Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: Demographic information of Intervertebral disc donors.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques:

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: Disc NLRP3 levels correlate with the pain and disc degeneration level in LDD patients. Disc specimens from 24 participants who had different levels of Thompson classification of the degeneration and scores for pain were analyzed. (A, B) The NLRP3 protein levels in disc tissue were quantified by ELISA in all 24 specimens. Reads of NLRP3 OD values at 450nm were presented. The correlation between NLRP3 protein levels and pain score ( A , r 2 = 0.85, p<0.0001) or Thompson classification of the degeneration level ( B , p=0.003) was assessed.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: NLRP3 is exclusively expressed in disc microglia. Single cell expression profile for mouse spinal cord was obtained from Panglaodb. (A–F) In all analyzed 6 mouse spinal cord samples, NLRP3 (blue rectangle) was exclusively expressed in disc microglia clusters (red rectangle).

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: Generation of microglia-specific NLRP3-KO or NLRP3-overexpressing mice. (A) Illustration of mice with microglia-specific depletion of NLRP3 (Tmem119p-CreERT2; NLRP3 (fx/fx)) and their control NLRP3(fx/fx) mice, as well as mice with microglia-specific persistent expression of NLRP3 (Tmem119p-CreERT2; NLRP3mut) and their control NLRP3mut mice. (B) NLRP3 staining was done in spinal discs from tamoxifen-challenged mice. (C, D) Dissociated cells from spinal discs of the mice were FAC sorted for CD68+Tmem119+ microglia, the NLRP3 levels of which were checked by ELISA. (C) The relative levels to those from NLRP3(fx/fx) (=1) were shown. (D) The presentative flow charts of FACS sorting CD68+Tmem119+ microglia. *p<0.05. ns, non-significant. Scale bars are 100µm.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Control, Expressing, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: NLRP3 depletion in microglia reduces phagocytosis potential and release of pro-inflammatory cytokines. (A) Phagocytosis for zymosan was assessed in sorted disc microglia from mice with microglia-specific alteration in NLRP3 expression. (B) ELISA for IL-1β, TNFα, IFNɣ, ARG1 and CD163 in sorted disc microglia from mice with microglia-specific alteration in NLRP3 expression. The relative levels to those from NLRP3mut (=1) were shown. *p<0.05. ns, non-significant.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: NLRP3 depletion in microglia reduces disc degeneration and associated pain. The effects of altering NLRP3 levels in microglia on disc degeneration and associated pain were examined in a mouse model for LDD. A total of 8 groups of mice were included in this experiment. Group 1, NLRP3 (fx/fx) mice received sham operation (Sham); Group 2: NLRP3 (fx/fx) mice received LDD induction (LDD); Group 3: NLRP3mut mice received sham operation; Group 4: NLRP3mut mice received LDD induction; Group 5, Tmem119p-CreERT2; NLRP3 (fx/fx) mice received sham operation; Group 6: Tmem119p-CreERT2; NLRP3 (fx/fx) mice received LDD induction; Group 7: Tmem119p-CreERT2; NLRP3mut mice received sham operation; Group 8: Tmem119p-CreERT2; NLRP3mut mice received LDD induction. Mice were analyzed 8 weeks after LDD or at age of 23-week-old. (A, B) Surgical induction of LDD and the quantification of disc degeneration were performed, shown by representative images (A) and by quantification for degenerative scores (B) . (C) A Von Frey filament test for pain evaluation, shown by the relative mechanically induced withdrawal threshold and by thermally induced withdrawal latency of the paw (normalized to those from NLRP3mut (=1)). *p<0.05. ns: no significance.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques:

Journal: Frontiers in Immunology

Article Title: Persistent expression of NLRP3 in spinal microglia promotes development of lumbar disc degeneration

doi: 10.3389/fimmu.2022.1064303

Figure Lengend Snippet: Reduction in disc degeneration and associated pain by NLRP3 depletion in microglia may result from an alleviation of neuroinflammation. (A–D) ELISA for NLRP3 (A) , IL-1β (B) , TNFα (C) and IFNɣ (D) levels in disc tissue sham/LDD-treated mice. The relative levels to those from NLRP3mut (=1) were shown. *p<0.05. ns, no significance.

