Journal: CNS Neuroscience & Therapeutics

Article Title: Alpha‐lipoic acid downregulates TRPV1 receptor via NF‐κB and attenuates neuropathic pain in rats with diabetes

doi: 10.1111/cns.13303

Figure Lengend Snippet: ALA inhibited TRPV1 expression via suppressing NF‐κB. A, Western blots for NF‐κB in DRGs innervating the hindpaw from control and STZ‐induced diabetic rats. Bar graph involved mean density relative of β‐actin and NF‐κB from control and STZ‐induced diabetic rats. STZ injection significantly enhanced expression of NF‐κB (N = 4 for each group; * P < .05, compared with CNT, two‐sample t test) in L4‐L6 DRGs. B, Western blots for TRPV1 in L4‐L6 DRGs from diabetic rats treated with p65 siRNA lentivirus and NC siRNA lentivirus, respectively. Graph showed mean density relative to GAPDH for TRPV1. The lentiviruses were intrathecally injected into rats. The expression of TRPV1 significantly reduced after p65 siRNA lentiviruses treatment compared with NC siRNA group (N = 4 for each group, * P < .05, compared with NC siR, two‐sample t test). C, Western blots for NF‐κB in L4‐L6 DRGs from diabetic rats treated with NS and ALA, respectively. Graph showed mean density relative to GAPDH for NF‐κB. ALA treatment strongly reduced the expression of NF‐κB (N = 4 for NS group, N = 3 for ALA group, ** P < .01, compared with NS, two‐sample t test)

Article Snippet: Anti‐TRPV1 (#ACC‐030, Alomone, 1:200), anti‐p65 (#6956, Cell Signaling Technology, 1:1000), and corresponding horseradish peroxidase‐conjugated secondary anti‐rabbit and anti‐mouse antibodies at dilutions of 1:2000 and 1:2000 were used to probe the proteins, respectively.

Techniques: Expressing, Western Blot, Injection

Journal: bioRxiv

Article Title: Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies

doi: 10.1101/2022.04.08.487599

Figure Lengend Snippet: Immunoblot of A549 human airway epithelial cells 2 hrs post-challenge with either IL-1β (10 ng/ml), TIGR4 (MOI 20) or 6B ST90 (MOI 20) (+/- IL-1β; 10 ng/ml). Whole cell lysates probed for p65, phosphorylated p65 at Serine 276, phosphorylated p65 at Serine 536 or Actin. A) Representative image of immunoblot. Actin normalized B) total p65, C) phosphorylated p65 at Serine 276 and D) phosphorylated p65 at Serine 536 (n=11 biological replicates). Dot blot with mean (red line). One-way ANOVA with repeated measures with mixed-effects analysis comparing all means with Tukey’s multiple comparison post-hoc test. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Full blots provided in Supplementary Information 1.

Article Snippet: Primary antibody to p65 (CST ref #6956 or CST ref# 8242), COMMD2 (Sigma ref# HPA044190-25UL; only works for immunofluorescence), or p62 (SQSTM1; abcam ref# ab109012) were diluted at 1:1,000 in 5% BSA 0.5% Tween20 and incubated overnight at 4°C.

Techniques: Western Blot, Dot Blot, Comparison

Journal: bioRxiv

Article Title: Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies

doi: 10.1101/2022.04.08.487599

Figure Lengend Snippet: Immunofluorescence confocal microscopy of paraformaldehyde fixed A549 cells 2 h post-challenge with either IL-1β (10 ng/ml) or TIGR4 (+/- IL-1β 10 ng/ml; MOI 20) stained for p65 (cyan) and nucleus (DAPI; gray). Scale bar = 100µm. B) Quantification of nuclear p65 normalized to the nuclei (n = 3 biological replicates with total nuclei counts for Uninfected n= 636, IL-1β n= 801, TIGR4 n=633, TIGR4 + IL-1β n=516). Tukey box and whisker plot with defined box boundaries being the upper and lower interquartile range (IQR), ‘whiskers’ (fences) being ± 1.5 times IQR and the median depicted by the middle solid line. Dots represent outliers. Two-way ANOVA comparing all means with Tukey’s multiple comparison post-hoc test. ****P ≤ 0.0001. C) RT-qPCR IL-6, IL-8, PTGS2 & CSF2 transcript profiles of A549 cells over a 2 hr time course challenged with either IL-1β or TIGR4 (+/- IL-1β 10 ng/ml; MOI 20). Graphed as the relative expression of each indicated transcript to matched uninfected/unstimulated control per time point (n = 3 biological replicates; 2 technicals per replicate). Displayed as a dot plot with each data point and a bar representing the mean. Chromatin was obtained from A549 cells either untreated (light gray), IL- 1β treated (10 ng/ml; dark gray) or 2 hrs post-challenge with TIGR4 (light blue; MOI 20). D) Schematic representation of PTGS2 promoter with ChIP-qPCR primer locations (P1 & P2) and NF-κB sites . E & F) ChIP-qPCR represented as % recovery against input of p65 at indicated NF-κB sites. Tukey box and whisker plot with defined box boundaries being the upper and lower interquartile range (IQR), ‘whiskers’ (fences) being ± 1.5 times IQR and the median depicted by the middle solid line (n=3 biological replicates with 2 technicals per replicate). One-way ANOVA comparing all means with Tukey’s multiple comparison post-hoc test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Article Snippet: Primary antibody to p65 (CST ref #6956 or CST ref# 8242), COMMD2 (Sigma ref# HPA044190-25UL; only works for immunofluorescence), or p62 (SQSTM1; abcam ref# ab109012) were diluted at 1:1,000 in 5% BSA 0.5% Tween20 and incubated overnight at 4°C.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Whisker Assay, Comparison, Quantitative RT-PCR, Expressing, Control, ChIP-qPCR

