pseudomonas 4 pseudomonas panipatensis lmg 24738 t  (ATCC)

Journal: Neurotherapeutics

Article Title: Oxidized mitochondrial DNA activates the cGAS-STING pathway in the neuronal intrinsic immune system after brain ischemia-reperfusion injury

doi: 10.1016/j.neurot.2024.e00368

Figure Lengend Snippet: Expression and cellular localization of key proteins of the cGAS-STING pathway in the ischemic penumbra after ischemia-reperfusion injury. A-C. WB analysis of the expression and phosphorylation levels of cGAS-STING pathway key proteins cGAS, STING, TBK1, IRF3, and P65 in sham rats and rats after ischemia-reperfusion injury at 4 ​h, 8 ​h, 12 ​h, and 24 ​h. The image is representative, and the statistical data are the mean ​± ​SD (n ​= ​3) of the ratios of the gray values of each protein band to the GAPDH band. D-G. Expression of pSTING in neurons, microglia, and astrocytes in sham rats and rats after ischemia-reperfusion injury at 4 ​h and 24 ​h. The images are representative of the ones observed by fluorescence microscopy. The statistical data mean ​± ​SD (n ​= ​3) represent the proportions of pSTING positive cells in neurons (NEUN), microglia (IBA1), and astrocytes (GFAP). ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001.

Article Snippet: The antibodies used were as follows: anti-GAPDH antibody (ab9485, Abcam), STING Antibody (2922D, Novus Biologicals), cGAS (31659, Cell Signaling Technology), IRF-3 Rabbit mAb (4302, Cell Signaling Technology), Rabbit Anti-NFKB p65 antibody (0465R, Bioss), TBK Rabbit mAb (38066, Cell Signaling Technology), Phospho-TMEM173/STING Antibody (AF7416, Affinity), Phospho-IRF-3 Rabbit mAb (29047, Cell Signaling Technology), Phospho–NF–κB p65 Rabbit mAb (3033, Cell Signaling Technology), Phospho-TBK1 Rabbit mAb (5483, Cell Signaling Technology), LONP1 Antibody (TD12119, Abmart), OGG1 Polyclonal Antibody (PA1-16505, Invitrogen), and flap FEN1 Polyclonal Antibody (PA5-27518, Invitrogen).

Techniques: Expressing, Fluorescence, Microscopy

Journal: Neurotherapeutics

Article Title: Oxidized mitochondrial DNA activates the cGAS-STING pathway in the neuronal intrinsic immune system after brain ischemia-reperfusion injury

doi: 10.1016/j.neurot.2024.e00368

Figure Lengend Snippet: Expression and phosphorylation of cGAS-STING proteins in HT22 ​cells after OGD/R A-D. WB analysis of the expression levels of cGAS-STING key proteins (cGAS, STING, TBK1, IRF3, P65), phosphorylated proteins (pSTING, pTBK1, pIRF3, pP65), and downstream cytokines (IL6, IFN, TNF) in NC and HT22 ​cells after OGD/R at 4, 8, 12, and 24 ​h. A representative image is shown. The statistical data are the ratios of the gray values of each protein band to the GAPDH band (n ​= ​3). E. Expression of pSTING in NC and HT22 ​cells after OGD/R at 24 ​h. Representative images observed by fluorescence microscopy are shown. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001.

Article Snippet: The antibodies used were as follows: anti-GAPDH antibody (ab9485, Abcam), STING Antibody (2922D, Novus Biologicals), cGAS (31659, Cell Signaling Technology), IRF-3 Rabbit mAb (4302, Cell Signaling Technology), Rabbit Anti-NFKB p65 antibody (0465R, Bioss), TBK Rabbit mAb (38066, Cell Signaling Technology), Phospho-TMEM173/STING Antibody (AF7416, Affinity), Phospho-IRF-3 Rabbit mAb (29047, Cell Signaling Technology), Phospho–NF–κB p65 Rabbit mAb (3033, Cell Signaling Technology), Phospho-TBK1 Rabbit mAb (5483, Cell Signaling Technology), LONP1 Antibody (TD12119, Abmart), OGG1 Polyclonal Antibody (PA1-16505, Invitrogen), and flap FEN1 Polyclonal Antibody (PA5-27518, Invitrogen).