Article Snippet: A mouse with CreERT2 knock-in under the microglia-specific Tmem119 promoter (Tmem119p-CreERT2; #031820, Jax Mice, Bar Harbor, ME, USA) ( ) was bred to a mouse with its NLRP3 exon 4 flanked by loxP sites (NLRP3 (fx/fx); #12935, Taconic Biosciences, Rensselaer, NY, USA) that allow constitutive knockout of NLRP3 in microglia after Cre-induced recombination (Tmem119p-CreERT2; NLRP3 (fx/fx)) induced by tamoxifen.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Mizoribine Ameliorates Renal Injury and Hypertension along with the Attenuation of Renal Caspase-1 Expression in Aldosterone-Salt-Treated Rats

doi: 10.1371/journal.pone.0093513

Figure Lengend Snippet: Aldosterone (ALD) enhanced the renal expression of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing (NLRP) 3 and caspase-1, and increased the number of TUNEL-positive cells—the hallmark of pyroptosis—whereas the increase in these factors was significantly suppressed by mizoribine (MZR). (A) Western blot analysis for NLRP3; caspase-1; and α-tubulin was performed. The total cell lysate was prepared from the rat kidney cortex. Graphs show the expression level quantified by densitometry and normalized with α-tubulin. (B) Representative photomicrographs of TUNEL staining of kidney sections. The graph shows the quantification of TUNEL-positive cells. Values are presented as mean ± SEM. * P <0.05.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-caspase-1 antibody (Millipore, Temecula, CA, USA); rabbit polyclonal anti-nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) antibody (Novus Biologicals, Littleton, CO, USA); mouse monoclonal anti-α-tubulin antibody (Sigma); rabbit polyclonal anti-αENaC antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-serum-glucocorticoid regulated kinase (SGK) antibody (Cell Signaling Technology, Beverly, MA, USA); and rabbit polyclonal anti-with-no-lysine 4 (WNK4) antibody (Millipore).

Techniques: Expressing, Binding Assay, TUNEL Assay, Western Blot, Staining

Journal: Molecular Medicine Reports

Article Title: Effects of PYRIN-containing Apaf1-like protein 1 on isoflurane-induced postoperative cognitive dysfunction in aged rats

doi: 10.3892/mmr.2020.11244

Figure Lengend Snippet: PYPAF1-targeting shRNA mediates the downregulation of PYPAF1 expression in the hippocampus. Sprague-Dawley rats were injected with lentivirus carrying PYPAF1-shRNA or NC-shRNA by intracerebroventricular injection. (A) mRNA and (B) protein levels of PYPAF1 in the hippocampus were measured using reverse transcription-quantitative PCR and western blot analysis, respectively. ***P<0.001. NC, negative control; PYPAF1, PYRIN-containing Apaf1-like protein 1; shRNA, short hairpin RNA.

Article Snippet: The sections were dehydrated at 60°C in an incubator for 2 h, deparaffinized and heated in citric acid/sodium citrate solution at 95°C for 10 min. After blocking with goat serum (cat. no. SL038; Beijing Solarbio Science and Technology Co., Ltd.) for 15 min at room temperature, tissue sections were washed and incubated with PYPAF1 antibody (1:200; cat. no. NBP2-12446SS; Novus Biologicals, LLC) and ionized calcium binding adapter molecule-1 (Iba-1) antibody (1:200; cat.no. ab15690; Abcam) at 4°C overnight.

Techniques: shRNA, Expressing, Injection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Negative Control

Journal: Molecular Medicine Reports

Article Title: Effects of PYRIN-containing Apaf1-like protein 1 on isoflurane-induced postoperative cognitive dysfunction in aged rats

doi: 10.3892/mmr.2020.11244

Figure Lengend Snippet: PYPAF1 silencing alleviates cognitive dysfunction induced by isoflurane in rats. The spatial learning ability of rats was assessed using the Morris water maze. (A) Escape time, ***P<0.001 vs. control; # P<0.05, ## P<0.01 vs. isoflurane + shRNA NC. (B) Τimes of platform crossing and (C) retention time at target quadrant were recorded and analyzed. ***P<0.001. NC, negative control; PYPAF1, PYRIN-containing Apaf1-like protein 1; shRNA, short hairpin RNA.

