Journal: Journal of Inflammation Research

Article Title: The AGEs/RAGE Signaling Pathway Regulates NLRP3-Mediated Neuronal Pyroptosis After MCAO Injury in Lepr −/− Obese Rats

doi: 10.2147/JIR.S476458

Figure Lengend Snippet: The Primer Sequences of NLRP3, GSDMD, RAGE, and GAPDH

Article Snippet: One hour before the MCAO surgery, the NLRP3 inhibitor MCC950 (5 mg/kg, Cat No. HY-12815A, Selleck Chemicals, USA) was administered via the tail vein.

Techniques:

Journal: Journal of Inflammation Research

Article Title: The AGEs/RAGE Signaling Pathway Regulates NLRP3-Mediated Neuronal Pyroptosis After MCAO Injury in Lepr −/− Obese Rats

doi: 10.2147/JIR.S476458

Figure Lengend Snippet: Excessive activation of NLRP3 inflammasome after MCAO in Lepr −/− obesity rats. ( A ) Protein expression of NLRP3 (n = 3 in each group, power value = 0.99, F (3, 8) = 139, P < 0.001), Pro-caspase1 (n = 4 in each group, power value = 0.92, F (3, 12) = 92.23, P < 0.001), Cleaved caspase1 (n = 4 in each group, power value = 0.91, F (3, 12) =584.8, P <0.001), Mature IL-18 (n = 4 in each group, power value = 0.93, F (3, 12) = 172.1, P < 0.001) and Mature IL-1β (n = 4 in each group, power value = 0.99, F (3, 12) = 229.2, P < 0.001) in the cortex penumbra region in each group. ( B ) qRT-PCR results showed the mRNA expression of NLRP3 in the cortex penumbra region in each group. (n = 4 in each group, power value = 0.81, F (3, 12) =108.1, P < 0.001). ( C and D ) Representative Immunofluorescence staining ( C ) and statistical comparison of Relative NLRP3 positive cells ( D ) in each group (scale bar = 40 μm) (n = 5 in each group, power value = 0.91, F (3, 16) =1398, P < 0.0001). Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s analysis. **** P < 0.0001, ** P < 0.01, * P < 0.05 vs WT Sham group, #### P < 0.0001 vs Lepr −/− Sham group, &&&& P < 0.0001, & P < 0.05 vs WT MCAO group.

Article Snippet: One hour before the MCAO surgery, the NLRP3 inhibitor MCC950 (5 mg/kg, Cat No. HY-12815A, Selleck Chemicals, USA) was administered via the tail vein.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Comparison

Journal: Journal of Inflammation Research

Article Title: The AGEs/RAGE Signaling Pathway Regulates NLRP3-Mediated Neuronal Pyroptosis After MCAO Injury in Lepr −/− Obese Rats

doi: 10.2147/JIR.S476458

Figure Lengend Snippet: Inhibition of NLRP3 reversed neuronal pyroptosis after MCAO in Lepr −/− obesity rats. ( A ) Zea-Longa score of Lepr −/− obesity rats in each group. (n = 10 in each group, power value = 0.87, KW = 12.567, P = 0.0019). Data are presented as medians±interquartile range. Data were analyzed using the Kruskal–Wallis test. ( B ) Representative images of HE staining in the cortex penumbra region, with the abnormality neurons indicated through the red arrows (scale bar = 20 μm). (n = 5 in each group). ( C ) Protein expression of Pro-caspase1 (n = 4 in each group, power value = 0.94, F (2, 9) =188.4, P < 0.001), Cleaved caspase1 (n = 3 in each group, power value = 0.93, F (2, 6) = 554, P < 0.001), GSDMD (n = 3 in each group, power value = 0.86, F (2, 6) =230.2, P < 0.001), N-terminal GSDMD (n = 3 in each group, power value = 0.99, F (2, 6) =28.27, P < 0.001), Mature IL-18 (n = 4 in each group, power value = 0.92, F (2, 9) =27.7, P < 0.001) and Mature IL-1β (n = 3 in each group, power value = 0.97, F (2, 6) = 22.17, P < 0.001) in the cortex penumbra region in each group. Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s analysis. **** P < 0.0001, *** P < 0.001, ** P < 0.01 vs DMSO+ Lepr −/− MCAO.