Journal: bioRxiv

Article Title: Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies

doi: 10.1101/2022.04.08.487599

Figure Lengend Snippet: Mass-spectrometry interactome (n=4 biological replicates per condition) of immunoprecipitated GFP-p65 from a stable A549 GFP-p65 cell line (1×10 7 cells total) 2 hrs post challenge with either 6B ST90 (MOI 20) or TIGR4 (MOI 20). A) Volcano plot of identified interacting partners with known NF-kB p65 partners in blue and general significant targets in yellow. Lines represent FDR and fold-change cutoffs with targets of interested denoted. B) Representative immunoblot of A549 whole cell lysates 2 hrs post-challenge with either IL-1β (10 ng/ml), TIGR4 (MOI 20) or 6B ST90 (MOI 20) (+/- IL-1β; 10 ng/ml) probed for RelB, HDAC6, or Actin. C & D) Quantification of RelB or HDAC6 levels normalized to Actin (n=4 biological replicates). Displayed as a dot blot with mean (red line). One-way ANOVA with repeated measures with mixed-effects analysis comparing all means. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Whole cell lysates from A549 cells 2 hrs post-challenge with either IL-1β (10 ng/ml) or TIGR4 (MOI 20; +/- IL-1β; 10 ng/ml) from E) Bafilomycin A1 (400nM) or G) SAR405 (500nM) pretreated cells (both 3 hrs) and immunoblots probed for p65 or actin (n=3 biological replicates). Quantified levels of total p65 normalized to actin from F) Bafilomycin A1 (400nM) or H) SAR405 (500nM). Dot blot with mean (red line). One-way ANOVA with repeated measures with mixed-effects analysis comparing all means. ns = not significant. Full blots provided in Supplementary Information 2

Article Snippet: Primary antibody to p65 (CST ref #6956 or CST ref# 8242), COMMD2 (Sigma ref# HPA044190-25UL; only works for immunofluorescence), or p62 (SQSTM1; abcam ref# ab109012) were diluted at 1:1,000 in 5% BSA 0.5% Tween20 and incubated overnight at 4°C.

Techniques: Mass Spectrometry, Immunoprecipitation, Western Blot, Dot Blot

Journal: bioRxiv

Article Title: Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies

doi: 10.1101/2022.04.08.487599

Figure Lengend Snippet: Immunoprecipitates using GFP-Trap agarose beads were collected from a stable A549 GFP-COMMD2 cell line 2 hrs post-challenge with either IL-1β (10 ng/ml) or TIGR4 (MOI 20; +/- IL-1β; 10 ng/ml). A) A single representative immunoblot from 3 biological replicates of GFP-COMMD2 immunoprecipitation lysates (input & IP) probed for p65 or GFP.. B) Representative immunoblot from 3 biological replicates of GFP-COMMD2 immunoprecipitation lysates (input & IP) probed for p62 or GFP.. Full blots provided in Supplementary Information 3. C) Immunofluorescence confocal microscopy of stable A549 GFP-COMMD2 cells 2 h post-challenge with either IL-1β (10 ng/ml) or TIGR4 (+/- IL-1β 10 ng/ml; MOI 20) stained for p62 (magenta) against GFP-COMMD2 (gray). Scale bar = 100 µm. Red inset images of single cell highlighting (white arrow) perinuclear punta. Inset scale bar 10 µm. Quantification of total cellular p62 D) normalized to cell area (GFP-COMMD2 signal) and nuclear p62 E) normalized to the area of the nucleus (DAPI signal; n = 3 biological replicates with total cell counts for Uninfected n= 548, IL-1β n= 670, TIGR4 n=356, TIGR4 + IL-1β n=271). Quantification of total cellular COMMD2 F) normalized to cell area (GFP-COMMD2 signal) and nuclear COMMD2 G) normalized to the area of the nucleus (DAP I signal; n = 3 biological replicates with total cell counts for Uninfected n= 864, IL-1β n= 1068, TIGR4 n=596, TIGR4 + IL-1β n=534). Tukey box and whisker plot with defined box boundaries being the upper and lower interquartile range (IQR), ‘whiskers’ (fences) being ± 1.5 times IQR and the median depicted by the middle solid line. Dots represent outliers. Two-way ANOVA comparing all means with Tukey’s multiple comparison post-hoc test. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. H) Cell fractions from a stable A549 GFP-COMMD2 cell line 2 hrs post-challenge with either IL-1β (10 ng/ml) or TIGR4 (MOI 20; +/- IL-1β; 10 ng/ml). Representative immunoblot probed for GFP (COMMD2) enrichment across cellular compartments. GapDH or histone H4 (H4) used to determine fraction purity. Full blots provided in Supplementary Information 3. I) Percent nuclear COMMD2 levels normalized to input (n=3 biological replicates). Graphed as mean ± STD with dots representing individual biological replicates. One-way ANOVA with repeated measures with mixed-effects analysis comparing all means with Tukey’s multiple comparison post-hoc test. ****P ≤ 0.0001.