Article Snippet: The sections were dehydrated at 60°C in an incubator for 2 h, deparaffinized and heated in citric acid/sodium citrate solution at 95°C for 10 min. After blocking with goat serum (cat. no. SL038; Beijing Solarbio Science and Technology Co., Ltd.) for 15 min at room temperature, tissue sections were washed and incubated with PYPAF1 antibody (1:200; cat. no. NBP2-12446SS; Novus Biologicals, LLC) and ionized calcium binding adapter molecule-1 (Iba-1) antibody (1:200; cat.no. ab15690; Abcam) at 4°C overnight.

Techniques: Control, shRNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: Effects of PYRIN-containing Apaf1-like protein 1 on isoflurane-induced postoperative cognitive dysfunction in aged rats

doi: 10.3892/mmr.2020.11244

Figure Lengend Snippet: PYPAF1 silencing inhibits the activation of microglia. (A) mRNA levels of PYPAF1, and (B and C) protein levels of PYPAF1 and ASC in the hippocampus were measured using reverse transcription-quantitative PCR and western blot analysis on days 3 and 7 following isoflurane treatment, respectively. (B) Lane 1, control; lane 2, isoflurane; lane 3, isoflurane + shRNA-PYPAF1; lane 4, isoflurane + shRNA-NC. (D) Expression of PYPAF1 and Iba-1 in the hippocampus were determined using immunofluorescence assay. ***P<0.001. ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; Iba-1, ionized calcium binding adapter molecule-1; Iso, isoflurane; NC, negative control; PYPAF1, PYRIN-containing Apaf1-like protein 1; shRNA, short hairpin RNA.

Article Snippet: The sections were dehydrated at 60°C in an incubator for 2 h, deparaffinized and heated in citric acid/sodium citrate solution at 95°C for 10 min. After blocking with goat serum (cat. no. SL038; Beijing Solarbio Science and Technology Co., Ltd.) for 15 min at room temperature, tissue sections were washed and incubated with PYPAF1 antibody (1:200; cat. no. NBP2-12446SS; Novus Biologicals, LLC) and ionized calcium binding adapter molecule-1 (Iba-1) antibody (1:200; cat.no. ab15690; Abcam) at 4°C overnight.

Techniques: Activation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, shRNA, Expressing, Immunofluorescence, Binding Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: Effects of PYRIN-containing Apaf1-like protein 1 on isoflurane-induced postoperative cognitive dysfunction in aged rats

doi: 10.3892/mmr.2020.11244

Figure Lengend Snippet: PYPAF1 knockdown inhibits the activity of caspase-1, and expression of IL-1β and IL-18. (A) mRNA levels and (B) concentration of IL-1β and IL-18 in the hippocampus was determined using reverse transcription-quantitative PCR and ELISA, respectively, on day 3 following isoflurane treatment. (C) Activity of caspase-1 in the hippocampus was assessed using a commercial caspase-1 activity assay kit on day 3 following isoflurane treatment. ***P<0.001. IL, interleukin; NC, negative control; PYPAF1, PYRIN-containing Apaf1-like protein 1; shRNA, short hairpin RNA.

Article Snippet: The sections were dehydrated at 60°C in an incubator for 2 h, deparaffinized and heated in citric acid/sodium citrate solution at 95°C for 10 min. After blocking with goat serum (cat. no. SL038; Beijing Solarbio Science and Technology Co., Ltd.) for 15 min at room temperature, tissue sections were washed and incubated with PYPAF1 antibody (1:200; cat. no. NBP2-12446SS; Novus Biologicals, LLC) and ionized calcium binding adapter molecule-1 (Iba-1) antibody (1:200; cat.no. ab15690; Abcam) at 4°C overnight.

