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: Cancer Immunology Research

Article Title: A. Muciniphila Suppresses Colorectal Tumorigenesis by Inducing TLR2/NLRP3-Mediated M1-Like TAMs

doi: 10.1158/2326-6066.cir-20-1019

Figure Lengend Snippet: Figure 3. NLRP3 is essential for A. muciniphila–mediated induction of M1-like macrophages. A, Protein expression levels of NLRP3 in the colon tumor of PBS control (Ctrl) or A. muciniphila–treated (Akk) ApcMin/þ mice were examined by immunofluorescence and Western blot; scale bars, 100 mm (n ¼ 3 per group). B, BMDMs or mouse macrophage Raw264.7 cells were incubated with PBS or A. muciniphila (Akk) or E. coli (MOI ¼ 100:1) for 24 hours. Protein levels of NLRP3, pro-IL1b, and IL1b (p17) were measured by Western blot. C, BMDMs isolated from wild-type (WT) mice or NLRP3/ mice were incubated with PBS or A. muciniphila (Akk; MOI ¼ 100:1) for 24 hours, the Raw264.7 cells were treated with the NLRP3 inhibitor MCC950 (10 nmol/L) for 2 hours followed by incubation with PBS or Akk, and then the expression of iNOS was tested by Western blot. D, BMDMs isolated fromwild-type mice or NLRP3/mice were incubated with PBS or A. muciniphila (Akk) for 24 hours, and then the percentage of M1 macrophages (F4/80þMHCIIþ) was determined by flow cytometry. E, The mRNA expressions of M1-like macrophage–related molecules, Inos, Tnfa, Il6, and Il12, were measured by qRT-PCR in BMDMs incubated with PBS or A. muciniphila (Akk; MOI ¼ 100:1). The independent experiment was repeated three times. Data are presented as mean SD, n ¼ 6. , P < 0.05; , P < 0.01; ANOVA test.

Article Snippet: To suppress NLRP3 in ApcMin/þ mice, the NLRP3 inhibitor MCC950 (10 mg/kg body weight, Cat. #CP-456773, Selleck) was intraperitoneally injected during the final 6 weeks of the experiment.

Techniques: Expressing, Control, Western Blot, Incubation, Isolation, Cytometry, Quantitative RT-PCR

Journal: Cancer Immunology Research

Article Title: A. Muciniphila Suppresses Colorectal Tumorigenesis by Inducing TLR2/NLRP3-Mediated M1-Like TAMs

doi: 10.1158/2326-6066.cir-20-1019

Figure Lengend Snippet: Figure 4. NLRP3 inhibition abrogates the tumor-suppressive effect of A. muciniphila in colorectal cancer. A, HCT116 and CT26 cells were cocultured with A. muciniphila (Akk)– treated wild-type or NLRP3/ BMDMs. Cancer cell proliferation was evaluated at 24, 48, and 72 hours by CCK-8 assay. B, Experimental model for ApcMin/þ mice treated with A. muciniphila (Akk) in the presence or absence of the NLRP3 inhibitor MCC950 (10 mg/kg body weight) and representative colon images are shown. Ctrl is ApcMin/þ mice treated with PBS. The blue arrows indicate the tumor locations (n ¼ 6 per group). C, Colon tumor number, colon tumor load, and small intestine tumor number were measured. D, Frequencies of M1-like TAMs from colon lamina propria by flow cytometry. E, HCT116 cells were injected into BALB/C nude mice combined with A. muciniphila (Akk; MOI ¼ 10:1); 6 days later, following NLRP3 inhibitor MCC950 (10 mg/kg body weight) intraperitoneal injection every 2 days, tumor volume and weight were recorded (n ¼ 5 per group). F, CT26 cells were injected into wild-type and NLRP3/ mice combined with A. muciniphila (Akk; MOI ¼ 10:1), n ¼ 5 per group. G, CT26 cells were injected into BALB/C nude mice as previous method described (n ¼ 4 per group). The independent experiment was repeated three times. Data are presented as mean SD or SEM. , P < 0.05; , P < 0.01; , P < 0.001; ANOVA test. CRC, colorectal cancer; MC, MCC950; WT, wild-type.

Article Snippet: To suppress NLRP3 in ApcMin/þ mice, the NLRP3 inhibitor MCC950 (10 mg/kg body weight, Cat. #CP-456773, Selleck) was intraperitoneally injected during the final 6 weeks of the experiment.

