Journal: Journal of Cellular and Molecular Medicine

Article Title: Stress‐induced phosphoprotein 1 restrains spinal cord ischaemia‐reperfusion injury by modulating NF‐κB signalling

doi: 10.1111/jcmm.17030

Figure Lengend Snippet: STIP1 restrains OGD‐induced inflammation in microglial cells. A, BV2 cell treatments; B, Western blotting determines STIP1 expression in BV2 cells after OGD treatment, C, Immunoprecipitation detects the binding between STIP1 and HSPA8 in BV2 cells; D, Western blotting determines STIP1 expression after STIP1 overexpression; E, Immunoprecipitation detects the interaction between HSPA8 and IκBβ in BV2 cells; F, Western blotting detects IκBβ expression in BV2 cells; G, Western blotting detects cytoplasmic and nuclear NF‐κB p65 levels in BV2 cells; H, Immunofluorescent staining detects the location of NF‐κB p65. Scale bar =50 μm; I, Immunofluorescent staining detects the expression of the microglia marker Iba‐1. Scale bar =50 μm; J, ELISA detects the secretion of inflammatory factors TNF‐α and IL‐6 in the cell culture supernatant; K, Western blotting determines TNF‐α and IL‐6 expression in BV2 cells after OGD treatment and STIP1 overexpression. NF‐κB, nuclear factor kappa B; OGD, oxygen and glucose deprivation; SCII, spinal cord ischaemia‐reperfusion injury; STIP1, stress‐induced phosphoprotein 1; TNF, tumour necrosis factor. ** p < 0.01, *** p < 0.001 versus the Sham group, # p < 0.05, ## p < 0.01, ### p < 0.001 versus the SCII + LV group

Article Snippet: The affinity agarose beads were pre‐coated with mouse anti‐human STIP1 monoclonal antibody (lot number: sc‐393475; Santa Cruz Biotechnology) and moue anti‐human HSPA8 (lot number: sc‐7298; Santa Cruz Biotechnology) or IgG antibody.

Techniques: Western Blot, Expressing, Immunoprecipitation, Binding Assay, Over Expression, Staining, Marker, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: Pharmacological inhibition of two pore domain potassium channels blocks NLRP3 inflammasome activation and IL‐1β processing. (ai–iii) IL‐1β ELISA of the supernatant of pBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) or K + channel inhibitors TEA (50 mM), TRAM‐34 (10 μM), TPA (50 μM), ML‐133 (20 μM), quinine (100 μM), Guangitoxin‐1E (25 nM), PAP‐1 (2 μM), or Dofetilide (1 μM) for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 4), silica (300 μg ml −1 , 4 h) ( n = 5) or imiquimod (75 μM, 2 h) ( n = 3). (Aiv) IL‐1β ELISA of the supernatant of pBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) or TEA (50 mM), TPA (50 μM), quinine (100 μM), for 15 min before stimulation with nigericin (10 μM, 1 h) ( n = 4). (b) Caspase‐1, IL‐ β and gasdermin D western blot of total cell lysates (cell lysate + supernatant) from LPS‐primed (1 μg ml −1 , 4 h) pBMDMs pretreated with vehicle control, TEA (50 mM), TPA (50 μM) or MCC950 (10 μM) for 15 min then stimulated with ATP (5 mM, 1 h). (c) IL‐1β ELISA of the supernatant of pBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) or TPA (3–300 μM) before stimulation with ATP (5 mM, 1 h) ( n = 5) or silica (300 μg ml −1 , 4 h) ( n = 3). (d) Caspase‐1 Glo assay to measure caspase‐1 activity of LPS‐primed (1 μg ml −1 , 4 h) pBMDMs pretreated with vehicle control, TEA (50 mM), TPA (50 μM) or MCC950 (10 μM) for 15 min then stimulated with ATP (5 mM, 1 h). **** p < .0001, *** p < .001, ** p < .01, * p < .05 determined by one‐way ANOVA with Dunnett's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Inhibition, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Glo Assay, Activity Assay