Techniques: Knockdown, Activity Assay, Expressing, Concentration Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Negative Control, shRNA

Journal: Molecular Medicine Reports

Article Title: Effects of PYRIN-containing Apaf1-like protein 1 on isoflurane-induced postoperative cognitive dysfunction in aged rats

doi: 10.3892/mmr.2020.11244

Figure Lengend Snippet: PYPAF1 silencing alleviates neuronal apoptosis. (A) Neuronal apoptosis in the hippocampus was determined using TUNEL-NeuN double staining on day 3 following isoflurane treatment. (B) Protein expression levels of Bcl-2, Bax and cleaved caspase-3 in the hippocampus were determined using western blot analysis. ***P<0.001. Iso, isoflurane; NC, negative control; PYPAF1, PYRIN-containing Apaf1-like protein 1; shRNA, short hairpin RNA.

Article Snippet: The sections were dehydrated at 60°C in an incubator for 2 h, deparaffinized and heated in citric acid/sodium citrate solution at 95°C for 10 min. After blocking with goat serum (cat. no. SL038; Beijing Solarbio Science and Technology Co., Ltd.) for 15 min at room temperature, tissue sections were washed and incubated with PYPAF1 antibody (1:200; cat. no. NBP2-12446SS; Novus Biologicals, LLC) and ionized calcium binding adapter molecule-1 (Iba-1) antibody (1:200; cat.no. ab15690; Abcam) at 4°C overnight.

Techniques: TUNEL Assay, Double Staining, Expressing, Western Blot, Negative Control, shRNA

Journal: PLoS ONE

Article Title: Staphylococcus aureus Activates the NLRP3 Inflammasome in Human and Rat Conjunctival Goblet Cells

doi: 10.1371/journal.pone.0074010

Figure Lengend Snippet: Human conjunctiva was analyzed by immunohistochemistry and NLRP3 was shown to be highly expressed in the epithelium as indicated by pink staining (A). Arrows indicate epithelial layer of conjunctiva; arrowheads indicate goblet cells. Isotype controls were negative. Rat conjunctiva was analyzed by immunofluorescence microscopy (B) and all three of the inflammasome components NLRP3, caspase 1, and ASC were identified as demonstrated by the red peri-nuclear staining. The green UEA staining indicates goblet cell secretory product, denoting the location of goblet cells in the conjunctiva. Arrows indicate goblet cells. The mouse isotype controls (B) were negative as were the rabbit isotype controls (not shown). Epi, epithelium.

Article Snippet: Antibodies used were mouse anti-human NLRP3 (1∶50, Enzo Life Sciences, catalog number 804–819-C100) and rabbit anti-rat caspase 1 (1∶20) (Enzo Life Sciences, Plymouth, PA); rabbit anti-rat ASC (1∶50) (Santa Cruz Biotechnology, CA, USA); anti-rabbit P2X4 and P2X7 (1∶50) (Alomone Labs, Jerusalem, Israel); anti-rabbit TLR2 (1∶100) (Santa Cruz Biotechnology, CA).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Microscopy

Journal: PLoS ONE

Article Title: Staphylococcus aureus Activates the NLRP3 Inflammasome in Human and Rat Conjunctival Goblet Cells

doi: 10.1371/journal.pone.0074010

Figure Lengend Snippet: Primary cultures of human or rat goblet cells were analyzed by immunofluorescence microscopy after staining with antibodies against inflammasome components NLRP3, caspase 1, and ASC ( A ). All three components were identified as indicated by the red peri-nuclear staining pattern. Rabbit isotype controls (shown) and mouse isotype controls (not shown) were negative. The presence of NLRP3 ( B ), caspase 1 ( C ), ASC ( D ) and IL-1β ( E ) were confirmed by western blot analysis in lysates from human goblet cells and rat conjunctiva and goblet cells. Please note that the positive controls for NLRP3 and ASC are transfected cells lines overexpressing the protein and thus the amount of protein loaded for the western blots was below the detection level of the β-actin antibody. In addition, the NLRP3 is tagged with His tag while the ASC is tagged with a FLAG tag causing these proteins to run at a higher molecular weight than the native molecules.