Article Snippet: Primary antibody to p65 (CST ref #6956 or CST ref# 8242), COMMD2 (Sigma ref# HPA044190-25UL; only works for immunofluorescence), or p62 (SQSTM1; abcam ref# ab109012) were diluted at 1:1,000 in 5% BSA 0.5% Tween20 and incubated overnight at 4°C.

Techniques: Western Blot, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Staining, Whisker Assay, Comparison

Journal: bioRxiv

Article Title: Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies

doi: 10.1101/2022.04.08.487599

Figure Lengend Snippet: Immunofluorescence confocal microscopy of stable A549 GFP-COMMD2 pretreated for 3 hrs with Leptomycin B (10 nM) prior to 2 hr challenge with either IL-1β (10 ng/ml) or TIGR4 (MOI 20). Paraformaldehyde fixed cells stained for p62 (magenta), or B) p65 (cyan) against GFP-COMMD2 (gray) and nuclei (DAPI; blue). Scale bar = 100 µm or 10 µm for uninfected and untreated single cell inserts. Nuclear levels of C) GFP-COMMD2 or D) p62 normalized to the segmented nuclei using DAPI signal (n = 3 biological replicates with total nuclei counts for Uninfected (-) n=1664, Uninfected n= 742, IL-1β (-) n=1068, IL-1β n= 920, TIGR4 (-) n=585, TIGR4 n=798). E) Nuclear levels of p65 normalized to the segmented nuclei using DAPI signal (n = 3 biological replicates with total nuclei counts for Uninfected (-) n=489, Uninfected n= 1492, IL-1β (-) n=576 IL-1β n= 1658, TIGR4 (-) n=680, TIGR4 n=1514). Tukey box and whisker plot with defined box boundaries being the upper and lower interquartile range (IQR), ‘whiskers’ (fences) being ± 1.5 times IQR and the median depicted by the middle solid line. Dots represent outliers. Two-way ANOVA comparing all means with Tukey’s multiple comparison post-hoc test. ***P ≤ 0.001, ****P ≤ 0.0001.

Article Snippet: Primary antibody to p65 (CST ref #6956 or CST ref# 8242), COMMD2 (Sigma ref# HPA044190-25UL; only works for immunofluorescence), or p62 (SQSTM1; abcam ref# ab109012) were diluted at 1:1,000 in 5% BSA 0.5% Tween20 and incubated overnight at 4°C.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Whisker Assay, Comparison

Journal: bioRxiv

Article Title: Pneumococcus triggers NFkB degradation in COMMD2 aggresome-like bodies

doi: 10.1101/2022.04.08.487599

Figure Lengend Snippet: Representative immunofluorescence confocal microscopy of stable A549 GFP-COMMD2 pretreated for 3 hrs with Leptomycin B (10nM) prior to 2 hr challenge TIGR4 (MOI 20; +/- IL-1β; 10 ng/ml). Paraformaldehyde fixed cells stained for (A) p62 (magenta) against GFP-COMMD2 (gray) and nuclei (DAPI; blue). Scale bar = 10µm B) Nuclear p62 puncta quantification (n = 3 biological replicates with total nuclei counts for Uninfected n=1041, IL-1β n=831, TIGR4 n=1164, TIGR4 + IL-1β n=1269). Graphed as mean ± STD with dots representing individual biological replicates. One-way ANOVA comparing all means with Tukey’s multiple comparison post-hoc test. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. C) Quantified immunofluorescence confocal microscopy of A549 GFP-COMMD2 cells pretreated with Bafilomycin A1 (400nM; 3 hrs) prior to 2 hr challenge with either IL-1β (10 ng/ml) or TIGR4 (MOI 20; +/- IL-1β; 10 ng/ml). Paraformaldehyde fixed and stained for C) p65, D) COMMD2 or E) p62. (n = 3 biological replicates with total cell count for Uninfected p62 & COMMD2 n=1648 & p65 n=1496, IL-1β p62 & COMMD2 n=2103 & p65 n=1703, TIGR4 p62 & COMMD2 n=2033 & p65 n=1597, TIGR4 + IL-1β p62 & COMMD2 n=1724 & p65 n=1759). Tukey box and whisker plot with defined box boundaries being the upper and lower interquartile range (IQR), ‘whiskers’ (fences) being ± 1.5 times IQR and the median depicted by the middle solid line. Dots represent outliers. Two-way ANOVA comparing all means with Tukey’s multiple comparison post-hoc test. ns=not significant, ***P ≤ 0.001,****P ≤ 0.0001.