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: Pharmacological inhibition of two pore potassium channels blocks priming of the NLRP3 inflammasome. (a) IL‐6 (i) and TNF (ii) ELISA and (b) LDH release assay of the supernatant of iBMDMs pretreated with Bay11(10 μM) or K + channel inhibitors TEA (50 mM), TRAM‐34 (10 μM), TPA (50 μM), ML‐133 (20 μM), quinine (100 μM), Guangitoxin‐1E (25 nM), PAP‐1 (2 μM) or Dofetilide (1 μM) for 15 min before priming with LPS (1 μg ml −1 , 4 h) ( n = 4). (c) NLRP3 and IL‐1β western blot of the supernatant and total cell lysates respectively of iBMDMs pretreated with TEA (50 mM), TPA (50 μM) or Bay11 (10 μM) for 15 min before priming with LPS (1 μg ml −1 , 4 h). **** p < .0001, ** p < .01 determined by one‐way ANOVA with Dunnett's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Western Blot

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: Potassium efflux is required for NLRP3 inflammasome activation but not ASC speck formation in response to ATP. (a, i) ASC speck formation measured in real time and (a, ii) ASC speck formation after 165 min of ATP stimulation from ASC‐mCherry iBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment vehicle control, TPA (50 μM) or MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM) ( n = 6). (a, iii) ASC speck formation after 165 min from ASC‐mCherry iBMDMS primed with LPS (1 μg ml −1 , 4 h) followed by treatment with vehicle control, TPA (50 μM) or MCC950 (10 μM) in the absence of ATP ( n = 6). (b, i) IL‐1β ELISA of the supernatant of iBMDMs primed with LPS (1 μg ml −1 , 4 h) followed by incubation in a control (145 mM NaCl/ 5 mM KCl), high K + and normal Cl − (150 mM KCl), high K + and Cl − free (150 mM KGluconate) or control and MCC950 (10 μM) solution for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 6). (b, ii) ASC speck formation measured in real time and (b, iii) ASC speck formation after 165 min of ATP stimulation from iBMDMs stably expressing ASC‐mCherry (ASC‐mCherry iBMDMs) primed with LPS (1 μg ml −1 , 4 h) followed by incubation in a control (145 mM NaCl/ 5 mM KCl), high K + and normal Cl − (150 mM KCl), high K + and Cl − free (150 mM KGluconate) or control and MCC950 (10 μM) solution for 15 min before stimulation with ATP (5 mM) ( n = 4). (b, iv) representative images of ASC‐mCherry iBMDMs after 165 min ATP stimulation (scale bar, 50 μm, arrows denote ASC specks). ASC speck experiments were performed in the presence of ac‐YVAD‐CMK (50 μM) to prevent pyroptosis and loss of ASC specks. **** p < .0001, * p < .05 determined by one‐way ANOVA with Dunnett's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Stable Transfection, Expressing

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: Inhibition of THIK‐1 blocks NLRP3 activation in mixed glia and isolated microglia. (a) IL‐1β ELISA of the supernatant of primary mouse mixed glia primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with vehicle control, TPA (50 μM) or MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 5), silica (300 μg ml −1 ,4 h) ( n = 3), imiquimod (75 μM, 2 h) ( n = 4) or nigericin (10 μM, 1 h) ( n = 3). (b) IL‐1β ELISA of the supernatant of mouse primary microglia primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with vehicle control, TPA (50 μM) or MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM, 1 h) or silica (300 μg ml −1 ,4 h) ( n = 3). **** p < .0001, *** p < .001, ** p < .01, * p < .05 determined by one‐way ANOVA with Dunnett's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Inhibition, Activation Assay, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: THIK‐1 specifically regulates ATP‐induced NLRP3 activation in bone‐marrow‐derived macrophages. (a) IL‐1β ELISA of the supernatant of primary wild‐type (WT) and Kcnk13 knockout (KO) BMDMs primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 7), silica (300 μg ml −1 ,4 h) ( n = 7), imiquimod (75 μM, 2 h) ( n = 4) or nigericin (10 μM, 1 h) ( n = 8). (b) Caspase‐1, IL‐1β and gasdermin D western blot of total cell lysates (cell lysate + supernatant) from LPS‐primed WT and Kcnk13 KO pBMDMs pretreated with vehicle control or MCC950 (10 μM) for 15 min before stimulated with ATP (5 mM, 1 h). (c and d) TNF and IL‐6 ELISA and NLRP3 and IL‐1β western blot of the supernatant and total cell lysates respectively of primary wild‐type (WT) and Kcnk13 knockout (KO) BMDMs pretreated with Bay11(10 μM) for 15 min before priming with LPS (1 μg ml −1 , 4 h) ( n = 7). **** p < .0001, *** p < .001, ** p < .01 determined by two‐way ANOVA with Bonferroni's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Activation Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Knock-Out, Western Blot