Article Snippet: Antibodies used were mouse anti-human NLRP3 (1∶50, Enzo Life Sciences, catalog number 804–819-C100) and rabbit anti-rat caspase 1 (1∶20) (Enzo Life Sciences, Plymouth, PA); rabbit anti-rat ASC (1∶50) (Santa Cruz Biotechnology, CA, USA); anti-rabbit P2X4 and P2X7 (1∶50) (Alomone Labs, Jerusalem, Israel); anti-rabbit TLR2 (1∶100) (Santa Cruz Biotechnology, CA).

Techniques: Immunofluorescence, Microscopy, Staining, Western Blot, Transfection, FLAG-tag, Molecular Weight

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: NLRP3 Inflammasome Activation and Altered Mitophagy Are Key Pathways in Inclusion Body Myositis

doi: 10.1002/jcsm.13672

Figure Lengend Snippet: Inclusion body myositis transcriptome. Transcriptomic analysis of muscle tissue from 38 patients with inclusion body myositis and 22 controls. (A) PCA (to the left) and heatmap (middle) showing clear separation between IBM patients and controls. Volcano plot (to the right) representing differentially expressed genes (blue). (B) Top 10 upregulated and top 10 downregulated pathways in IBM. The NLRP3 inflammasome and mitophagy pathways are detailed in heatmaps, with downregulated genes shown in blue and upregulated genes shown in red. Transcriptomic sex differences are shown in C and D. (C) Volcano plot showing the signed ‐log 10 ( p value) for each gene in females on the x ‐axis and in males on the y‐axis. Differentially expressed genes that are consistent in both sexes are shown in black, female‐unique genes are shown in red, male‐unique genes are shown in blue and genes with reverse trends are shown in purple. Genes that did not reach fold change or p value cutoffs in either sex are shown in grey (not significant). (D) The top 10 upregulated and downregulated pathways for sex‐specific genes.

Article Snippet: Frozen muscle sections (10 μm thick) were stained with haematoxylin and eosin, ATPase (pH 4.3 and 4.6), cytochrome c oxidase, NLRP3 (Novus Biologicals, NBP2–12446) and p‐S65 Ub antibodies.

Techniques:

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: NLRP3 Inflammasome Activation and Altered Mitophagy Are Key Pathways in Inclusion Body Myositis

doi: 10.1002/jcsm.13672

Figure Lengend Snippet: NLRP3 inflammasome activation in inclusion body myositis. (A) Western blot of muscle lysates from IBM patients and controls showing increased NLRP3 and ASC protein expression in samples from both males and females with IBM. The Mann–Whitney test was performed for group comparisons. Western blot results comparing the IBM group to the control group were as follows (Mann–Whitney U, two‐tailed p value): NLRP3 for both sexes (20, p = 0.0009), NLRP3 for males (6, p = 0.045), NLRP3 for females (3, p = 0.011), ASC for both sexes (4, p < 0.0001), ASC for males (3, p = 0.0047) and ASC for females (0, p = 0.0007). (B) NLRP3 immunohistochemistry showing increased NLRP3 immunoreactivity in scattered muscle fibres (examples shown as white arrows) as well as in inflammatory cells (examples shown as black arrowheads). (C) Radar maps displaying top coexpressed and inversely coexpressed genes with NLRP3 according to Spearman rank correlation analysis. The correlation coefficient corresponds to the distance from the centre. All represented genes had a p value less than 0.0001.

Article Snippet: Frozen muscle sections (10 μm thick) were stained with haematoxylin and eosin, ATPase (pH 4.3 and 4.6), cytochrome c oxidase, NLRP3 (Novus Biologicals, NBP2–12446) and p‐S65 Ub antibodies.

