Journal: Cells

Article Title: Calcineurin B1 Deficiency Reduces Proliferation, Increases Apoptosis, and Alters Secretion in Enteric Glial Cells of Mouse Small Intestine in Culture

doi: 10.3390/cells12141867

Figure Lengend Snippet: Reduced proliferation of CNB1 -deficient EGCs. ( A – F ) Immunocytochemistry against BrdU (white), GFAP (EGC marker; green), and αSMA (fibroblast marker; red) and nuclear staining with DAPI (blue) of primary myenteric cultures from the small intestine of control ( A , C , E ) and CNB1 -CKO ( B , D , F ) mice. The cultures were treated with 20 μM BrdU for 18 h before fixation and fixed at 3 ( A , B ), 6 ( C , D ), and 10 ( E , F ) days in vitro (DIV). Arrows indicate proliferative fibroblasts (αSMA+ and BrdU+ cells), which were excluded from the analysis. Scale bar in A1 (applies to all images) = 50 µm. ( G –I) Ratios of proliferative EGCs (GFAP+ and BrdU+ cells to GFAP+ cells) in primary myenteric cultures from the small intestine of control and CNB1 -CKO mice at 3 ( G ), 6 ( H ), and 10 (I) DIV. n = 125 (( G ), control), 103 (( G ), CKO), 106 (( H ), control), 116 (( H ), CKO), 112 (( I ), control), and 99 (( I ), CKO) images from three independent experiments. Error bars represent SEM. The ratio was significantly lower in CNB1 -deficient EGCs than in control EGCs at 3 DIV (G; * p < 0.05, two-tailed unpaired t -test).

Article Snippet: CNB1 -floxed (#006581) and GFAP - Cre (#004600) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Immunocytochemistry, Marker, Staining, Control, In Vitro, Two Tailed Test

Journal: Cells

Article Title: Calcineurin B1 Deficiency Reduces Proliferation, Increases Apoptosis, and Alters Secretion in Enteric Glial Cells of Mouse Small Intestine in Culture

doi: 10.3390/cells12141867

Figure Lengend Snippet: Increased apoptosis of CNB1 -deficient EGCs. ( A – F ) Live staining with Annexin V-FITC (green), PI (red), and Hoechst 33342 (blue) and immunocytochemistry against GFAP (white) of primary myenteric cultures from the small intestine of control ( A , C , E ) and CNB1 -CKO ( B , D , F ) mice. The cultures were stained with Annexin V-FITC, PI, and Hoechst 33342 as a cell death assay at 3 ( A , B ), 6 ( C , D ), and 10 ( E , F ) DIV. Immediately after fluorescent images were acquired, they were fixed and processed for immunocytochemistry against GFAP, and the same optical fields were imaged again. Arrows indicate late apoptotic/necrotic EGCs (GFAP+, Annexin V+, PI+ cells). Scale bar in A1 (applies to all images) = 50 µm. ( G – L ) Ratios of early apoptotic EGCs (GFAP+, Annexin V+, PI- cells to GFAP+ cells) and late apoptotic/necrotic EGCs (GFAP+, Annexin V+, PI+ cells to GFAP+ cells) in primary myenteric cultures from the small intestine of control and CNB1 -CKO mice at 3 ( G , H ), 6 ( I , J ), and 10 ( K , L ) DIV. n = 110 (( G , H ); control), 109 (( G , H ); CKO), 82 (( I , J ); control), 97 (( I , J ); CKO), 75 (( K , L ); control), and 82 (( K , L ); CKO) images from three independent experiments. Error bars represent SEM. Ratios of early apoptotic EGCs were significantly higher in CNB1 -deficient EGCs than in control EGCs at 3 and 6 DIV (G, I; * p < 0.05, two-tailed unpaired t -test).

Article Snippet: CNB1 -floxed (#006581) and GFAP - Cre (#004600) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Staining, Immunocytochemistry, Control, Two Tailed Test

Journal: Cells

Article Title: Calcineurin B1 Deficiency Reduces Proliferation, Increases Apoptosis, and Alters Secretion in Enteric Glial Cells of Mouse Small Intestine in Culture

doi: 10.3390/cells12141867

Figure Lengend Snippet: Increases in expression of S100B, iNOS, and GFAP and phosphorylation of NF-κB p65 in CNB1 -deficient EGCs. ( A , C , D ) Representative Western blots of S100B ( A ), iNOS ( C ), GFAP ( D ), and β-actin ( A , C , D ) and quantification of the band intensity of S100B ( A ), iNOS ( C ), and GFAP ( D ) for purified EGC cultures from the small intestine of control and CNB1 -CKO mice. For quantification, the band intensity of each protein was normalized by that of β-actin and is shown as the ratio relative to the control. n = 8 ( A ), 3 ( C ), and 11 ( D ) pairs of control and CNB1 -deficient EGCs. Error bars represent SEM. The band intensities of S100B ( A ), iNOS ( C ), and GFAP ( D ) were significantly stronger for CNB1 -deficient EGCs than for control EGCs (* p < 0.05, two-tailed paired t -test). ( B ) Representative Western blots of NF-κB p65 and phospho-NF-κB p65 and quantification of the band intensity of phospho-NF-κB p65/NF-κB p65 for purified EGC cultures from the small intestine of control and CNB1 -CKO mice. For quantification, the band intensity of phospho-NF-κB p65 was normalized by that of NF-κB p65 and is shown as the ratio relative to the control. n = 5 pairs of control and CNB1 -deficient EGCs. Error bars represent SEM. The band intensity of phospho-NF-κB p65/NF-κB p65 was significantly stronger for CNB1 -deficient EGCs than for control EGCs (* p < 0.05, two-tailed paired t -test).

Article Snippet: CNB1 -floxed (#006581) and GFAP - Cre (#004600) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Purification, Control, Two Tailed Test