Journal: Frontiers in Physiology

Article Title: Impact of KvLQT1 potassium channel modulation on alveolar fluid homeostasis in an animal model of thiourea-induced lung edema

doi: 10.3389/fphys.2022.1069466

Figure Lengend Snippet: Impact of KvLQT1 activation on alveolar type I (AT1) and II (ATII) epithelial cell markers in thiourea-challenged WT mice. Representative immunofluorescence images (left panels) of lung sections (5 μm, Scale: 100 μm) from WT control mice (PBS/PBS), and WT mice challenged with thiourea (TU, 5 mg/kg i.p., PBS/TU) and treated or not (PBS) with the KvLQT1 activator R-L3 (R-L3/TU) stained with the ATI markers podoplanin (A) , n = 50 images, AQP5 (B) , n = 59–60 or ATII marker pro-SPC (C) , n = 48–51). Nuclei were stained by DAPI. Quantification (right panels) of podoplanin, AQP5, and pro-SPC marker intensities was made with a protocol exploited by ICY software. Values are presented as means ± SEM. Non-parametric Mann-Whitney t-test (Agostino/Pearson normality test: negative) for panel (A) . One-way ANOVA non-parametric comparison test (normality Agostino/Pearson test: negative) for panel (B , C) . * p < .05, ** p < .01 or **** p < .0001 vs . PBS/PBS, ## p < .01 vs . PBS/TU.

Article Snippet: For AQP5 detection by immunostaining, tissue sections were processed for heat-induced antigenic retrieval (with citrate buffer, pH 3.45) and then blocked with a solution of PBS +10% FBS (Saradigm, United States) + 10% BSA (Sigma-Aldrich) + .01% Triton X-100 (Amersham Biosciences, Sweden) for 1 h. Slides were then incubated overnight at 4°C with an anti-AQP5 rabbit polyclonal antibody (1:100, #AQP-005, Alomone Labs, Israël).

Techniques: Activation Assay, Immunofluorescence, Staining, Marker, Software, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands

doi: 10.3390/ijms22020619

Figure Lengend Snippet: Spatiotemporal expression of CXC-chemokine receptor 4 (CXCR4) and its ligand, CXCL12, and developmental genes in embryonic organs. ( A ) Schematic diagram of embryonic submandibular gland (eSMG) isolation and ex vivo culture. ( B ) Ex vivo branching morphogenesis of eSMGs from embryonic day (E) 13 to 17, showing epithelial growth and retraction of mesenchyme. Scale bars: 500 µm. ( C ) Temporal mRNA expression patterns of keratin 7 ( Krt7 ), aquaporin 5 ( Aqp5 ), e-cadherin ( Cdh1 ), Krt15 , Cxcr4 , and Cxcl12 were measured from E13 to E17 by qPCR ( n = 3). ( D ) Epithelial (Epi) and mesenchymal (Mes) expression of Cxcr4 , Cxcl12 , odd-skipped related transcription factor 1 ( Osr1 ), and Cdh1 were quantified by qPCR at E13. The comparative C t values are expressed as fold increase relative to the epithelium ( n = 3). ( E ) Representative images showing expression of CXCR4 and CXCL12 in eSMG (upper) and their colocalization (lower) ( n = 3, scale bar: 500 µm). ( F ) Representative immunofluorescence images of CXCR4 and CXCL12 expression in E12 embryonic lung and pancreas ( n = 4); whole view (left two panels; scale bar: 500 µm) and magnified lumen structures (right two panels; scale bar: 50 µm). Data are presented as the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS: not significant; two-way ANOVA ( C ); t -test ( D ).

Article Snippet: The following primary antibodies were used in the procedures: rat monoclonal anti-CXCR4 antibody (R&D Systems, MAB21651), rabbit monoclonal anti-KRT7 antibody (Abcam, ab181598; Cambridge, UK); rabbit monoclonal anti-CDH1 antibody (CST, 3195; Beverly, MA, USA), rabbit monoclonal anti-H2AK119ub antibody (CST, 8240); mouse monoclonal anti-CXCL12 antibody (Novus Biologicals, MAB350; Littleton, CO, USA), rabbit polyclonal anti-AQP5 antibody (Alomone Labs, AQP-005; Jerusalem, Israel), rabbit monoclonal anti-CASP3 antibody (CST, 9664), and rat monoclonal anti-KI67 antibody (ThermoFisher, 14-5698-82).