Article Snippet: One hour before the MCAO surgery, the NLRP3 inhibitor MCC950 (5 mg/kg, Cat No. HY-12815A, Selleck Chemicals, USA) was administered via the tail vein.

Techniques: Inhibition, Staining, Expressing

Journal: Journal of Inflammation Research

Article Title: The AGEs/RAGE Signaling Pathway Regulates NLRP3-Mediated Neuronal Pyroptosis After MCAO Injury in Lepr −/− Obese Rats

doi: 10.2147/JIR.S476458

Figure Lengend Snippet: FPS-ZM1 improved the neuronal pyroptosis mediated by NLRP3 in Lepr −/− obese rats after MCAO. ( A ) Zea-Longa score of Lepr −/− obesity rats in each group. (n = 10 in each group, power value = 0.89, KW = 13.596, P = 0.0011). Data are presented as medians±interquartile range. Data were analyzed using the Kruskal–Wallis test. ( B ) Representative images of HE staining in the cortex penumbra region, with the abnormality neurons indicated through the red arrows (scale bar = 20 μm). (n = 5 in each group). ( C and D ) Protein expression of NLRP3 (n = 4 in each group, power value = 0.99, F (2, 9) =153.7, P < 0.001), Pro-caspase1 (n = 4 in each group, power value = 0.91, F (2, 9) =224.9, P < 0.001), Cleaved caspase1 (n = 3 in each group, power value = 0.96, F (2, 6) =160.5, P < 0.001), Mature IL-1β (n = 4 in each group, power value = 0.94, F (2, 9) =14.45, P = 0.0016) ( C ) and GSDMD (n = 4 in each group, power value = 0.97, F (2, 9) =248.6, P < 0.00), N-terminal GSDMD (n = 3 in each group, power value = 0.97, F (2, 6) =27.43, P < 0.001), Mature IL-18 (n = 3 in each group, power value = 0.93, F (2, 6) =9.062, P < 0.001) ( D ) in the cortex penumbra region in each group. Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s analysis. **** P < 0.0001,*** P < 0.001,** P < 0.01,* P < 0.05 vs Lepr −/− MCAO+DMSO group.

Article Snippet: One hour before the MCAO surgery, the NLRP3 inhibitor MCC950 (5 mg/kg, Cat No. HY-12815A, Selleck Chemicals, USA) was administered via the tail vein.

Techniques: Staining, Expressing

Journal: Journal of Inflammation Research

Article Title: The AGEs/RAGE Signaling Pathway Regulates NLRP3-Mediated Neuronal Pyroptosis After MCAO Injury in Lepr −/− Obese Rats

doi: 10.2147/JIR.S476458

Figure Lengend Snippet: FPS-ZM1 inhibited the expression of NLRP3 and GSDMD in Lepr −/− obesity rats after MCAO. ( A and B ) Representative Immunofluorescence staining ( A ) and statistical comparison of Relative NLRP3 positive cells ( B ) in each group (scale bar = 40 μm) (n = 5 in each group, power value = 0.81, F (2,12) =269.8, P < 0.0001). ( C and D ) Representative Immunofluorescence staining ( C ) and statistical comparison of Relative GSDMD positive cells ( D ) in each group (scale bar = 40 μm) (n = 5 in each group, power value = 0.84, F (2, 12) =239.5, P < 0.0001). Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s analysis. **** P < 0.0001 vs Lepr −/− MCAO+DMSO group.

Article Snippet: One hour before the MCAO surgery, the NLRP3 inhibitor MCC950 (5 mg/kg, Cat No. HY-12815A, Selleck Chemicals, USA) was administered via the tail vein.