Article Snippet: Primary antibody to p65 (CST ref #6956 or CST ref# 8242), COMMD2 (Sigma ref# HPA044190-25UL; only works for immunofluorescence), or p62 (SQSTM1; abcam ref# ab109012) were diluted at 1:1,000 in 5% BSA 0.5% Tween20 and incubated overnight at 4°C.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Comparison, Cell Counting, Whisker Assay

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: Beta cell-specific p65 knockout dampens islet inflammation but impairs insulin secretion in response to a glucose challenge. ( a ) Real-time quantitative PCR analysis of Rela mRNA in islets isolated from littermate mice wild-type for p65 (p65fl/fl) or with beta cell-specific knockout of p65 (βp65KO). ( b ) Immunoblot of lysates from islets isolated from p65fl/fl or βp65KO littermate mice and stimulated with recombinant TNF for the times indicated. Proteins (kDa) assessed included components of the canonical NF-κB signalling pathway, phosphorylated and total JNK (p-JNK and T-JNK, respectively) and a β-actin loading control. Representative of three independent experiments. ( c ) Cumulative densitometry (relative units) of immunoblots represented in ( b ), illustrated as heat maps. Data compared against wild-type floxed (fl) 0 h sample in each blot. The flow diagram of major signalling nodes illustrates the position of each signalling event with respect to the transcription factor Rela /p65 (red). ( d ) Insulin-stained pancreatic sections (scale bar: 100 µm) from 8 week old female mice of the indicated genotypes. ( e ) Real-time quantitative PCR analysis of inflammatory mRNAs in islets isolated from littermate p65fl/fl or βp65KO mice and stimulated with TNF for the times indicated. Data are fold change relative to no TNF stimulation. ( f ) Weight and ( g ) fasting blood glucose levels of mice with or without beta cell-specific knockout of p65. F, female; M, male. ( h , i ) Blood glucose levels were monitored following an ( h ) i.p. GTT (2 g/kg glucose) (p65fl/fl, n =17; βp65KO, n =17) or ( i ) i.v. GTT (1 g/kg glucose) (p65fl/fl, n =9; βp65KO, n =9) in female mice. ( j ) Blood insulin levels (pmol/l) were measured following i.v. injection in ( i ) (p65fl/fl, n =9; βp65KO, n =9). ( k ) Beta cell mass and ( l ) in vitro GSIS assay (20 mmol/l) in islets isolated from mice with or without beta cell-specific knockout of p65. ( m ) Blood glucose levels before (day 0) and following minimal mass transplant of islets from mice with or without beta cell-specific knockout of p65 under the kidney capsule of wild-type syngeneic diabetic recipients (βp65KO, n =7; p65fl/fl, n =4). ( n ) Insulin-stained sections of islet graft 30 days post transplant (scale bar: 100 µm). Statistical analysis was performed using Student’s t tests ( a , e–g , k , l ) or AUCs ( h–j , m ). Data are means ± SEM. * p <0.05, ** p <0.01, *** p <0.001. BGL, blood glucose level; TAB, TAK1-binding protein; TAK1, TGF-β-activated kinase 1

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Isolation, Western Blot, Recombinant, Control, Staining, Injection, In Vitro, Binding Assay

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: Islet-derived inflammatory genes harbour p65 binding sites. ( a–c ) Representative plots of p65 ChIP-seq and corresponding accessibility signal enrichment at the ( a ) CXCL10 , ( b ) ICAM1 and ( c ) TNFAIP3 locuses. ChIP-seq and ATAC-seq peaks are shown underneath the respective signal coverage tracks. ( d ) p65 ChIP-seq enrichment, epigenomic annotations and high-confidence pcHi-C interactions from islet samples encompassing the CXCL1 locus. p65 ChIP-seq data were obtained from ENCODE. ATAC-seq data were obtained from Bysani et al . All browser views were generated using the WashU Epigenome Browser. Only the significant pcHi-C loops within the broadcast region are shown. ChIP, chromatin immunoprecipitation

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: Derivative Assay, Binding Assay, ChIP-sequencing, Generated, Chromatin Immunoprecipitation