Techniques: Activation Assay, Western Blot, Expressing, MANN-WHITNEY, Control, Two Tailed Test, Immunohistochemistry

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: NLRP3 Inflammasome Activation and Altered Mitophagy Are Key Pathways in Inclusion Body Myositis

doi: 10.1002/jcsm.13672

Figure Lengend Snippet: Altered mitophagy in inclusion body myositis patients. (A) Measurement of p‐S65‐Ub levels via sandwich ELISA in muscle samples from IBM patients and controls. The results comparing the IBM samples to the controls were as follows (Mann–Whitney U, two‐tailed p value): both sexes (43, p = 0.0054), males only (7, p = 0.0136) and females only (16, p = 0.31). MSD‐ECL:Meso Scale Discovery‐Electrochemiluminescence. (B and C) Frozen muscle sections from IBM samples and controls were reacted to p‐S65‐Ub, NLRP3 and ATPase at pH 4.3 (Type 1 fibres: dark brown, Type 2 fibres: pink) and at pH 4.6 (Type 1 fibres: dark brown; Type 2a fibres: light brown; Type 2b fibres: in between shade), and cytochrome c oxidase. (B) Top row: muscle samples from controls; p‐S65‐Ub shows clear differentiation of fibre types, reflecting the difference in baseline mitophagy and mitochondrial content between fibre types. Type 1 fibres, dark brown on ATPase 4.3, had increased p‐S65‐Ub and CCO reactivity. Middle and bottom rows: muscle samples from male and female IBM patients, respectively, demonstrating p‐S65‐Ub‐positive aggregates in scattered muscle fibres (examples shown as arrows). These fibres were almost exclusively Type 2 fibres (ATPase stains) that were also devoid of CCO activity (CCO‐negative fibres). Additionally, all CCO‐negative fibres, with or without observable abnormal p‐S65‐Ub reactivity, were Type 2 fibres. Scale bar for Panel B: top row: 50 μm; middle row: 50 μm; bottom row: 200 μm. (C) Muscle samples from IBM patients were reacted for NLRP3 and p‐S65‐Ub, demonstrating muscle fibres with increased NLRP3 and p‐S65‐Ub reactivity (arrows). Scale bar: top row 50 μm, bottom row 20 μm.

Article Snippet: Frozen muscle sections (10 μm thick) were stained with haematoxylin and eosin, ATPase (pH 4.3 and 4.6), cytochrome c oxidase, NLRP3 (Novus Biologicals, NBP2–12446) and p‐S65 Ub antibodies.

Techniques: Sandwich ELISA, MANN-WHITNEY, Two Tailed Test, Electrochemiluminescence, Activity Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: NLRP3 Inflammasome Activation and Altered Mitophagy Are Key Pathways in Inclusion Body Myositis

doi: 10.1002/jcsm.13672

Figure Lengend Snippet: Correlation of NLRP3 inflammasome activation with altered mitophagy and muscle weakness. Pearson correlation plot demonstrating correlation coefficient values for the NLRP3 RNA level, p‐S65‐Ub level, manual muscle test (MMT) score and disease duration by sex. Both sexes: NLRP3 and p‐S65‐Ub, p ‐value = 0.059; NLRP3 and MMT score, p = 0.072; NLRP3 and disease duration, p = 0.543; p‐S65‐Ub and MMT score, p = 0.58; p‐S65‐Ub and disease duration, p = 0.699. Males: NLRP3 and p‐S65‐Ub, p = 0.116; NLRP3 and MMT score, p = 0.032; NLRP3 and disease duration, p = 0.699; p‐S65‐Ub and MMT score, p = 0.359; p‐S65‐Ub and disease duration, p = 0.49. Females: NLRP3 and p‐S65‐Ub, p = 0.106; NLRP3 and MMT score, p = 0.706; NLRP3 and disease duration, p = 0.781; p‐S65‐Ub and MMT score, p = 0.642; p‐S65‐Ub and disease duration, p = 0.898.

Article Snippet: Frozen muscle sections (10 μm thick) were stained with haematoxylin and eosin, ATPase (pH 4.3 and 4.6), cytochrome c oxidase, NLRP3 (Novus Biologicals, NBP2–12446) and p‐S65 Ub antibodies.