Techniques: Expressing, Immunofluorescence, Staining, Comparison

Journal: Immunity, Inflammation and Disease

Article Title: The role of autophagy in SIM mediated anti‐inflammatory osteoclastogenesis through NLRP3 signaling pathway

doi: 10.1002/iid3.1145

Figure Lengend Snippet: Simvastatin (SIM) suppresses lipopolysaccharide (LPS)‐induced osteoclast (OC)‐associated gene expression. RAW264.7 cells were stimulated with NOD‐like receptor family pyrin Domain‐containing protein 3 (NLRP3) inhibitor MCC950 (30 µM) and SIM, then treated them with 100 ng/mL of LPS for 12 h. (A–C) The expression of OC‐specific genes ( RANK , CTSK , and c‐Fos ) were detected by reverse transcription‑quantitative polymerase chain reaction (RT‐qPCR). Bars represent the mean ± SD of three independent experiments. ## p < .01 and ### p < .001 compared with control group; * p < .05, ** p < .01, and *** p < .001 compared with LPS‐treated group.

Article Snippet: MCC950 (CP‐456773, Cat# GC31644) was purchased from Glpbio Technology.

Techniques: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: Immunity, Inflammation and Disease

Article Title: The role of autophagy in SIM mediated anti‐inflammatory osteoclastogenesis through NLRP3 signaling pathway

doi: 10.1002/iid3.1145

Figure Lengend Snippet: MCC950 is involved in the inhibition of Simvastatin (SIM) on autophagy‐related proteins in lipopolysaccharide (LPS)‐treated RAW264.7 macrophages. (A) MCC950 was pretreated into LPS‐stimulated RAW264.7 macrophages in the presence or absence of SIM. (B–D) The band intensities were quantified using ImageJ software. Bars represent the mean ± SD of three independent experiments. # p < .05 and ## p < .01 compared with control group; * p < .05 and ** p < .01 compared with LPS‐treated group.

Article Snippet: MCC950 (CP‐456773, Cat# GC31644) was purchased from Glpbio Technology.

Techniques: Inhibition, Software, Control

Journal: Science (New York, N.Y.)

Article Title: HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation

doi: 10.1126/science.aas8995

Figure Lengend Snippet: (A) The NLRP3, pyrin, and AIM2 inflammasome pathways triggered by nigericin or MSU, TcdB, and dsDNA, respectively. As shown below, NLRP3 and pyrin inflammasome puncta localize at the MTOC. Inflammasome activation culminates in pro-caspase-1 and pro-IL-1β processing. Upward arrows indicate processing sites. (B) Immunofluorescence images showing the co-localization of NLRP3 and ASC puncta with the centrosomal markers ninein and GTU in THP-1 cells. Blue represents nuclear staining by Hoechst 33342. (C) Line scan of intensity distribution profiles of puncta a, b, and c from (B). (D–E) Live-cell images of iBMDM-Casp-1 (D) and iBMDM-IL-1β (E) at 30 min (top panel) and 60 min (bottom panel) post-nigericin stimulation, showing co-localization of inflammasome puncta (depicted by mNeonGreen) with the MTOC (depicted by SiR-Tubulin staining that labels the microtubule network). (F) FRET analysis of caspase-1 cleavage and IL-1β processing at MTOC as a function of time for areas inside and outside the puncta in iBMDM-Casp-1 (left) and iBMDM-IL-1β (right) cells. FRET was calculated by dividing the FRET channel fluorescence intensity (donor excitation with acceptor emission) with mTurquoise2 channel fluorescence intensity (donor excitation with donor emission). Values are mean±SD for n=10–15 cells. (G–H) Recruitment of IL-1β to a region in proximity to the MTOC imaged using 3D lattice light-sheet microscopy (LLSM). iBMDM-IL-1β cells stained with SiR-Tubulin were exposed to nigericin for 12 min (G), and 23 min (H). (a–c) Representative images deconvolved using the Richardson–Lucy algorithm corresponding to a single optical plane section. The arrows highlight the MTOC and the nearby locations where IL-lβ was recruited. (d–f) Enlarged images of the regions indicated by the arrows. (I–J) Lack of co-localization of AIM2 inflammasome puncta with the MTOC in iBMDM-Casp-1 (I) and iBMDM-IL-1β (J) cells activated by dsDNA for 6 hours. (K–L) Co-localization of pyrin inflammasome puncta with the MTOC in iBMDM-Casp-1 (K) and iBMDM-IL-1β (L) cells activated by TcdB toxin for 1 hour. Images are representative of three or more independent experiments and arrowheads indicate puncta or MTOC (B, D–E, G–L). Scale bars: 10 μm (B, D–E), 5 μm (Ga–c, Ha–c, I–L), and 1 μm (Gd–f, Hd–f).

