Journal: International Journal of Molecular Sciences

Article Title: ZnT2 Is Critical for TLR4-Mediated Cytokine Expression in Colonocytes and Modulates Mucosal Inflammation in Mice

doi: 10.3390/ijms231911467

Figure Lengend Snippet: Infection with Citrobacter rodentium caused inflammation and oxidative stress in wild-type (WT) but not ZnT2-null (KO) mice. Data represent mean fold-change in TNFα ( A ), IL17 ( B ), IL22 ( C ) and TGFβ ( D ) and iNOS ( E ) mRNA levels normalized to b-actin relative to WT mice pre-infection ± SD in WT and KO mice pre-infection (d0), and 10 (d10) and 21 (d21) post-infection; n = 5 mice/genotype; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( F ) Number of 8OHdG + cells/crypt ± SD in WT and KO mice pre-infection (d0), and 10 (d10) and 21 (d21) post-infection; n = 30 crypts from 3 mice/genotype; ** p < 0.01, **** p < 0.0001. ( G ) Number of neutrophils/20× field of view ± SD in WT and KO mice pre-infection (d0), and 10 (d10) and 21 (d21) post-infection; n = 2–3 fields of view from 3 mice/genotype; * p < 0.05, ** p < 0.01. ns: not significant.

Article Snippet: Where indicated, membranes were stripped and re-probed using rabbit anti-βactin (1:10,000; ThermoFisher, Waltham, MA, USA, MA5-15379) or anti-TATA binding protein (TBP) antibody (1:1000; Cell Signaling Technologies, Danvers, MA, USA 44059) as total protein and nuclear loading controls, respectively.

Techniques: Infection

Journal: International Journal of Molecular Sciences

Article Title: ZnT2 Is Critical for TLR4-Mediated Cytokine Expression in Colonocytes and Modulates Mucosal Inflammation in Mice

doi: 10.3390/ijms231911467

Figure Lengend Snippet: ZnT2 is vital for expression of Toll-like receptor 4 (TLR4) and pathogen-stimulated expression of TNFα and IL8 in the colon. ( A ) Data represent mean fold-change in TLR4 mRNA levels normalized to β-actin relative to WT mice ± SD; n = 5 mice/genotype; * p < 0.05. ( B ) Representative images of TLR4 expression (green) and DAPI (blue) in the colon of WT (i) and KO (ii) mice. Magnification ×20; scale bars = 50 µm. ( C ) Data represent mean fold-change in TLR4 mRNA levels normalized to β-actin relative to Mock transfected HT29 cells ± SD in ZnT2-expressing (Mock) and ZnT2-attenuated (ZnT2KD) HT29 cells under control conditions or treated with TPEN; n = 6 samples/genotype, experiment was repeated twice; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ( D ) ZnT2-expressing (Mock) and ZnT2-attenuated (ZnT2KD) HT29 cells were treated with LPS (1 µg/mL for 6 h) or left untreated. Data represent mean fold-change in TNFα and IL-8 mRNA levels normalized to β-actin relative to Mock transfected untreated (control) HT29 cells ± SD; n = 6 samples/genotype, experiment was repeated twice; * p < 0.05, ** p < 0.01. ( E ) Representative immunoblot of NF-κβ in the nucleus of Mock-transfected (Control) or ZnT2-attenuated (ZnT2KD) HT29 cells in response to LPS stimulation over 60 min. Membranes were stripped and reprobed for TBP as a normalization control.

Article Snippet: Where indicated, membranes were stripped and re-probed using rabbit anti-βactin (1:10,000; ThermoFisher, Waltham, MA, USA, MA5-15379) or anti-TATA binding protein (TBP) antibody (1:1000; Cell Signaling Technologies, Danvers, MA, USA 44059) as total protein and nuclear loading controls, respectively.