Techniques: Activation Assay

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: NLRP3 Inflammasome Activation and Altered Mitophagy Are Key Pathways in Inclusion Body Myositis

doi: 10.1002/jcsm.13672

Figure Lengend Snippet: The vicious cycle of inflammasome activation‐mitochondrial dysfunction/altered mitophagy in inclusion body myositis. The release of mitochondrial damage‐associated molecular patterns (DAMPs) results in the activation of the NLRP3 inflammasome. Under normal conditions, damaged mitochondria and the NRLP3 inflammasome are both subsequently removed by mitophagy and autophagy, reestablishing cellular homeostasis. In IBM, mitophagy and autophagy are altered, establishing a feedforward loop in which the inflammatory milieu results in additional oxidative stress and mitochondrial dysfunction with further release of mitochondrial DAMPs and subsequent aberrant NLRP3 inflammasome activation.

Article Snippet: Frozen muscle sections (10 μm thick) were stained with haematoxylin and eosin, ATPase (pH 4.3 and 4.6), cytochrome c oxidase, NLRP3 (Novus Biologicals, NBP2–12446) and p‐S65 Ub antibodies.

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: A full-spectrum Boswellia serrata extract with enhanced bioavailability, and its co-delivered system with curcumin alleviate pain and stiffness associated with moderate spondylitis: a randomized double-blind, placebo-controlled, 3-arm study

doi: 10.3389/fphar.2025.1577429

Figure Lengend Snippet: Mean change in (a) IL-1β and (b) NLRP3. Values are expressed as Mean ±SD, 2 × 3 repeated measures ANOVA was analysed to perform statistical significance.

Article Snippet: Serum concentrations of IL-1β (catalog no: E-EL-H0149) and NLRP3 (E-EL-H2557) were analyzed with the respective ELISA kits following the manufacturer’s instruction, (Elabscience, Biotechnology Co., Limited.

Techniques:

Journal: Diabetes & Vascular Disease Research

Article Title: Baicalin ameliorates atherosclerosis by inhibiting NLRP3 inflammasome in apolipoprotein E-deficient mice

doi: 10.1177/1479164120977441

Figure Lengend Snippet: Baicalin inhibited NLRP3 inflammasome activation in aortas: (a) Western blot analysis of NLRP3 and Caspase-1 p20, (b) relative mRNA levels of NLRP3 and caspase-1 were detected by qRT-PCR. Relative protein levels of NLRP3 (c), and caspase-1 (d) from western blot were quantified. Data were shown as mean ± SD. n = 7 for each group. * p < 0.05, ** p < 0.01 compared with the control group; # p < 0.05 compared with the AS group.

Article Snippet: Lentiviral vector expressing Nlrp3 shRNA or control shRNA was purchased from Origene (Beijing, China).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR

Journal: Diabetes & Vascular Disease Research

Article Title: Baicalin ameliorates atherosclerosis by inhibiting NLRP3 inflammasome in apolipoprotein E-deficient mice

doi: 10.1177/1479164120977441

Figure Lengend Snippet: Effects of NLRP3 silencing on the progression of atherosclerosis in ApoE −/− mice: (a) relative mRNA levels of NLRP3, caspase-1, ICAM-1, and VCAM-1 were detected by qRT-PCR, (b) IL-1β and IL-18 levels were measured by the ELISA assay, (c) mtROS and ROS levels were detected, and (d) atherosclerotic plaques area of aortic root was quantified. Scale bar = 50 μm. Data were shown as mean ± SD. n = 7 for each group. * p < 0.05, ** p < 0.01 compared with the control group; # p < 0.05 compared with the AS+sh-Control group. Mice of Control and AS+sh-Control group were treated with lentivirus expressing control shRNA, and mice of AS+sh-NLRP3 group were treated with lentivirus expressing NLRP3 shRNA.

Article Snippet: Lentiviral vector expressing Nlrp3 shRNA or control shRNA was purchased from Origene (Beijing, China).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, shRNA