Article Snippet: Pharmacological inhibition We pre-treated LPS-primed iBMDMs for 1 hour with HDAC6 inhibitors 10 μM Tubastatin A (Sigma-Aldrich, Cat. no: SML0044), 30 μM Rocilinostat (Selleckchem, Cat. no: S8001), or 20 μM, or 5–40 μM Tubacin (Enzo Life Sciences, Cat. no: BML-GR362–0500), 10 μM microtubule polymerization inhibitors Colchicine (Sigma-Aldrich, Cat. no: C9754–100MG) or 10 μM Nocodazole (Sigma-Aldrich, Cat. no: M1404–2MG), or with 0.1–20 μM NLRP3 inhibitor MCC950 (CP-456773) (Cayman Chemicals Cat. no: 210826–40-7 and Sigma-Aldrich, Cat. no: PZ0280).

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Microscopy

Journal: Science (New York, N.Y.)

Article Title: HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation

doi: 10.1126/science.aas8995

Figure Lengend Snippet: (A–C) NLRP3 inflammasome activation under various inhibition conditions analyzed by caspase-1 processing (p20) (A), quantification of propidium iodide (PI) permeability by flow cytometry (B), and secreted IL-1β quantified by ELISA (C). Colchicine and nocodazole: microtubule polymerization inhibitors; Ciliobrevin A: dynein ATPase inhibitor; Rocilinostat, tubacin and tubastatin A: HDAC6 inhibitors. (D) Caspase-1 processing (p20) upon NLRP3 inflammasome activation with pre-treatment of increasing concentrations of tubacin (left to right: 5 μM, 10 μM, 20 μM and 40 μM) or the NLRP3 inhibitor MCC950 (left to right: 0.1 μM, 0.5 μM, 1 μM, 10 μM and 20 μM). Anti-acetylated α-tubulin and anti-β-actin immunoblots are shown for tubulin acetylation and as the loading control, respectively. (E–G) Pyrin inflammasome activation under various pharmacological conditions analyzed by caspase-1 processing (p20) (E), PI permeability (F), and secreted IL-1β (G). (H–J) AIM2 inflammasome activation under various pharmacological conditions analyzed by caspase-1 processing (p20) (H), PI permeability (I) and secreted IL-1β (J). Data are presented as the mean±SD for 3–4 wells from three or more independent experiments.

Article Snippet: Pharmacological inhibition We pre-treated LPS-primed iBMDMs for 1 hour with HDAC6 inhibitors 10 μM Tubastatin A (Sigma-Aldrich, Cat. no: SML0044), 30 μM Rocilinostat (Selleckchem, Cat. no: S8001), or 20 μM, or 5–40 μM Tubacin (Enzo Life Sciences, Cat. no: BML-GR362–0500), 10 μM microtubule polymerization inhibitors Colchicine (Sigma-Aldrich, Cat. no: C9754–100MG) or 10 μM Nocodazole (Sigma-Aldrich, Cat. no: M1404–2MG), or with 0.1–20 μM NLRP3 inhibitor MCC950 (CP-456773) (Cayman Chemicals Cat. no: 210826–40-7 and Sigma-Aldrich, Cat. no: PZ0280).

Techniques: Activation Assay, Inhibition, Permeability, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Science (New York, N.Y.)