Techniques: Expressing, Isolation, Ex Vivo, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands

doi: 10.3390/ijms22020619

Figure Lengend Snippet: AMD3100-induced precocious differentiation of epithelial cells. ( A , B ) Representative contour tracing ( A ) and bud number changes ( B ) of control and AMD3100-treated eSMGs during 48 h at 6-h intervals ( n = 3). ( C ) EdU staining results at 6 and 24 h after AMD3100 treatment. EdU in green and PNA in gray ( n = 4, scale bar: 500 µm). ( D ) Immunostaining results of Ki67 (red) and F-actin (green) in acini and duct of eSMGs 24 h after AMD3100 treatment. Morphologies of acinar buds and duct cells are outlined with white dotted lines. Scale bar: 50 µm. ( E ) Duct widths of control and AMD3100-treated eSMGs were visualized via F-actin-based intensity profiles of horizontal sectioning of ducts 24 h after the treatment. ( F ) Duct widths of control and AMD3100-treated eSMGs were quantified 24 h after the treatment ( n = 9). ( G ) Immunostaining results of AQP5 (green) and KRT7 (red). Magnified regions of acinar buds are marked with white dotted squares. The white arrows (middle panels) indicate areas with the highest AQP5 expression ( n = 4, scale bar: left, 100 µm; middle and right, 50 µm). Data are presented as the mean ± SEM; * p < 0.05, **** p < 0.0001; t -test.

Article Snippet: The following primary antibodies were used in the procedures: rat monoclonal anti-CXCR4 antibody (R&D Systems, MAB21651), rabbit monoclonal anti-KRT7 antibody (Abcam, ab181598; Cambridge, UK); rabbit monoclonal anti-CDH1 antibody (CST, 3195; Beverly, MA, USA), rabbit monoclonal anti-H2AK119ub antibody (CST, 8240); mouse monoclonal anti-CXCL12 antibody (Novus Biologicals, MAB350; Littleton, CO, USA), rabbit polyclonal anti-AQP5 antibody (Alomone Labs, AQP-005; Jerusalem, Israel), rabbit monoclonal anti-CASP3 antibody (CST, 9664), and rat monoclonal anti-KI67 antibody (ThermoFisher, 14-5698-82).

Techniques: Staining, Immunostaining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands

doi: 10.3390/ijms22020619

Figure Lengend Snippet: eSMGs lacking PRC1 develop precocious differentiation in acini and ducts. ( A ) Schematic diagram of AMD3100-induced effect on organogenesis. Down-regulation of PRC1 genes leads to the inability of methylation site recognition by chromobox 8 (CBX8), a polycomb group protein ( 1 ), resulting in failure of the ubiquitin-ligating activity of ring finger protein 1 (RING1) ( 2 ) and thus, disabled DNA condensation ( 3 ). Hence, the developmental genes are readily transcribed by RNA polymerase II. ( B ) Representative images showing ubiquitin expression at Lys119 of histone 2A (H2AK119ub) in control and AMD3100-treated eSMGs 24 h after the treatment ( n = 4, scale bar: 50 µm). ( C ) Mean fluorescence intensities of H2AK119ub in epithelial buds per z -stack of 0.91 µm (25 stacks per end bud). False discovery rate (FDR) q -values of the differences in H2AK119ub expression at each of the 25 stacks are shown in the heat map, in which 23 out of 25 stacks marked a significant difference between control and AMD3100-treated eSMGs. -log( q -value) > 1.3 is considered significant ( q < 0.05; t -test with 5% FDR correction). The heat map (right) shows the color profile ( n = 4). ( D ) Methyl-seq results from control and AMD3100-treated eSMGs. Ten eSMGs per group were homogenized and analyzed. ( E ) Relative DAPI intensities of end buds in control, AMD3100-, and UNC3866-treated groups are expressed as a colored heat map (0–65,535 gray-value). Magnified regions are marked with white dotted squares ( n = 4, scale bar: 20 µm). ( F ) Quantification of DAPI intensities in nuclei of control (CON), AMD3100 (AMD)-, and UNC3866 (UNC)-treated eSMGs ( n = 18). ( G ) Immunostaining results of AQP5 (yellow) and KRT7 (gray) in eSMGs treated with AMD3100 or UNC3866. eSMGs were fixed and immunostained 24 h after the treatment ( n = 4, scale bar: upper panel, 50 µm; lower panel, 500 µm). ( H ) Quantification of AQP5 intensities in acinar buds of eSMGs treated with AMD3100 (AMD) or UNC3866 (UNC) ( n = 30). ( I ) Quantification of KRT7 intensities in ducts of eSMGs treated with AMD3100 (AMD) or UNC3866 (UNC) ( n = 4). Data are presented as the mean ± SEM; ** p < 0.01, *** p < 0.001, **** p < 0.0001; one-way ANOVA.