Journal: Glia

Article Title: The two pore potassium channel THIK ‐1 regulates NLRP3 inflammasome activation

doi: 10.1002/glia.24174

Figure Lengend Snippet: THIK‐1 regulates NLRP3 activation in primary adult microglia. (a) IL‐1β ELISA of the supernatant of primary wild‐type (WT) and Kcnk13 knockout (KO) adult microglia primed with LPS (1 μg ml −1 , 4 h) followed by pretreatment with MCC950 (10 μM) for 15 min before stimulation with ATP (5 mM, 1 h) ( n = 4), silica (300 μg ml −1 ,4 h) ( n = 3), imiquimod (75 μM, 2 h) ( n = 3) or nigericin (10 μM, 1 h) ( n = 4). (b) TNF ELISA of the supernatant of primary wild‐type (WT) and Kcnk13 knockout (KO) primary adult microglia pretreated with Bay11(10 μM) for 15 min before priming with LPS (1 μg ml −1 , 4 h) ( n = 5). **** p < .0001, *** p < .001, ** p < .01, * p < .05 determined by two‐way ANOVA with Bonferroni's post hoc analysis. Values shown are the mean ± SEM

Article Snippet: Specific antibodies were used targeting: mouse IL‐1β (AF‐401, R&D), caspase‐1 p10 (EPR16883, Abcam), gasdermin D (ab209845, Abcam), NLRP3 (G‐20B‐0014‐C100, Adipogen), and β‐actin (Sigma).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: bioRxiv

Article Title: A cross–nearest neighbor/Monte Carlo algorithm for single-molecule localization microscopy defines interactions between p53, Mdm2, and MEG3

doi: 10.1101/857912

Figure Lengend Snippet: MEG3 was induced by treatment of U2OS-MEG3 cells for 20 h with 1 μg/mL doxycycline and/or 10 μM nutlin-3a. Cells were stained for Mdm2 with a secondary antibody conjugated to ATTO 488 (green) and for p53 with a secondary antibody conjugated to Alexa Fluor 647 (magenta). From left to right: Merged image; RNA channel; p53 channel; dSTORM localization map; 30x inset of dSTORM localizations in the black box, with shaded circles indicating “molecules”. Scale bars are 10 μm, or 200 nm (right column).

Article Snippet: Primary antibodies (rabbit anti-p53 [7F5] (Cell Signaling 2527S, Lot 8), mouse anti-Mdm2 [2A10] (Abcam ab16895, Lot GR324625-5)) were applied at 1:1000 and 1:200 dilutions, respectively, in blocking buffer and incubated overnight at 4 °C.

Techniques: Staining

Journal: bioRxiv

Article Title: A cross–nearest neighbor/Monte Carlo algorithm for single-molecule localization microscopy defines interactions between p53, Mdm2, and MEG3

doi: 10.1101/857912

Figure Lengend Snippet: MEG3 was induced by treatment of U2OS-MEG3 cells for 20 h with or without 1 μg/mL doxycycline and/or 10 μM nutlin-3a. Cells were then fixed and stained for 2-color dSTORM of p53 and Mdm2. For each condition, single molecule localizations were collected from 10 randomly chosen cells in 3 separate experiments. (A) Fraction of pairs associated, as defined by a probability of chance association < 0.1 (i.e., correction for local density) and distance < 200 nm (upper limit for binding distance, accounting for error). (B) Median distance between pairs for each cell (nm). Boxes indicate median +/- upper and lower quartile; whiskers indicate the range excluding outliers. Data points are colored by replicate. Means for each replicate are indicated by same-colored squares. * indicates p < 0.05 by ANOVA.

Article Snippet: Primary antibodies (rabbit anti-p53 [7F5] (Cell Signaling 2527S, Lot 8), mouse anti-Mdm2 [2A10] (Abcam ab16895, Lot GR324625-5)) were applied at 1:1000 and 1:200 dilutions, respectively, in blocking buffer and incubated overnight at 4 °C.

Techniques: Staining, Binding Assay