Article Title: HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation

doi: 10.1126/science.aas8995

Figure Lengend Snippet: (A) Immunoblotting shows the absence of HDAC6 protein in CRISPR/Cas9 Hdac6−/− iBMDMs as compared to WT iBMDMs. Loading control was provided by the anti-β-actin antibody. The loss of HDAC6 leads to an increase in acetylated α-tubulin depicted using anti-acetylated α-tubulin antibody. (B–D) Compromised NLRP3 inflammasome activation in Hdac6−/− iBMDMs challenged with nigericin, shown for caspase-1 processing (B), PI permeability (C), and secreted IL-1β (D). Data are presented as the mean±SD for triplicate wells from three or more independent experiments (C, D). (E) Domain architecture of human HDAC6 with important mutations (DA, Ub1 and Ub2) labeled. DA: H216A/H611A on catalytic residues, deacetylase mutant; Ub1: mutations H1160A/H1164A on zinc-coordinating residues; Ub2: mutation W1182A on the surface that binds ubiquitin. (F) Rescue of nigericin-mediated caspase-1 processing in Hdac6−/− iBMDMs by reconstituting with WT human HDAC6. (G) Analysis of nigericin-mediated caspase-1 processing in Hdac6−/− iBMDMs reconstituted with WT HDAC6, and the DA, Ub1 and Ub2 mutants. (H) Sensitivity to rocilinostat in Hdac6−/− iBMDMs reconstituted with WT HDAC6, but not the DA mutant, as depicted by inhibition of p20 processing. (I) Rescue of nigericin-induced punctum formation in Hdac6−/− iBMDM-Casp-1 cells transfected with WT HDAC6-mRuby3. Arrowheads indicate puncta. Cells containing puncta had HDAC6 expression (a and b), whereas cells that do not contain puncta did not have HDAC6 expression (c and d). (J) Rescue of nigericin-induced punctum formation in Hdac6−/− iBMDM-Casp-1 cells stably reconstituted with WT and DA mutant of HDAC6-mRuby3, but not with Ub1 and Ub2 mutants of HDAC6-mRuby3. Arrowheads indicate puncta. HDAC6 WT reconstituted cells failed to form puncta upon pretreatment by rocilinostat. (K–L) Inflammasome puncta formation and its link to autophagy analyzed by immunofluorescence of ASC and the autophagy marker LC3b before (K) and after (L) NLRP3 inflammasome stimulation. Arrowheads indicate puncta. Images are representative of three or more independent experiments. Scale bars: 10 μm.

Article Snippet: Pharmacological inhibition We pre-treated LPS-primed iBMDMs for 1 hour with HDAC6 inhibitors 10 μM Tubastatin A (Sigma-Aldrich, Cat. no: SML0044), 30 μM Rocilinostat (Selleckchem, Cat. no: S8001), or 20 μM, or 5–40 μM Tubacin (Enzo Life Sciences, Cat. no: BML-GR362–0500), 10 μM microtubule polymerization inhibitors Colchicine (Sigma-Aldrich, Cat. no: C9754–100MG) or 10 μM Nocodazole (Sigma-Aldrich, Cat. no: M1404–2MG), or with 0.1–20 μM NLRP3 inhibitor MCC950 (CP-456773) (Cayman Chemicals Cat. no: 210826–40-7 and Sigma-Aldrich, Cat. no: PZ0280).

Techniques: Western Blot, CRISPR, Activation Assay, Permeability, Labeling, Histone Deacetylase Assay, Mutagenesis, Inhibition, Transfection, Expressing, Stable Transfection, Immunofluorescence, Marker

Journal: Science (New York, N.Y.)

Article Title: HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation

doi: 10.1126/science.aas8995

Figure Lengend Snippet: (A–B) Non-canonical inflammasome activated by intracellular delivery of LPS (electroporation) quantified by PI permeability (A) and FAM-FLICA™ substrate cleavage by active caspase-11 (B). (C–D) NLRC4 inflammasome activation triggered by active FlaTox (inactive FlaTox served as a control) analyzed for caspase-1 processing (p20) (C), and PI permeability (D). Data are presented as the mean±SD for triplicate wells from three or more independent experiments. (E) Immunofluorescence analysis of NLRC4 punctum formation in Nlrp3−/− (top) and Nlrp3−/−Hdac6−/− (bottom) iBMDMs upon treatment with FlaTox. Blue represents nuclear staining by Hoechst 33342. The NLRC4 inflammasome punctum represented by ASC staining is distinctly apart from the centrosomal marker ninein. Arrowheads depict puncta or MTOC. Images are representative of three or more independent experiments. Scale bars: 5 μm. (F) Summary of location of punctum formation and HDAC6-dependence in the different inflammasomes.