Techniques: Inhibition, CCK-8 Assay, Cytometry, Injection

Journal: Cancer Immunology Research

Article Title: A. Muciniphila Suppresses Colorectal Tumorigenesis by Inducing TLR2/NLRP3-Mediated M1-Like TAMs

doi: 10.1158/2326-6066.cir-20-1019

Figure Lengend Snippet: Figure 5. TLR2/NF-kB activation is involved in M1-like macrophage modulation by A. muciniphila. A, BMDMs or Raw264.7 cells were incubated with PBS, A. muciniphila (Akk), or E. coli (MOI ¼ 100:1) for 24 hours. The mRNA expressions of Tlr2, Tlr4, Tlr5, and Tlr9 were measured by qRT-PCR. B, Protein expression levels of TLR2, p-p65, and IkB-a were examined by Western blot in BMDMs and TLR2 protein was also measured in the colon tumor from ApcMin/þ mice (n ¼ 3 per group). C, BMDMs were treated with the TLR2 inhibitor CPT22 (10 mmol/L) for 2 hours followed by incubation with PBS or A. muciniphila (Akk) for 24 hours, and then the percentage of M1-like macrophages (F4/80þMHCIIþ) was determined by flow cytometry. D, Protein levels of p-p65, NLRP3, pro-IL1b, and iNOS were tested by Western blot in both BMDMs and Raw264.7 cells after coculture in the presence (Akk) or absence (Ctrl) of A. muciniphila. E, The mRNA expression levels of M1-like macrophage–related molecules Inos, Tnfa, Il6, and Il12 were measured by qRT-PCR in BMDMs. The independent experiment was repeated three times. Data are presented as mean SD. , P < 0.05; , P < 0.01; and , P < 0.001; ANOVA test.

Article Snippet: To suppress NLRP3 in ApcMin/þ mice, the NLRP3 inhibitor MCC950 (10 mg/kg body weight, Cat. #CP-456773, Selleck) was intraperitoneally injected during the final 6 weeks of the experiment.

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

Journal: Cancer Immunology Research

Article Title: A. Muciniphila Suppresses Colorectal Tumorigenesis by Inducing TLR2/NLRP3-Mediated M1-Like TAMs

doi: 10.1158/2326-6066.cir-20-1019

Figure Lengend Snippet: Figure 6. A. muciniphila abundance correlates with the expression of NLRP3 and TLR2 in patients with colorectal cancer. A, Representative image of immunostaining for iNOS in tumor tissue with low or high A. muciniphila (Akk) abundance from cohort 2; scale bars, 100 mm. B, Representative images of immunofluorescence stain- ing of NLRP3 (CD68þNLRP3þ dual positive) in cancer tissues from cohort 2; scale bars, 100 mm. The yellow arrows indicate the positively stained cells. C, Transcript levels of NLRP3 and TLR2 in 64 matched fresh-frozen colorectal cancer and normal tissues from cohort 2 were examined by qRT-PCR, and the correlation of NLRP3 and TLR2 was analyzed. D, The mRNA levels of NLRP3 and TLR2 in tumor tissue with low or high A. muciniphila (Akk) abundance from cohort 3, and the correlation of NLRP3 and TLR2 was analyzed. E, Expression of NLRP3 and TLR2 in colorectal cancer tissue and normal tissue in TCGA TARGET GTEx datasets. Data are presented as mean SD. , P < 0.05; , P < 0.01; , P < 0.0001; Student t test (A), Wilcoxon matched-pairs signed-rank test (C), Mann–Whitney test (D and E), and Spearman correlation analysis (C–E). CRC, colorectal cancer.

Article Snippet: To suppress NLRP3 in ApcMin/þ mice, the NLRP3 inhibitor MCC950 (10 mg/kg body weight, Cat. #CP-456773, Selleck) was intraperitoneally injected during the final 6 weeks of the experiment.

Techniques: Expressing, Immunostaining, Staining, Quantitative RT-PCR, MANN-WHITNEY

Journal: Cancer Immunology Research

Article Title: A. Muciniphila Suppresses Colorectal Tumorigenesis by Inducing TLR2/NLRP3-Mediated M1-Like TAMs

doi: 10.1158/2326-6066.cir-20-1019

Figure Lengend Snippet: Figure 7. A. muciniphila regulation of colorectal cancer progression. Resident A. muciniphila specifically activates native macro- phage into M1-like TAMs that release IL1b to suppress colorectal cancer progression in the tumor microenviron- ment. TLR2, as the macrophage pattern recognition receptor to A. muciniphila, activates the NF-kB/NLRP3 pathway and stimulates the acquisition of the M1 macrophage phenotype.