Techniques: Expressing, Transfection, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: ZnT2 Is Critical for TLR4-Mediated Cytokine Expression in Colonocytes and Modulates Mucosal Inflammation in Mice

doi: 10.3390/ijms231911467

Figure Lengend Snippet: Loss of ZnT2 is associated with fewer and smaller lysosomes but does not affect basal autophagy in HT29 cells. ( A ) Representative image of Lysotracker Red (red) in ZnT2-expressing (Mock) and ZnT2-attenuated (ZnT2KD) HT29 cells. Magnification ×20; scale bar = 25 µm. ( B ) Data represent mean number of lysosomes/cell ± SD; n = 25 cells/genotype, experiment was repeated twice; *** p < 0.001. ( C ) Data represent mean lysosome area (µm 2 ) ± SD; n = 100 lysosomes/genotype, experiment was repeated three times; **** p < 0.0001. ( D ) Representative immunoblot of LC3 and p62 in ZnT2-expressing (Mock) or ZnT2-attenuated (ZnT2KD) HT29 cells. Membranes immunoblotted for p62 were stripped and reprobed for β-actin as a normalization control. ( E ) Data represent mean ratio of LC3II/LC3I ± SD; n = 6 samples/genotype, experiment was repeated twice; **** p < 0.0001. ( F ) Data represent mean p62 abundance normalized to β-actin ± SD; n = 6 samples/genotype, experiment was repeated twice; ** p < 0.01.

Article Snippet: Where indicated, membranes were stripped and re-probed using rabbit anti-βactin (1:10,000; ThermoFisher, Waltham, MA, USA, MA5-15379) or anti-TATA binding protein (TBP) antibody (1:1000; Cell Signaling Technologies, Danvers, MA, USA 44059) as total protein and nuclear loading controls, respectively.

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: ZnT2 Is Critical for TLR4-Mediated Cytokine Expression in Colonocytes and Modulates Mucosal Inflammation in Mice

doi: 10.3390/ijms231911467

Figure Lengend Snippet: ZnT2 is vital for lysosome biogenesis and the activation of autophagy in HT29 cells. ( A ) Representative images of Lysotracker Red (red) in ZnT2-expressing (Mock) and ZnT2-attenuated (ZnT2KD) HT29 cells treated with LPS (+LPS; 1 µg/mL for 6 h) or left untreated (−LPS). Magnification ×20; scale bar = 25 µm. ( B ) Data represent mean number of lysosomes/cell ± SD in Mock or ZnT2KD cells treated with LPS or left untreated; n = 20cells/genotype, experiment was repeated four times; * p < 0.05, ** p < 0.01, **** p < 0.0001. ( C ) Data represent mean lysosome area (µm 2 ) ± SD in Mock or ZnT2KD cells treated with LPS or left untreated; n = 100 lysosomes/group, experiment was repeated four times; * p < 0.05, **** p < 0.0001. ( D ) Representative immunoblot of LC3 in ZnT2-expressing (Mock) or ZnT2-attenuated (ZnT2KD) HT29 cells treated with LPS (+) or left untreated (−). Membranes immunoblotted for p62 were stripped and reprobed for β-actin as a normalization control. ( E ) Data represent mean ratio of LC3II/LC3I ± SD; n = 6 samples/genotype, experiment was repeated twice; * p < 0.05, ** p < 0.01, **** p < 0.0001. ( F ) Representative immunoblot of p62 in ZnT2-expressing (Mock) or ZnT2-attenuated (ZnT2KD) HT29 cells treated with LPS (+) or left untreated (−). Membranes were stripped and reprobed for β-actin as a normalization control. ( G ) Data represent mean p62 abundance normalized to β-actin ± SD; n = 12–18 samples/genotype, experiment was repeated four times; ** p < 0.01, *** p < 0.001. ns: not significant.

Article Snippet: Where indicated, membranes were stripped and re-probed using rabbit anti-βactin (1:10,000; ThermoFisher, Waltham, MA, USA, MA5-15379) or anti-TATA binding protein (TBP) antibody (1:1000; Cell Signaling Technologies, Danvers, MA, USA 44059) as total protein and nuclear loading controls, respectively.

Techniques: Activation Assay, Expressing, Western Blot, Control