Article Snippet: Pharmacological inhibition We pre-treated LPS-primed iBMDMs for 1 hour with HDAC6 inhibitors 10 μM Tubastatin A (Sigma-Aldrich, Cat. no: SML0044), 30 μM Rocilinostat (Selleckchem, Cat. no: S8001), or 20 μM, or 5–40 μM Tubacin (Enzo Life Sciences, Cat. no: BML-GR362–0500), 10 μM microtubule polymerization inhibitors Colchicine (Sigma-Aldrich, Cat. no: C9754–100MG) or 10 μM Nocodazole (Sigma-Aldrich, Cat. no: M1404–2MG), or with 0.1–20 μM NLRP3 inhibitor MCC950 (CP-456773) (Cayman Chemicals Cat. no: 210826–40-7 and Sigma-Aldrich, Cat. no: PZ0280).

Techniques: Electroporation, Permeability, Activation Assay, Immunofluorescence, Staining, Marker

Journal: Science (New York, N.Y.)

Article Title: HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation

doi: 10.1126/science.aas8995

Figure Lengend Snippet: (A–C) Immunofluorescence analysis of TGN38 and NLRP3 in Asc−/− iBMDMs (A), WT iBMDMs (B) and Hdac6−/− iBMDMs (C). (D) Immunofluorescence analysis of NEK7 and NLRP3 distribution in Hdac6−/− iBMDMs. Images are representative of three or more independent experiments containing ~100 cells. Arrowheads depict puncta. Scale bars: 10 μm.

Article Snippet: Pharmacological inhibition We pre-treated LPS-primed iBMDMs for 1 hour with HDAC6 inhibitors 10 μM Tubastatin A (Sigma-Aldrich, Cat. no: SML0044), 30 μM Rocilinostat (Selleckchem, Cat. no: S8001), or 20 μM, or 5–40 μM Tubacin (Enzo Life Sciences, Cat. no: BML-GR362–0500), 10 μM microtubule polymerization inhibitors Colchicine (Sigma-Aldrich, Cat. no: C9754–100MG) or 10 μM Nocodazole (Sigma-Aldrich, Cat. no: M1404–2MG), or with 0.1–20 μM NLRP3 inhibitor MCC950 (CP-456773) (Cayman Chemicals Cat. no: 210826–40-7 and Sigma-Aldrich, Cat. no: PZ0280).

Techniques: Immunofluorescence

Journal: Science (New York, N.Y.)

Article Title: HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation

doi: 10.1126/science.aas8995

Figure Lengend Snippet: (A–F) A mouse model of lethal LPS-induced endotoxemia. (A) Experimental layout with indicated timing and dose. (B–D) Effects of tubastatin A (HDAC6 inhibitor) (B), Hdac6−/− (C), or MCC950 (NLRP3 inhibitor) (D) on IL-1β secretion. Values are mean±SD (n=3–5/group). (E) Effects of tubastatin A on acute lung injury (ALI). Representative histopathological images from harvested lung tissues are shown. Scale bar: 50 μm. (F) Quantified lung injury depicted by defined clinical parameters in ALI score. ALI scores are mean±SD (n=3–4/group). (G–K) A mouse model of MSU-induced peritonitis. (G) Experimental layout with indicted timing and dose. (H–I) Effects of Hdac6−/− or Hdac6−/− + MCC950 on peritoneal IL-1β production (H) and neutrophil recruitment (I) upon MSU challenge. Values are mean±SD (n=6–7/group). (J–K) Effects of MCC950 on peritoneal IL-1β production (J) and neutrophil recruitment (K) upon MSU challenge. Values are mean±SD (n=5–8/group). For (B–D, F, H–K), one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was performed for data analysis as used previously (64, 65). *: P≤0.05, **: P≤0.01, ***: P≤0.001 and ****: P≤0.0001.

