Journal: International Journal of Molecular Sciences

Article Title: Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation

doi: 10.3390/ijms24065653

Figure Lengend Snippet: CO inhibits ASC speck formation during AIM2 inflammasome activation. ( A – D ) LPS (100 ng/mL)-primed BMDMs were pre-treated with CO for 30 min, then transfected with poly(dA:dT) (1 μg/mL) for 2 h. Western blots of ( A ) ASC translocation from Triton X-100 soluble fraction to insoluble fraction, ( B ) ASC oligomerization in the insoluble fraction (pellet), and ( C ) ASC speck images at 400× magnification upon stimulation with poly(dA:dT). ASC and nuclei are depicted in red and blue (DAPI), respectively. White arrowheads point to ASC speck. ( D ) Quantification of cells with ASC specks showed the percentage of positive cells. At least three fields and more than 100 cells were counted for each condition. Data represent the means ± SDs of three independent experiments; ** p < 0.01.

Article Snippet: The following reagents and materials were purchased for the study: LPS (L2630), from Pierce Chemical (Rockford, IL, USA); nigericin (tlrl-nig), nano-SiO2 (tlrl-sol), poly(deoxyadenylic-deoxythymidylic) acid (poly(dA:dT), tlrl-patn), Pam3CSK4 (tlrl-pm2s-1), lipofectamine 2000 (11668019), YVAD-CMK (Inh-yvad), and a mouse IL-1 beta ELISA kit (88-7013-88), from Invivogen (San Diego, CA, USA); a human IL-1 beta ELISA kit (DY201), from R&D systems (Minneapolis, MN, USA); flagellin (AG-40B-0095), from Adipogen International (San Diego, CA, USA); ATP (A7699), thiazolyl blue tetrazolium bromide (M5655), DMSO (D2660), KCl (P5405), and glycine (G7126), from Sigma-Aldrich (St. Louis, MO, USA); disuccinimidyl suberate (DSS, #21555) and a BCA assay kit (#23225), from Thermo Fisher Scientific (Walthan, MA, USA); RIPA lysis buffer (#89900), from iNtRON (Seoul, Republic of Korea); protease inhibitor (P3100-005), from GenDEPOT (Katy, TX, USA); RPMI1640 (11875119), DMEM (11995073), Opti-MEM (31985088), fetal bovine serum (16000-044), antibiotic-antimitotic (15240062), and trypsin/EDTA (15400-054), from Gibco (Grand Island, NY, USA); TNF-alpha (315-01A) and human interferon gamma (300-02), from PeproTech (Rocky Hil, NJ, USA); antibodies against ASC (AL177) and caspase-1 (p20) (AG-20B-0042), from Adipogen International (San Diego, CA, USA); antibodies against mouse IL-1 beta/IL-1F2 (AF401-NA) and NLRP3 (Cryo2), from R&D systems (Minneapolis, MN, USA); antibodies against β-actin (sc-1616), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against AIM2 (63660S) and GAPDH (sc-365062), from Cell Signaling Technology (Beverly, MA, USA); antibodies against FLAG (F1804), from Sigma-Aldrich (St. Louise, MO, USA); an LDH assay kit (DG-LDH1000) and a Western blot chemiluminescence reagent kit (EZ-Western Lumi Pico Kit, DG-WP250), from DoGenBio (Seoul, Republic of Korea); Nitrocellulose membranes (HATE00010), from Millipore Corporation (Bedford, MA, USA); and an ELISA MAXTM Deluxe Set Mouse IL-17A kit (432504), from BioLegend (San Diego, CA, USA).

Techniques: Activation Assay, Transfection, Western Blot, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation

doi: 10.3390/ijms24065653

Figure Lengend Snippet: CO inhibits AIM2 speck formation resulting from poly(dA:dT) stimulation. ( A , B ) HEK293T cells were transfected with a FLAG-tagged AIM2 expression vector 24 h prior to the experiment. Cells were pre-treated with CO (10 μg/mL) for 30 min, then stimulated with poly(dA:dT) (1 μg/mL) for 4 h. ( A ) AIM2 speck images at 400× magnification upon stimulation with poly(dA:dT) in cells expressing FLAG-tagged AIM2. AIM2 and nuclei are depicted in red and blue (DAPI), respectively. White arrowheads point to AIM2 speck. ( B ) Statistical analysis of the AIM2 specks showing the percentage of positive cells. At least three fields and more than 100 cells were counted for each condition. Data represent the means ± SDs of three independent experiments; *** p < 0.001.