Article Snippet: The following primary antibodies were used in the procedures: rat monoclonal anti-CXCR4 antibody (R&D Systems, MAB21651), rabbit monoclonal anti-KRT7 antibody (Abcam, ab181598; Cambridge, UK); rabbit monoclonal anti-CDH1 antibody (CST, 3195; Beverly, MA, USA), rabbit monoclonal anti-H2AK119ub antibody (CST, 8240); mouse monoclonal anti-CXCL12 antibody (Novus Biologicals, MAB350; Littleton, CO, USA), rabbit polyclonal anti-AQP5 antibody (Alomone Labs, AQP-005; Jerusalem, Israel), rabbit monoclonal anti-CASP3 antibody (CST, 9664), and rat monoclonal anti-KI67 antibody (ThermoFisher, 14-5698-82).

Techniques: Methylation, Activity Assay, Expressing, Fluorescence, Immunostaining

Journal: International Journal of Environmental Research and Public Health

Article Title: Keratinocyte Growth Factor-1 Protects Radioiodine-Induced Salivary Gland Dysfunction in Mice

doi: 10.3390/ijerph17176322

Figure Lengend Snippet: Histological analysis of salivary glands. ( A ) Representative histological images of PAS and MT staining. More mucin-containing acini and less periductal fibrosis were observed in the RI+KGF-1 group than in the RI group. ( B ) Representative immunohistochemical images showing HIF1a, salivary epithelial cells (AQP5 stained) and endothelial cells (CD31 stained). The expression of HIF1a was higher and the expressions of AQP5 and CD31 were lower in the RI group than in treatment naïve controls. HIF1a staining intensities was lesser, and AQP5 and CD31 staining intensities were greater in the RI+KGF-1 group than in the RI group. (N; treatment naïve control group, RI; RI group, KGF; RI+KGF-1 group). Results are presented as means ± SDs; *, vs. treatment naïve controls; # , vs. the RI group. * p < 0.05, # p < 0.05 ( n = 6 mice per group) (N; treatment naïve controls, RI; RI group, KGF; RI+KGF-1 group), Scale bar: 50 μm. ( C ) More mucin-containing acini and less periductal fibrosis were observed in the RI+KGF-1 group than in the RI group. The expression of HIF1a was higher and the expressions of AQP5 and CD31 were lower in the RI group than in treatment naïve controls. HIF1a staining intensities was lesser, and AQP5 and CD31 staining intensities were greater in the RI+KGF-1 group than in the RI group. (N; treatment naïve control group, RI; RI group, KGF; RI+KGF-1 group). Results are presented as means ± SDs; *, vs. treatment naïve controls; # , vs. the RI group. * p < 0.05, # p < 0.05 ( n = 6 mice per group) (N; treatment naïve controls, RI; RI group, KGF; RI+KGF-1 group), Scale bar: 50 μm.

Article Snippet: They were incubated overnight at 4 °C with rabbit polyclonal antibodies against aquaporin 5 (AQP5) (diluted 1:200; Alomone Labs, Jerusalem, Israel), cluster of differentiation 31 (CD31), and hypoxia-inducible factor 1-alpha (HIF1a) (diluted 1:200; Santa Cruz, CA, USA) using an LSAB kit (Dako, Carpinteria, CA, USA) counterstained with Haemotoxylin and Eosin (H&E), dehydrated, and mounted.

Techniques: Staining, Immunohistochemical staining, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Influence of Different Feed Physical Forms on Mandibular Gland in Growing Pigs

doi: 10.3390/ani10050910

Figure Lengend Snippet: Pig mandibular gland. AQP5 binding sites at duct (*) and demilune (↑) level in CM, FP and CP groups.