Article Snippet: Pharmacological inhibition We pre-treated LPS-primed iBMDMs for 1 hour with HDAC6 inhibitors 10 μM Tubastatin A (Sigma-Aldrich, Cat. no: SML0044), 30 μM Rocilinostat (Selleckchem, Cat. no: S8001), or 20 μM, or 5–40 μM Tubacin (Enzo Life Sciences, Cat. no: BML-GR362–0500), 10 μM microtubule polymerization inhibitors Colchicine (Sigma-Aldrich, Cat. no: C9754–100MG) or 10 μM Nocodazole (Sigma-Aldrich, Cat. no: M1404–2MG), or with 0.1–20 μM NLRP3 inhibitor MCC950 (CP-456773) (Cayman Chemicals Cat. no: 210826–40-7 and Sigma-Aldrich, Cat. no: PZ0280).

Techniques:

Journal: Journal of Inflammation Research

Article Title: Benidipine Hydrochloride Inhibits NLRP3 Inflammasome Activation by Inhibiting LPS-Induced NF-κB Signaling in THP-1 Macrophages

doi: 10.2147/JIR.S467796

Figure Lengend Snippet: Benidipine hydrochloride decreases IL-1β production in LPS and ATP-stimulated THP-1 macrophages. ( A ) The IL-1β levels of THP-1 macrophages were detected after pretreatment with benidipine hydrochloride or MCC950 for 1 h, incubation with LPS (1 μg/mL) for 4 h, and ATP (5 mM) incubation for 2 h. ( B ) Analysis of IL-1β mRNA levels by qRT-PCR. ( C ) Western blot of IL-1β and caspase-1 levels in cell lysates (Lys) and supernatants (SN). ( D ) Quantitative analysis results of ( C ). The results represent the mean ± SD for three experiments. # p<0.05, ### p< 0.001 vs the control group. * p<0.05, ** p<0.01 and *** p< 0.001 vs the LPS+ATP group.

Article Snippet: Benidipine hydrochloride (BH) (Cat# T6227) and MCC950 (Cat# T3701) were purchased from Topscience.

Techniques: Incubation, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of Inflammation Research

Article Title: Benidipine Hydrochloride Inhibits NLRP3 Inflammasome Activation by Inhibiting LPS-Induced NF-κB Signaling in THP-1 Macrophages

doi: 10.2147/JIR.S467796

Figure Lengend Snippet: Benidipine hydrochloride inhibited LPS+ATP-stimulated NLRP3 inflammasome activation in THP-1 macrophages. ( A ) THP-1 macrophages were pretreated with benidipine hydrochloride or MCC950 for 1 h, then incubated with LPS (1 μg/mL) for 4 h, followed by ATP (5 mM) for 2 h, and qRT-PCR was used to analyze the mRNA expression levels of ( A ) nlrp3, ( B ) asc, ( C ) caspase 1. ( D ) The expression level of GSDMD was detected by Western blotting. ( E ) Quantitative analysis results of ( D ). The results represent the mean ± SD for three experiments. # p<0.05, ### p< 0.001 vs the control group. * p<0.05, ** p<0.01 and *** p< 0.001 vs the LPS+ATP group.

Article Snippet: Benidipine hydrochloride (BH) (Cat# T6227) and MCC950 (Cat# T3701) were purchased from Topscience.

Techniques: Activation Assay, Incubation, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Brain, behavior, and immunity

Article Title: Maternal P2X7 receptor inhibition prevents autism-like phenotype in male mouse offspring through the NLRP3-IL-1β pathway.

doi: 10.1016/j.bbi.2022.01.015

Figure Lengend Snippet: Fig. 1. Experimental design of the study. Timeline of experiments and treatments. In Experiment 1 and 3, we examined the ASD-related behaviours following two doses of maternal poly(I:C) treatment at E12.5 and E17.5. Maternal pretreatment (MCC950) was used in Experiment 1, and repeated offspring treatment (JNJ47965567) was used in Experiment 3. In Experiment 2, maternal and fetal tissues were collected after a single-dose poly(I:C) and prior maternal pretreatment (MCC950/JNJ47965567).