Article Snippet: To suppress NLRP3 in ApcMin/þ mice, the NLRP3 inhibitor MCC950 (10 mg/kg body weight, Cat. #CP-456773, Selleck) was intraperitoneally injected during the final 6 weeks of the experiment.

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: Cell reports

Article Title: L-plastin enhances NLRP3 inflammasome assembly and bleomycin-induced lung fibrosis

doi: 10.1016/j.celrep.2022.110507

Figure Lengend Snippet: (A) LPS-primed or nigericin-activated NLRP3-stimulated BMDM cell lysates were subjected to Pyk2 immunoprecipitation (IP) using a specific antibody. Pulled down Pyk2 and co-precipitated ASC and NLRP3 were probed by immunoblotting. Here, initial cell lysate is used as an input control, and immunoglobulin G (IgG) isotype is used as a negative control for anti-Pyk2 IP. Results representing 2 independent experiments are shown.. (B and C) BMDMs were cultured on cover glass in serum-free medium for 1.5 h prior to serum addition to induce podosome formation (B, green dots). Cells were stained for F-actin with phalloidin-AF488 (green) and anti-Pyk2-IgG-DyLight594 (red). A 60× image (scale bars, 10μm) is shown here, with the insets showing podosomes (arrows; scale bars, 5 μm). Results represent 2 independent experiments. In (C), WT BMDMs were primed with LPS and stimulated for NLRP3 activation by ATP (30 min) in the absence and presence of z-VAD-fmk (50 μM, 1 h), MCC-950 (50 μM, 1 h), and PF-431396 (25 μM) for 1 h or 4.5 h. Cell lysates were probed for Pyk2 phosphorylation and IL-1β products by immunoblotting. The density of each band is shown below. Here, β-actin is the total cell control.. (D) WT and LPL −/− BMDMs were LPS primed and activated with ATP (30 min) in the presence of PF-431396 (25 μM, 1 h), and cell lysates were analyzed by immunoblotting for Pyk2 phosphorylation and IL-1β processing. Here, NLRP3 is the priming control, and density is shown below each band. (C and D) The results represent 2 independent experiments.. (E) Unstimulated (US), LPS-primed, and NLRP3-activated (nigericin) WT and LPL −/− BMDM cell lysates were analyzed for phosphorylated (Tyr-402) Pyk2 and processed gasdermin-D and IL-1β (cleaved). Here, NLRP3 expression, showing priming signal and β-actin, serves as loading control. Densitometry values are shown below each protein band. The control immunoblot for LPL is derived from a separate immunoblot run (not normalized) of the same lysates. A representative immunoblot from 3 independent experiments is shown.. (F) The ratio of phosphorylated Pyk2 (Tyr-402) to total Pyk2 is measured by densitometry from multiple experiments. Conditions include US, LPS priming, and after NLRP3 activation (nigericin or ATP). Here, each dot represents an individual data point, showing median (solid line) and interquartile range (dashed line). Data points (n = 3) are from , , and and replicate independent experiments (data not shown). Statistical significance was measured by Mann-Whitney test.

Article Snippet: Pharmacological inhibitors e.g., Pan-caspase-1 inhibitor; Z-vad-fmk (Enzo LifeSciences; Cat# BML-P416-000), NLRP3 inhibitor; MCC-950 (Selleckchem; Cat#S8930) and Pyk2 inhibitor; PF-431396 (Sigma-Aldrich; Cat#PZ0185) were added 30 min before NLRP3 activation.

Techniques: Immunoprecipitation, Western Blot, Control, Negative Control, Cell Culture, Staining, Activation Assay, Phospho-proteomics, Expressing, Derivative Assay, MANN-WHITNEY

Journal: Cell reports

Article Title: L-plastin enhances NLRP3 inflammasome assembly and bleomycin-induced lung fibrosis

doi: 10.1016/j.celrep.2022.110507

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Pharmacological inhibitors e.g., Pan-caspase-1 inhibitor; Z-vad-fmk (Enzo LifeSciences; Cat# BML-P416-000), NLRP3 inhibitor; MCC-950 (Selleckchem; Cat#S8930) and Pyk2 inhibitor; PF-431396 (Sigma-Aldrich; Cat#PZ0185) were added 30 min before NLRP3 activation.

Techniques: Virus, Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Software