Article Snippet: Subsequently, once the excess of reagent has been removed from the sections, they were incubated, overnight at RT, each with one of the primary antibodies: rabbit polyclonal antibody anti-APLN (1:100, NBP2-31176, Novus Biologicals, Littleton, CO, USA), mouse monoclonal antibody anti-APLNR (1:100, sc-517300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal antibody anti-AQP5 (1:100, AQP-005, Alomone Labs, Jerusalem 9104201, Israel).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Cell-specific expression of aquaporin-5 ( Aqp5 ) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation

doi: 10.1038/s41598-017-03152-7

Figure Lengend Snippet: HDAC inhibitor increases AQP5 expression. Representative western blot (WB) ( A ) and corresponding quantitative analysis ( B ) shows that treatment of MLE-15 cells with HDAC inhibitor SAHA for 24 h increases AQP5 (27 kD) protein expression (normalized to eIF-2α (36 kD)) (n = 4, *p < 0.05 compared to DMSO). SAHA increases Aqp5 luciferase activity in a dose-dependent manner in both NIH3T3 ( C ) and MLE-15 cells ( D ) transfected with -358- Aqp5 -Luc following 18 h treatment. Protein concentrations were used for sample normalization (n = 3, *p < 0.05 compared to DMSO). Twenty-four h of SAHA (1 µM) treatment had no cytotoxic effects on NIH3T3 ( E ) and MLE-15 cells ( F ). ( G ) GATA6 (pCDNA3/GATA6), Sp1 (pCMV/Sp1) and their corresponding empty vectors were transfected individually or in combination into NIH3T3 cells, followed by treatment with SAHA (0.8 µM) for 18 h. SAHA significantly increases Sp1-activated -358 Aqp5 promoter/enhancer activity. Protein concentrations were used for sample normalization. n = 3 except for column 2 (empty vectors with SAHA), where n = 2, * § p < 0.05 shows significant difference between control (column 1, empty vectors with DMSO) and all other groups except column 2. Since column 2 is only n = 2, we did not perform statistical analysis for comparison with control. *p < 0.05 compared to column 5 (Sp1 with DMSO). ChIP with anti-acetyl-H3 (H3Ac) and anti-acetyl-H4 (H4Ac) Abs demonstrates enrichment of H3 acetylation ( H ) and decreased H4 acetylation ( I ) at the Aqp5 promoter/enhancer region homologous to the proximal 358-bp of the rat Aqp5 promoter following SAHA treatment (1 µM, 24 h) in MLE-15 cells (n = 3, *p < 0.05). ChIP efficiency was calculated relative to untreated cells precipitated with H3Ac and H4Ac Ab, respectively, which was set as 1. Rabbit IgG pull-down is used as control.

Article Snippet: Primary antibodies (Abs) used included rabbit polyclonal anti-AQP5 (#AQP005, Alomone Labs, Jerusalem, Israel), -p300 (#SC584, Santa Cruz Biotechnology, Dallas, Texas), -HDAC2 (#SC7899, Santa Cruz Biotechnology) and -HDAC3 (SC11417, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Transfection

Journal: Scientific Reports

Article Title: Cell-specific expression of aquaporin-5 ( Aqp5 ) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation

doi: 10.1038/s41598-017-03152-7

Figure Lengend Snippet: HDAC3 modulates expression of AQP5. NIH3T3 cells were co-transfected with 358- Aqp5 -Luc, Sp1 and increasing amounts of HDAC1 ( A ), HDAC2 ( B ) and HDAC3 ( C ) expression vectors or empty vector (pCDNA3). HDAC2 and HDAC3, but not HDAC1 inhibited Sp1-activated Aqp5 transcription. Protein concentrations were used for sample normalization (n = 3, *p < 0.05 compared to empty vector). WB ( D ) and corresponding quantitative analysis ( E – G ) show that AQP5 expression (normalized to GAPDH (37 kD)) is significantly upregulated ( D , G ) following knockdown of HDAC3 (59 kD) with shRNA ( D , E ) but not HDAC2 (49 kD) with siRNA ( D , F ) (n = 3, *p < 0.05 compared to non-silencing shRNA and GFP siRNA) in MLE-15 cells. ( H ) ChIP with anti-acetyl-H3 Ab (H3Ac) demonstrates enrichment of H3 acetylation at the Aqp5 promoter/enhancer region homologous to the proximal 358-bp of the rat Aqp5 promoter following HDAC3 knockdown in MLE-15 cells (n = 3, *p < 0.05). ChIP efficiency was calculated relative to non-silencing shRNA (NS-shRNA)-treated cells precipitated with H3Ac Ab, which was set as 1. Rabbit IgG pull-down is used as control.