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: Human islets exhibit a large network of accessible p65 binding sites linked to genes governing metabolism. ( a ) Venn diagram showing the enrichment of islet-accessible p65 footprints in the islet enhancer hubs. p65 footprints were identified from islet ATAC-seq samples ( n =8 healthy donors) available from Bysani et al and intersected with islet enhancer hubs reported by Miguel-Escalada et al . ( b ) Top 30 significantly enriched GO terms or gene sets for p65 footprints identified within the islet enhancer hubs. The footprint regions ( n =1684 within the 821 enhancer hubs) were assigned to the nearest transcription start site to perform GO term ontology and gene set enrichment using the ChIP-Enrich tool

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: Binding Assay

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: SLC2A2 and CAPN9 locuses form part of the islet enhancer hubs linked with p65 regulation. ( a , b ) p65 ChIP-seq enrichment, epigenomic annotations and high-confidence pcHi-C interactions from islet samples at the two candidate loci: ( a ) SLC2A2 and ( b ) CAPN9 . p65 ChIP-seq data were obtained from ENCODE. Islet pcHi-C interactions and super enhancers were obtained from Miguel-Escalada et al . All browser views were generated using the WashU Epigenome Browser . Only the significant pcHi-C loops within the broadcast region are shown. ChIP, chromatin immunoprecipitation. ( c , d ) Real-time PCR analysis of ( c ) Slc2a2 and ( d ) Capn9 in islets isolated from p65fl/fl and βp65KO mice. Statistical analysis was performed using Student’s t tests. Data are means ± SEM. * p <0.05, *** p <0.001

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: ChIP-sequencing, Generated, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: Annotation of p65 binding sites in proximal promoters and enhancers of glycolytic genes, with impacts on gene expression

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Targeted inhibition of RAGE in substantia nigra of rats blocks 6-OHDA–induced dopaminergic denervation

doi: 10.1038/s41598-017-09257-3

Figure Lengend Snippet: FPS-ZM1 blocked the 6-OHDA-induced nuclear translocation of NF-κB-p65. Rats were prepared for immunofluorescence 15 days after 6-OHDA administration. ( a ) Representative co-immunofluorescence images of SN immunostained for NF-κB-p65, RAGE and DAPI ( n = 10 per group). The ipsilateral sides are showed. The microscopy images were taken with 50 μm of magnification. ( b ) Cell details. ( c ) p65+ nucleus quantification/picture with 50 μm magnification area. Values represent mean ± SD from 6 rats per group. One-way analysis of variance and Bonferroni Multiple Comparison post-hoc test were applied to all data. p values are embedded in the figure.

Article Snippet: Anti-NF-κB -p65 (1:200; 6956) and Anti-TH (1:200; 2792 S) were from Cell Signaling Technology ® (MA, USA).

Techniques: Translocation Assay, Immunofluorescence, Microscopy, Comparison

Journal: PLoS ONE

Article Title: NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

doi: 10.1371/journal.pone.0073409

Figure Lengend Snippet: (A) Dose dependent activation of MyD88 by IL1B in AGS cells. Total RNA was extracted from AGS cells treated with increasing concentration of recombinant IL1B (0, 5, 10ng/ml) for 10 min. Real time PCR analysis for MyD88 was performed in those RNA samples. β-actin was taken as the endogenous control. The graph represents the mean of relative quantification measured from three different experiments +/- SD. (B) Analysis of IL1B induced phosphorylation of MAP3K TAK1. AGS cells were treated with 10ng/ml recombinant IL1B protein for 0, 15 and 30 min and then lysed for western blot analysis with p-TAK1, TAK1 and β–actin antibodies. A representative blot is shown. The band intensities were scanned by imageJ and the normalized mean band intensities of three independent experiments with +/-SD values graphically plotted. (C) Dose dependent activation of NFkB p50 and p65 by TAK1. AGS cells were co-transfected in a dose dependent manner with pCMVTAK1 along with its activator pCMVTAB1 and western blot analysis was done for NFkB p50 and p65 respectively. A representative blot is shown. The band intensities were scanned by imageJ and the normalized mean band intensities of three independent experiments with +/-SD values graphically plotted. (D and E) Effect of TAK1/TAB1on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) and either with (D) varying concentrations of pCMVTAK1 along with 0.2 µg of pCMVTAB1 or (E) varying concentrations of pCMVTAB1 along with 0.5 µg of pCMVTAK1.The IL1B (10ng/ml) treatment as control has been included in panel E. Cells were harvested after 48 hr of transfection for luciferase assay. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (F) Knock down of TAK1 in IL1B-treated AGS cells releases gastrin repression. AGS cells were first transfected with either TAK1 siRNA (80 nM) or control siRNA(80 nM). After twenty four hr these cells were transfected with 0.5 µg of pGAS-Luc.Forty six hr post pGAS-Luc transfection, cells were treated with IL1B (10ng/ml) for two hr and subsequently harvested. Control experiments with only pGAS-Luc transfected and IL1B treated pGAS-Luc transfected AGS cells are also shown. (G) A cartoon showing that IL1B induces NFkB via MyD88/TAK1 to regulate gastrin expression. Stars in panel C indicate statistical significance of the observations.

