Journal: Cell Discovery

Article Title: Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

doi: 10.1038/s41421-021-00259-0

Figure Lengend Snippet: a Schematic representation of hPANX1 heptamer in light blue docked into the cryo-EM density map. The middle panel shows the top view of hPANX1 with glycosylation indicated. The right panel shows a magnified view of the N-linked glycans (including two N-Acetylglucosamine and eight Mannose). b Representative images of the localization of WT and N255Q hPANX1 in THP-1-derived macrophages (MPs). The images were collected using the super-resolution STED mode. OE is the abbreviation of overexpression in all the figures. Scale bar = 5 µm. c Western blot analysis of the NLRP3 and Pro-IL-1β in WT hPANX1 overexpressing and N255Q hPANX1 overexpressing MPs. MPs were treated with LPS (1 µg/ml) and ATP (3 mM) for 24 h. GAPDH was used as a loading control. d IL-1β release from mock, WT hPANX1 overexpressing and N255Q hPANX1 overexpressing MPs. MPs were primed with LPS (1 µg/ml) overnight and stimulated with nigericin (10 µM) for 0.5, 1, or 2 h. IL-1β release was measured by ELISA. Error bars represent s.e.m. for n = 3. * P < 0.05, NS not significant (one-way ANOVA followed by Dunnett’s test, OE WT set as comparison control). e Phase-contrast images of MPs stimulated by LPS and ATP. Images were acquired with an IncusyteS3 system on day 6. f Time course of live cell confluence of MPs. Image data were collected every 2 h and analyzed with an IncusyteS3 system. Error bars represent s.d. for n = 4. *** P < 0.001, **** P < 0.0001 (one-way ANOVA followed by Dunnett’s test, OE WT set as comparison control).

Article Snippet: To differentiate THP-1 cells into macrophages (MPs), 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, P1585–1MG) was added to the growth medium and maintained for 24 or 48 h. THP-1 derived MPs were stimulated for the indicated time with 30 μM R848, pHrodo E. coli bioparticles (Life Technologies, P35361), 1 μg/ml LPS (Novus Biologicals, NBP2-25295), R848 plus bioparticles, LPS plus 3 mM ATP (Sigma-Aldrich, A2383-5G), or primed with LPS and stimulated with 10 μM nigericin (MERCK Sigma-Aldrich, 481990).

Techniques: Cryo-EM Sample Prep, Glycoproteomics, Derivative Assay, Over Expression, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Cell Discovery

Article Title: Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis

doi: 10.1038/s41421-021-00259-0

Figure Lengend Snippet: a Representative images of the localization of WT and mutant hPANX1 (D379A and S424A) in MPs. The images were collected using the super-resolution STED mode. Scale bar = 5 µm. b Western blot analysis of the NLRP3, Pro-IL-1β in WT and mutant (D379A and S424A) hPANX1 overexpressing MPs. MPs were treated with LPS (1 µg/ml) and ATP (3 mM) for 24 h. GAPDH was used as a loading control. c IL-1β release from mock, WT overexpressing and mutant (D379A and S424A) overexpressing MPs. MPs were primed with LPS (1 µg/ml) overnight and stimulated with nigericin (10 µM) for 0.5, 1, or 2 h. IL-1β release was measured by ELISA. Error bars represent s.e.m. for n = 3. * P < 0.05, ** P < 0.01, NS not significant (one-way ANOVA followed by Dunnett’s test, OE WT set as comparison control). d Phase-contrast images of MPs stimulated by LPS and ATP. Images were acquired with an IncusyteS3 system on day 6. e Time course of live cell confluence of MPs. Image data were collected every 2 h and analyzed with an IncusyteS3 system. Error bars represent s.d. for n = 4. *** P < 0.001, **** P < 0.0001 (one-way ANOVA followed by Dunnett’s test, OE WT set as comparison control).

Article Snippet: To differentiate THP-1 cells into macrophages (MPs), 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, P1585–1MG) was added to the growth medium and maintained for 24 or 48 h. THP-1 derived MPs were stimulated for the indicated time with 30 μM R848, pHrodo E. coli bioparticles (Life Technologies, P35361), 1 μg/ml LPS (Novus Biologicals, NBP2-25295), R848 plus bioparticles, LPS plus 3 mM ATP (Sigma-Aldrich, A2383-5G), or primed with LPS and stimulated with 10 μM nigericin (MERCK Sigma-Aldrich, 481990).

Techniques: Mutagenesis, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Altered Intestinal Morphology and Microbiota Composition in the Autism Spectrum Disorders Associated SHANK3 Mouse Model

doi: 10.3390/ijms20092134

Figure Lengend Snippet: Altered gut morphology in Shank3αβ knock-out (KO) mice. ( A – D ) Histological evaluation of GI tract from wild type and Shank3αβ KO mice. ( A ) Longitudinal cross sections of Shank3αβ KO mice and wild type (WT) mice were stained with hematoxylin/eosin (HE) (upper panels) and periodic acid schiff (PAS) reaction (lower panels). Exemplary images are shown. ( B – D ) Morphological analysis of ( B ) villi length and ( C ) width, and ( D ) crypt depth reveals a significantly decreased villi length (Mann-Whitney U -test, p = 0.009; n = 5 animals per group) but not width ( p = 0.534), and normal crypt depth ( p = 0.983) in Shank3αβ KO mice. ( E , F ) Immunohistochemistry was performed on 5 mice per group and 5 optic fields of view each from 3 sections per mouse were analyzed. ( E ) A slight but non-significant decrease in FABP2 signal intensity was observed in Shank3αβ KO mice compared to wild types (left panel). Significantly higher ZONULIN-1 levels were found in Shank3αβ KO mice (right panel) ( t -test, p = 0.0413). ( F ) The levels of CLAUDIN3 and lipopolysaccharide (LPS) were not significantly different between Shank3αβ KO mice and wild types in gut epithelium. ( G ) Significantly higher ZONULIN-1 levels in Shank3αβ KO mice were confirmed by western blotting using gut epithelium protein lysate ( t -test, p = 0.0434, n = 3 per group). ( H ) Protein lysate from liver tissue from WT and Shank3αβ KO mice ( n = 3 per group) were analyzed for E. coli LPS levels using Western Blotting. The results show significantly higher LPS levels in the liver of Shank3αβ β KO mice ( t -test, p = 0.0452). * p < 0.05, ** p < 0.01.

Article Snippet: Zonulin 1 antibody was purchased from Thermo Fisher Scientific (Invitrogen) (Waltham, MA, USA); Claudin3 antibody from Abcam (Berlin, Germany); FABP2 antibody from Thermo Fisher Scientific (Invitrogen); LPS antibody from Origene (Rockville, MD, USA); and IL6 antibody was purchased from Cell signaling Technologies (Danvers, MA, USA); GFAP antibody was purchased from Sigma Aldrich (St. Louis, MO, USA); Cytokeratin and Vimentin antibody from Abcam.

Techniques: Knock-Out, Staining, MANN-WHITNEY, Immunohistochemistry, Western Blot