Article Snippet: In Experiment 1, a cohort of pregnant dams was pretreated with 50 mg/kg of NLRP3 selective antagonist MCC950 (CRID3 sodium salt, Tocris Bioscience, Cat. No. 5479) or with 25 μg/kg goat anti-mouse IL-1 beta /IL-1F2 antigen affinity-purified polyclonal IgG (R&D Systems, AF-401-NA) on E12.5, two hours before poly(I:C) injection.

Techniques:

Journal: Brain, behavior, and immunity

Article Title: Maternal P2X7 receptor inhibition prevents autism-like phenotype in male mouse offspring through the NLRP3-IL-1β pathway.

doi: 10.1016/j.bbi.2022.01.015

Figure Lengend Snippet: Fig. 3. Maternal poly(I:C) treatment did not elicit autism-like phenotype in either VEH or MCC950 treated P2rx7−/−animals. (A) The experimental protocol shown in a flowchart (Experiment 1). (B) No social deficit appeared in either group. (C-F) Repetitive behaviors (C-D) and sensorimotor coordination (E-F) showed no difference by MIA in either PIC group compared to the control groups. Corresponding to this, no difference appeared in the density of Purkinje cells in lobule VII (G- H). Scale bar 100 μm. All data are expressed as mean ± s.e.m. Statistical tests used with exact n, F, and p values are provided in Supplementary Table. *p < 0.05. **p < 0.01. ***p < 0.001.

Article Snippet: In Experiment 1, a cohort of pregnant dams was pretreated with 50 mg/kg of NLRP3 selective antagonist MCC950 (CRID3 sodium salt, Tocris Bioscience, Cat. No. 5479) or with 25 μg/kg goat anti-mouse IL-1 beta /IL-1F2 antigen affinity-purified polyclonal IgG (R&D Systems, AF-401-NA) on E12.5, two hours before poly(I:C) injection.

Techniques: Control

Journal: Brain, behavior, and immunity

Article Title: Maternal P2X7 receptor inhibition prevents autism-like phenotype in male mouse offspring through the NLRP3-IL-1β pathway.

doi: 10.1016/j.bbi.2022.01.015

Figure Lengend Snippet: Fig. 5. Pharmacological inhibition of NLRP3 prevented significant IL-1β elevation by poly(I:C) in placenta and fetal brain samples. (A) The experimental protocol shown in a flowchart (Experiment 2). (B) PIC repeatedly increased the IL-1β level in maternal plasma of P2rx7+/+ mice 1 h following PIC injection both in NLRP3 antagonist pretreated and saline-treated mice (B). Different dots correspond to samples of different animals (5–10 animals per group). (C-D) PIC also induced elevation in IL-1β concentrations 24 h after MIA induction in placental (C) and fetal brain (D) samples of wild-type mice, while MCC950-pretreated mice showed no significant elevation following poly(I:C) administration. 2 intact placenta and 2–3 fetal brain samples of similar size and different uterine locations were collected from 5 pregnant animals per group. Cytokine and chemokine values measured in the plasma are expressed in picograms per milliliter, in the fetal brains, and placenta in picograms per mg of total protein. All data are expressed as mean ± s.e.m. Statistical tests used with exact n, F, and p values are provided in Supplementary Table. *p < 0.05. **p < 0.01. ***p < 0.001.

Article Snippet: In Experiment 1, a cohort of pregnant dams was pretreated with 50 mg/kg of NLRP3 selective antagonist MCC950 (CRID3 sodium salt, Tocris Bioscience, Cat. No. 5479) or with 25 μg/kg goat anti-mouse IL-1 beta /IL-1F2 antigen affinity-purified polyclonal IgG (R&D Systems, AF-401-NA) on E12.5, two hours before poly(I:C) injection.

Techniques: Inhibition, Clinical Proteomics, Injection, Saline