Article Snippet: IL1B treated AGS cells (2.0×10 6 cells) were subjected to western blot analysis using antibodies against TAK1(1:500 dilution, #4505, Cell Signaling, USA), p-TAK1(1:100 dilution, #4536, Cell Signaling, USA), TAB1(1:500 dilution, #3226, Cell Signaling, USA) NFkB p50 antibody (1:500 dilution, #3035, Cell Signaling, USA), NFkB p65 antibody(1:500 dilution, Sc-7151,Santa Cruz Biotechnology Inc, Santa Cruz, USA and #6956, Cell Signaling, USA), NFkB phosphop65S536 antibody(1:100 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, USA), or Beta -actin (Sigma Aldrich, St. Louis, USA).

Techniques: Activation Assay, Concentration Assay, Recombinant, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Activity Assay, Luciferase, Expressing

Journal: PLoS ONE

Article Title: NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

doi: 10.1371/journal.pone.0073409

Figure Lengend Snippet: (A) Effect of NFkB p65 on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) along with 0.2µg of NFkB p65 (pWTp65NFkB). Forty six hr after transfection, cells were either treated or left untreated with 10ng/ml of IL1B for two hr and then harvested for luciferase assay. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (B and C) Effect of NFkB p65 mutants on gastrin promoter activity. AGS cells were co-transfected with gastrin luciferase (pGAS-Luc) along with (B) either NFkB p65 (pWTp65NFkB) or the lysine NFkB p65 mutants (pK310R p65 NFkB and pK221R p65 NFkB) and (C) with either NFkB p65 (pWTp65NFkB) or with the serine NFkB p65 mutants (pS536A p65 NFkB, pS529A p65 NFkB and pS276A p65 NFkB). Forty six hr after transfection, cells were either treated or left untreated with 10 ng/ml of IL1B for two hr and then harvested for luciferase assay. 0.2µg of wild type or mutant NFkB p65 were used for transfection. The normalized mean Relative Luciferase Unit/µg protein +/- SD of three different experiments was plotted. (D) Analysis of IL1B induced phosphorylation of NFkB p65 at ser536 residue. AGS cells were treated with varying concentrations of recombinant IL1B protein for two hr and then lysed and immunoblotted with anti p-p65NFkB (S536) and anti α-tubulin antibodies. A representative blot is shown. The star in panel C indicates that the repression of transcription of pGAS-Luc with pWTp65NFkB is significantly alleviated when p65S536ANFkB was used both in presence and absence of IL1B.NS indicates that the differences between the repressive activity of pWTp65NFkB and that of the mutant clones on gastrin promoter are not significant.

Article Snippet: IL1B treated AGS cells (2.0×10 6 cells) were subjected to western blot analysis using antibodies against TAK1(1:500 dilution, #4505, Cell Signaling, USA), p-TAK1(1:100 dilution, #4536, Cell Signaling, USA), TAB1(1:500 dilution, #3226, Cell Signaling, USA) NFkB p50 antibody (1:500 dilution, #3035, Cell Signaling, USA), NFkB p65 antibody(1:500 dilution, Sc-7151,Santa Cruz Biotechnology Inc, Santa Cruz, USA and #6956, Cell Signaling, USA), NFkB phosphop65S536 antibody(1:100 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, USA), or Beta -actin (Sigma Aldrich, St. Louis, USA).

Techniques: Activity Assay, Transfection, Luciferase, Mutagenesis, Recombinant, Clone Assay

Journal: PLoS ONE

Article Title: NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

doi: 10.1371/journal.pone.0073409

Figure Lengend Snippet: (A) Effect of HDAC1 on gastrin promoter activity. AGS cells were co-transfected with pGAS-Luc construct and increasing amount of HDAC1 expression vector. Cells were harvested 48 hr after transfection and luciferase activity was measured. The mean luciferase activity of three different experiments +/- SD is graphically plotted. (B and C) Combined effect of NFkB p65 and HDAC1 on gastrin promoter activity. (B) AGS cells were co-transfected with pGAS-Luc construct along with pHDAC1 or pWTp65NFkB or both. Transfected cells were then incubated with either NBD (150µM) for 24 hr or TSA (100nM) for 48 hr or both or left untreated. Cells were harvested after 48 hr of transfection. The mean luciferase activity of three different experiments is represented as fold repression with respect to gastrin promoter alone and graphically plotted. (C) AGS cells were co-transfected with pGAS-Luc in combination with either pWTp65NFkB or pS536A p65 NFkB with and without pHDAC1 expression vector. Cells were harvested after 48 hr of transfection. The mean luciferase activity of three different experiments is represented as Relative Luciferase Unit/µg protein with respect to gastrin promoter alone and graphically plotted. (D) Co-immunoprecipitation assay confirms the association of NFkB p65 with either p300 or HDAC1 in IL1B stimulus specific manner. AGS cells were either treated with 10ng/ml of recombinant IL1B or left untreated for two hr. Lysates were prepared for immunoprecipitation with either p300 or HDAC1. Immunoprecipitated protein complexes were then immunoblotted with NFkB p65 antibody. (E) The band intensities of co-immunoprecipitation experiments were scanned by imageJ and their mean normalized density of three independent experiments with +/-SD values graphically plotted.

