated as shown and co-stained with anti-8OHdG and anti-TFAM antibodies. Bars, 10 µm. Alternatively, cell lysates were subjected to IP with anti-TFAM antibody and the immunoprecipitates were analyzed by dot blot. (B–D) Neutrophils were treated as shown and cell lysates were analyzed by Western blot with antibodies against TFAM (B), PGC1α (C), Mitofilin or TOMM20 (D), and GAPDH as control. Total mitochondrial mass was assessed by MitoTracker Green (D). (E) Neutrophils were treated as shown and cell lysates were subjected to IP with anti-TFAM antibody. The immunoprecipitates were then blotted with antibodies against PKA-phosphorylated peptides (α-PKA sub) or TFAM (loading control). The PKA inhibitor H89 was used as a control. (F) Neutrophils were treated with αRNP ± IFN in the presence or absence of the cAMP analogue 8Br-cAMP or the PDE inhibition IBMX. Extruded mtDNA was analyzed by dot blot (left) and supernatants were assessed for their interferogenic capacity on pDCs (right). (G) Neutrophils were treated as shown and cAMP levels were measured in the enriched mitochondrial fraction. cAMP levels were normalized to the total protein content and expressed in arbitrary units (AU). Bars represent mean ± SD from at least three independent experiments, with n = 3–5. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibody against 8OHdG (J-1; rabbit polyclonal IgG2b) and all chemicals were purchased from Santa Cruz Biotechnology, Inc. Antibodies against LL-37, TFAM (Clone 18G102B2E11), Mitofilin (Clone 2E4AD5), γH2A.X, TOMM20 (Clone 4F3), Pyruvate Dehydrogenase E2/E3bp (PDH), VDAC/Porin, Glyceraldehyde 3 - phosphate dehydrogenase (GADPH), dsDNA, LAMP1 (Clone H4A3), and Rab7 were purchased from Abcam.

Techniques: Staining, Dot Blot, Western Blot, Control, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus

doi: 10.1084/jem.20151876

Figure Lengend Snippet: Intramitochondrial Ox mtDNA is a feature of SLE blood neutrophils. (A) Freshly isolated neutrophils from, healthy, JDM, or SLE blood were co-stained with anti-8OHdG and anti-TOMM20 antibodies. Bars, 10 µm. Quantification of 8OHdG staining in neutrophils from healthy donors ( n = 6), JDM ( n = 6), and SLE ( n = 14) patients is also shown. (B) Correlation between Ox mtDNA levels in SLE neutrophils and IFN-α production by pDCs upon activation with corresponding SLE neutrophil supernatants. ( n = 15). (C) Healthy ( n = 6), JDM ( n = 6), or SLE ( n = 15) sera were assessed for the presence of anti-Ox mtDNA autoantibodies. Anti-8OHdG antibody was used as a control. *, P < 0.05; **, P < 0.01.

Article Snippet: Antibody against 8OHdG (J-1; rabbit polyclonal IgG2b) and all chemicals were purchased from Santa Cruz Biotechnology, Inc. Antibodies against LL-37, TFAM (Clone 18G102B2E11), Mitofilin (Clone 2E4AD5), γH2A.X, TOMM20 (Clone 4F3), Pyruvate Dehydrogenase E2/E3bp (PDH), VDAC/Porin, Glyceraldehyde 3 - phosphate dehydrogenase (GADPH), dsDNA, LAMP1 (Clone H4A3), and Rab7 were purchased from Abcam.

Techniques: Isolation, Staining, Activation Assay, Control