Techniques: Expressing, Fluorescence, Microscopy

Journal: Neurotherapeutics

Article Title: Oxidized mitochondrial DNA activates the cGAS-STING pathway in the neuronal intrinsic immune system after brain ischemia-reperfusion injury

doi: 10.1016/j.neurot.2024.e00368

Figure Lengend Snippet: Inhibition of OGD/R-induced activation of cGAS-STING pathway and ox-mtDNA-Related Proteins in HT22 ​cells by mitochondrial DNA depletion A. PCR validation of mitochondrial DNA levels in mtDNA-depleted HT22 ​cell model induced by EtBr treatment. Data are presented as the mean ​± ​SD, n ​= ​3. B-D. WB detection of the expression levels of key cGAS pathway proteins (cGAS, STING, TBK1, IRF3, and P65) and phosphorylated proteins (pSTING, pTBK1, pIRF3, pP65) in HT22 ​cells in the blank control group, EtBr treatment group, OGD/R 24 ​h group, and EtBr treatment ​+ ​OGD/R 24 ​h group. Data are presented as mean ​± ​SD, n ​= ​3. E, F. WB detection of LONP1, OGG1, and FEN1 expression levels in HT22 ​cells from the control, EtBr, OGD/R, and EtBr treatment ​+ ​OGD/R groups. B and C Data are presented as mean ​± ​SD, n ​= ​3. G. Immunofluorescence microscopy observation of changes in mitochondrial membrane potential labeled by the JC-1 kit in HT22 ​cells of the control, OGD/R, and CsA treatment ​+ ​OGD/R groups. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001.

Article Snippet: The antibodies used were as follows: anti-GAPDH antibody (ab9485, Abcam), STING Antibody (2922D, Novus Biologicals), cGAS (31659, Cell Signaling Technology), IRF-3 Rabbit mAb (4302, Cell Signaling Technology), Rabbit Anti-NFKB p65 antibody (0465R, Bioss), TBK Rabbit mAb (38066, Cell Signaling Technology), Phospho-TMEM173/STING Antibody (AF7416, Affinity), Phospho-IRF-3 Rabbit mAb (29047, Cell Signaling Technology), Phospho–NF–κB p65 Rabbit mAb (3033, Cell Signaling Technology), Phospho-TBK1 Rabbit mAb (5483, Cell Signaling Technology), LONP1 Antibody (TD12119, Abmart), OGG1 Polyclonal Antibody (PA1-16505, Invitrogen), and flap FEN1 Polyclonal Antibody (PA5-27518, Invitrogen).

Techniques: Inhibition, Activation Assay, Expressing, Control, Immunofluorescence, Microscopy, Membrane, Labeling

Journal: Neurotherapeutics

Article Title: Oxidized mitochondrial DNA activates the cGAS-STING pathway in the neuronal intrinsic immune system after brain ischemia-reperfusion injury

doi: 10.1016/j.neurot.2024.e00368

Figure Lengend Snippet: CsA inhibits the release of ox-mtDNA and the activation of the cGAS-STING pathway after cerebral ischemia-reperfusion, reduces infarct size, and improves neurological function A. Immunofluorescent observation of the colocalization of 8-OHdG with neurons, microglia, and astrocytes in the rat brain at 24 ​h after tMCAO with or without CsA treatment. B. Immunofluorescent observation of the colocalization of pSTING with neurons, microglia, and astrocytes in the rat brain at 24 ​h after tMCAO with or without CsA treatment. C,D. WB was performed to detect the expression and phosphorylation levels of TBK1, cGAS, STING, and IRF3 molecules in the sham group, the tMCAO group without drug treatment at 24 ​h after ischemia, and the tMCAO group with CSA treatment at 24 ​h after ischemia. The figure shows the representative images. The data analysis is presented as mean ​± ​SD, n ​= ​3. E,F. Open-field test comparison of the total walking distance in 5 ​min by rats in the sham, tMCAO, and CsA-treated tMCAO groups (cm). G,I. TTC staining shows changes in the brain infarct volume of rats in the tMCAO and CsA-treated tMCAO groups. H,J. MRI measurement of changes in brain infarct volume in rats in the tMCAO and CsA-treated tMCAO groups. C-E, data are presented as mean ​± ​SD, n ​= ​3. ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001.

Article Snippet: The antibodies used were as follows: anti-GAPDH antibody (ab9485, Abcam), STING Antibody (2922D, Novus Biologicals), cGAS (31659, Cell Signaling Technology), IRF-3 Rabbit mAb (4302, Cell Signaling Technology), Rabbit Anti-NFKB p65 antibody (0465R, Bioss), TBK Rabbit mAb (38066, Cell Signaling Technology), Phospho-TMEM173/STING Antibody (AF7416, Affinity), Phospho-IRF-3 Rabbit mAb (29047, Cell Signaling Technology), Phospho–NF–κB p65 Rabbit mAb (3033, Cell Signaling Technology), Phospho-TBK1 Rabbit mAb (5483, Cell Signaling Technology), LONP1 Antibody (TD12119, Abmart), OGG1 Polyclonal Antibody (PA1-16505, Invitrogen), and flap FEN1 Polyclonal Antibody (PA5-27518, Invitrogen).

Techniques: Activation Assay, Expressing, Comparison, Staining