Article Snippet: IL1B treated AGS cells (2.0×10 6 cells) were subjected to western blot analysis using antibodies against TAK1(1:500 dilution, #4505, Cell Signaling, USA), p-TAK1(1:100 dilution, #4536, Cell Signaling, USA), TAB1(1:500 dilution, #3226, Cell Signaling, USA) NFkB p50 antibody (1:500 dilution, #3035, Cell Signaling, USA), NFkB p65 antibody(1:500 dilution, Sc-7151,Santa Cruz Biotechnology Inc, Santa Cruz, USA and #6956, Cell Signaling, USA), NFkB phosphop65S536 antibody(1:100 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, USA), or Beta -actin (Sigma Aldrich, St. Louis, USA).

Techniques: Activity Assay, Transfection, Construct, Expressing, Plasmid Preparation, Luciferase, Incubation, Co-Immunoprecipitation Assay, Recombinant, Immunoprecipitation

Journal: PLoS ONE

Article Title: NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin

doi: 10.1371/journal.pone.0073409

Figure Lengend Snippet: (A) Direct binding of NFkB heterodimers to gastrin promoter. EMSA was performed with nuclear extracts prepared from IL1B treated (10ng/ml for 2hr.) AGS cells. Supershift assay was performed by incubating the nuclear lysates first with either NFkB p50 or p65 antibody for 30 min and then with radiolabelled gastrin EMSA probe. The chase was performed using corresponding unlabelled oligo probes. NE stands for Nuclear Extract. (B) Gastrin promoter occupancy by NFkB heterodimers and other co-factors. ChIP qPCR analysis was done with anti p50, p65, HDAC1 and p300 antibodies in IL1B (10ng/ml) treated and untreated AGS cells. (C) NFkB associates with HDAC1 and NCoR on the gastrin promoter to bring about histone methylation. ChIP qPCR analysis was done with anti NFkB p65, anti-H3K9Me3 and anti-NCoR in IL1B (10ng/ml) treated and untreated AGS cells.

Article Snippet: IL1B treated AGS cells (2.0×10 6 cells) were subjected to western blot analysis using antibodies against TAK1(1:500 dilution, #4505, Cell Signaling, USA), p-TAK1(1:100 dilution, #4536, Cell Signaling, USA), TAB1(1:500 dilution, #3226, Cell Signaling, USA) NFkB p50 antibody (1:500 dilution, #3035, Cell Signaling, USA), NFkB p65 antibody(1:500 dilution, Sc-7151,Santa Cruz Biotechnology Inc, Santa Cruz, USA and #6956, Cell Signaling, USA), NFkB phosphop65S536 antibody(1:100 dilution, Santa Cruz Biotechnology Inc, Santa Cruz, USA), or Beta -actin (Sigma Aldrich, St. Louis, USA).

Techniques: Binding Assay, Methylation

Journal: Journal for immunotherapy of cancer

Article Title: Clofarabine induces tumor cell apoptosis, GSDME-related pyroptosis, and CD8 + T-cell antitumor activity via the non-canonical P53/STING pathway.

doi: 10.1136/jitc-2024-010252

Figure Lengend Snippet: Figure 3 Clofarabine activates the P53-induced non-canonical STING/NF-κB pathway and induces apoptosis, pyroptosis, and immunogenic cell death in melanoma and lung cancer cells. (A) Western blotting revealed the protein expression of P53, p-P53, cGAS, STING, p-IkBα, BAX, Cleaved Caspase-3, Cleaved Caspase-6, and Cleaved PARP in lysates collected from A375 and A549 treated with Clo. (B) ELISA was used to analyze the concentration of cGAMP in the lysate of A375 and A549 treated with Clo for 48 hours. n=2. (C) Western blotting revealed the nuclear protein expression of NF-κB p50 and p65 in lysates collected from A375 and A549 cells treated with Clo. (D) Immunoprecipitation was conducted to detect the interaction between P53 and STING. (E) Western blotting revealed the protein expression of GSDME-FL and GSDME-N in lysates collected from A375 and A549 treated with Clo. (F) The images of A375 treated with Clo for 48 hours show PI uptake. The statistical analysis was displayed. n=3. (G) qRT-PCR measurement of CCL5, CXCL10, HLA-A, HLA-B, HLA-C, and BAX mRNA expression in A375 and A549 treated with Clo for 48 hours. n=3. (H) ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of A375 and A549 treated with Clo for 48 hours. n=2. The p value was obtained by multiple comparisons in an ordinary one-way analysis of variance (B, F, G, H), and the results were presented as the mean±SD. ***p<0.001, ****p<0.0001. cGAS, cyclic GMP-AMP synthase; Clo, clofarabine; IgG, immunoglobulin G; PI, propidium iodidemRNA, messenger RNA; qRT- PCR, quantitative reverse transcription-PCR; STING, stimulator of interferon genes.