Article Snippet: The following reagents and materials were purchased for the study: LPS (L2630), from Pierce Chemical (Rockford, IL, USA); nigericin (tlrl-nig), nano-SiO2 (tlrl-sol), poly(deoxyadenylic-deoxythymidylic) acid (poly(dA:dT), tlrl-patn), Pam3CSK4 (tlrl-pm2s-1), lipofectamine 2000 (11668019), YVAD-CMK (Inh-yvad), and a mouse IL-1 beta ELISA kit (88-7013-88), from Invivogen (San Diego, CA, USA); a human IL-1 beta ELISA kit (DY201), from R&D systems (Minneapolis, MN, USA); flagellin (AG-40B-0095), from Adipogen International (San Diego, CA, USA); ATP (A7699), thiazolyl blue tetrazolium bromide (M5655), DMSO (D2660), KCl (P5405), and glycine (G7126), from Sigma-Aldrich (St. Louis, MO, USA); disuccinimidyl suberate (DSS, #21555) and a BCA assay kit (#23225), from Thermo Fisher Scientific (Walthan, MA, USA); RIPA lysis buffer (#89900), from iNtRON (Seoul, Republic of Korea); protease inhibitor (P3100-005), from GenDEPOT (Katy, TX, USA); RPMI1640 (11875119), DMEM (11995073), Opti-MEM (31985088), fetal bovine serum (16000-044), antibiotic-antimitotic (15240062), and trypsin/EDTA (15400-054), from Gibco (Grand Island, NY, USA); TNF-alpha (315-01A) and human interferon gamma (300-02), from PeproTech (Rocky Hil, NJ, USA); antibodies against ASC (AL177) and caspase-1 (p20) (AG-20B-0042), from Adipogen International (San Diego, CA, USA); antibodies against mouse IL-1 beta/IL-1F2 (AF401-NA) and NLRP3 (Cryo2), from R&D systems (Minneapolis, MN, USA); antibodies against β-actin (sc-1616), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against AIM2 (63660S) and GAPDH (sc-365062), from Cell Signaling Technology (Beverly, MA, USA); antibodies against FLAG (F1804), from Sigma-Aldrich (St. Louise, MO, USA); an LDH assay kit (DG-LDH1000) and a Western blot chemiluminescence reagent kit (EZ-Western Lumi Pico Kit, DG-WP250), from DoGenBio (Seoul, Republic of Korea); Nitrocellulose membranes (HATE00010), from Millipore Corporation (Bedford, MA, USA); and an ELISA MAXTM Deluxe Set Mouse IL-17A kit (432504), from BioLegend (San Diego, CA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation

doi: 10.3390/ijms24065653

Figure Lengend Snippet: CO reduces IMQ-induced serum IL-17A elevation and the expression of AIM2 inflammasome components in skin tissue. CO and Aldara cream were applied as described in C to obtain serum and prepare skin tissue extracts. ( A ) Serum IL-17A levels measured by an ELISA assay. Data represent the means ± SDs; *** p < 0.001. ( B ) Western blot analysis for the levels of AIM2 inflammasome components in skin extracts.

Article Snippet: The following reagents and materials were purchased for the study: LPS (L2630), from Pierce Chemical (Rockford, IL, USA); nigericin (tlrl-nig), nano-SiO2 (tlrl-sol), poly(deoxyadenylic-deoxythymidylic) acid (poly(dA:dT), tlrl-patn), Pam3CSK4 (tlrl-pm2s-1), lipofectamine 2000 (11668019), YVAD-CMK (Inh-yvad), and a mouse IL-1 beta ELISA kit (88-7013-88), from Invivogen (San Diego, CA, USA); a human IL-1 beta ELISA kit (DY201), from R&D systems (Minneapolis, MN, USA); flagellin (AG-40B-0095), from Adipogen International (San Diego, CA, USA); ATP (A7699), thiazolyl blue tetrazolium bromide (M5655), DMSO (D2660), KCl (P5405), and glycine (G7126), from Sigma-Aldrich (St. Louis, MO, USA); disuccinimidyl suberate (DSS, #21555) and a BCA assay kit (#23225), from Thermo Fisher Scientific (Walthan, MA, USA); RIPA lysis buffer (#89900), from iNtRON (Seoul, Republic of Korea); protease inhibitor (P3100-005), from GenDEPOT (Katy, TX, USA); RPMI1640 (11875119), DMEM (11995073), Opti-MEM (31985088), fetal bovine serum (16000-044), antibiotic-antimitotic (15240062), and trypsin/EDTA (15400-054), from Gibco (Grand Island, NY, USA); TNF-alpha (315-01A) and human interferon gamma (300-02), from PeproTech (Rocky Hil, NJ, USA); antibodies against ASC (AL177) and caspase-1 (p20) (AG-20B-0042), from Adipogen International (San Diego, CA, USA); antibodies against mouse IL-1 beta/IL-1F2 (AF401-NA) and NLRP3 (Cryo2), from R&D systems (Minneapolis, MN, USA); antibodies against β-actin (sc-1616), from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against AIM2 (63660S) and GAPDH (sc-365062), from Cell Signaling Technology (Beverly, MA, USA); antibodies against FLAG (F1804), from Sigma-Aldrich (St. Louise, MO, USA); an LDH assay kit (DG-LDH1000) and a Western blot chemiluminescence reagent kit (EZ-Western Lumi Pico Kit, DG-WP250), from DoGenBio (Seoul, Republic of Korea); Nitrocellulose membranes (HATE00010), from Millipore Corporation (Bedford, MA, USA); and an ELISA MAXTM Deluxe Set Mouse IL-17A kit (432504), from BioLegend (San Diego, CA, USA).

Techniques: Expressing, Cream, Enzyme-linked Immunosorbent Assay, Western Blot