Article Snippet: Nuclei preparation and chromatin digestion were then performed, followed by obtaining 10 μg of chromatin and immunoprecipitation using the corresponding antibodies (NF-κB p65, 6956#, CST; rabbit IgG, #2729S, CST).

Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Immunoprecipitation, Quantitative RT-PCR, Reverse Transcription

Journal: Journal for immunotherapy of cancer

Article Title: Clofarabine induces tumor cell apoptosis, GSDME-related pyroptosis, and CD8 + T-cell antitumor activity via the non-canonical P53/STING pathway.

doi: 10.1136/jitc-2024-010252

Figure Lengend Snippet: Figure 5 Clofarabine regulates tumor cell death and downstream MHC-I/CCL5/CXCL10/BAX expression through NF-κB. (A) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then the cell viability of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours was measured by CCK8 assay. n=5. (B) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then flow cytometry was conducted to determine the apoptosis of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. n=3. (C) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then western blotting revealed the protein expression of GSDME-FL and GSDME-N in lysates collected from A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. (D–E) Pretreated A375 and A549 with JSH- 23 10 µM for 24 hours, then ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. n=2. (F) ChIP assay detects the binding of NF-κB p65 to the CCL5/CXCL10/HLA-B/BAX promoter of A549 after Clo 1.5 µM treatment for 24 hours. n=3. (G) ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of B16F10 with or without Gsdme knockdown after Clo 1.5 µM treatment for 48 hours. n=2. The p value was obtained by multiple comparisons in an ordinary one-way analysis of variance (A, B, D, E, F, G), and the results were presented as the mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ChIP, chromatin immunoprecipitation; Clo, clofarabine; IgG, immunoglobulin G; MHC, major histocompatibility complex; NC, negative control.

Article Snippet: Nuclei preparation and chromatin digestion were then performed, followed by obtaining 10 μg of chromatin and immunoprecipitation using the corresponding antibodies (NF-κB p65, 6956#, CST; rabbit IgG, #2729S, CST).

Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Binding Assay, Knockdown, Chromatin Immunoprecipitation, Immunopeptidomics, Negative Control

Journal: Journal for immunotherapy of cancer

Article Title: Clofarabine induces tumor cell apoptosis, GSDME-related pyroptosis, and CD8 + T-cell antitumor activity via the non-canonical P53/STING pathway.

doi: 10.1136/jitc-2024-010252

Figure Lengend Snippet: Figure 6 Clofarabine enhances the recruitment and cytotoxicity of CD8+ T cells through STING-NF-κB. (A) The chemotactic ability of supernatants of B16F10 with or without Sting1 knockdown after Clo 1.5 µM treatment for 48 hours was detected by the chemotaxis experiment. n=3. (B) The killing ratio of CD8+ T cells against B16F10 with or without Sting1 knockdown after Clo treatment for 24 hours was detected by the T cell killing assay. n=3. (C) The impact of supernatants of B16F10 with or without Sting1 knockdown after Clo 1.5 µM treatment for 48 hours on CD8+ T cell cytotoxicity was detected by flow cytometry. n=3. (D) Pretreated A549 with JSH-23 10 µM for 24 hours, then treated A549 with Clo 1.5 µM alone or Clo 1.5 µM and JSH-23 10 µM for 48 hours. The chemotactic ability of collected supernatants on CD8+ T cells was detected by the chemotaxis experiment. n=3. (E) Pretreated A549 with JSH-23 10 µM for 24 hours, then treated A549 with Clo 1.5 µM alone or Clo 1.5 µM and JSH-23 10 µM for 24 hours. The killing ratio of CD8+ T cells against A549 was detected by the T cell killing assay. n=4. (F) Pretreated A549 with JSH-23 10 µM for 24 hours, then treated A549 with Clo 1.5 µM alone or Clo 1.5 µM and JSH-23 10 µM for 48 hours. The impact of collected supernatants on CD8+ T cell cytotoxicity was detected by flow cytometry. n=3. (G–H) Correlation analysis of the expression of STING1 and NF-κB and its downstream molecules in melanoma and lung cancer. (I) Schematic diagram of the molecular mechanism of Clo. The p value was obtained by multiple comparisons in an ordinary one-way analysis of variance (A, B, C, D, E, F), and the results were presented as the mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Clo, clofarabine; FSC-A, forward scatter area; MHC, major histocompatibility complex; STING, stimulator of interferon genes.

Article Snippet: Nuclei preparation and chromatin digestion were then performed, followed by obtaining 10 μg of chromatin and immunoprecipitation using the corresponding antibodies (NF-κB p65, 6956#, CST; rabbit IgG, #2729S, CST).

Techniques: Knockdown, Chemotaxis Assay, Flow Cytometry, Expressing, Immunopeptidomics