Article Snippet: Primary antibodies (Abs) used included rabbit polyclonal anti-AQP5 (#AQP005, Alomone Labs, Jerusalem, Israel), -p300 (#SC584, Santa Cruz Biotechnology, Dallas, Texas), -HDAC2 (#SC7899, Santa Cruz Biotechnology) and -HDAC3 (SC11417, Santa Cruz Biotechnology).

Techniques: Expressing, Transfection, Plasmid Preparation, shRNA

Journal: Scientific Reports

Article Title: Cell-specific expression of aquaporin-5 ( Aqp5 ) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation

doi: 10.1038/s41598-017-03152-7

Figure Lengend Snippet: GATA6 regulation of AQP5 expression involves H3 acetylation. RNA and protein were harvested from MLE-15 cells transduced with lentivirus expressing Gata6 or control non-silencing shRNA (MOI = 2) for 3 days. qRT-PCR shows that knockdown of Gata6 ( A ) significantly decreases Aqp5 mRNA ( B ) and protein (AQP5 normalized to β-actin (42 kD)) ( C , D ) expression (n = 3, *p < 0.05 compared to non-silencing shRNA (NS-shRNA)). ( E ) ChIP with anti-acetyl-H3 Ab (H3Ac) demonstrates enrichment of H3 acetylation at the Aqp5 promoter/enhancer region homologous to the proximal 358-bp of the rat Aqp5 promoter using chromatin harvested from MLE-15 cells transduced with GATA6-expressing lentivirus or control (GFP) (MOI = 2) (n = 3, *p < 0.05). ChIP efficiency was calculated relative to control virus-transduced cells precipitated with H3Ac Ab, which was set as 1. Rabbit IgG pull-down is used as control.

Article Snippet: Primary antibodies (Abs) used included rabbit polyclonal anti-AQP5 (#AQP005, Alomone Labs, Jerusalem, Israel), -p300 (#SC584, Santa Cruz Biotechnology, Dallas, Texas), -HDAC2 (#SC7899, Santa Cruz Biotechnology) and -HDAC3 (SC11417, Santa Cruz Biotechnology).

Techniques: Expressing, Transduction, shRNA, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Cell-specific expression of aquaporin-5 ( Aqp5 ) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation

doi: 10.1038/s41598-017-03152-7

Figure Lengend Snippet: p300 increases GATA6/Sp1-mediated -358- Aqp5 -Luc activity. ( A – C ) NIH3T3 cells were co-transfected with -358- Aqp5 -Luc and different combinations of GATA6, Sp1, p300 and CBP expression vectors. Luciferase assays were performed 48 h after transfection. p300 and CBP have no effect on Aqp5 -Luc activity ( A ), while p300 (but not CBP) augments both GATA6- ( B ) and Sp1- ( C ) mediated Aqp5 transcriptional activity. Protein concentration was used to normalize samples for transfection efficiency (n = 3, *p < 0.05 compared to GATA6 or Sp1 only). qRT-PCR shows knockdown of p300 mRNA using two different shRNAs (V2LMN_102133 indicated as p300-1 and V2LMN_90586 indicated as p300-2) ( D ) decreases Aqp5 mRNA expression following p300 knockdown ( E ) (n = 4, *p < 0.05 compared to non-silencing shRNA control (NS shRNA)). WB shows knockdown of p300 ( F , G ) decreases AQP5 protein expression ( F , H ). (n = 4, *p < 0.05 compared to NS shRNA control).

Article Snippet: Primary antibodies (Abs) used included rabbit polyclonal anti-AQP5 (#AQP005, Alomone Labs, Jerusalem, Israel), -p300 (#SC584, Santa Cruz Biotechnology, Dallas, Texas), -HDAC2 (#SC7899, Santa Cruz Biotechnology) and -HDAC3 (SC11417, Santa Cruz Biotechnology).

Techniques: Activity Assay, Transfection, Expressing, Luciferase, Protein Concentration, Quantitative RT-PCR, shRNA

Journal: Scientific Reports

Article Title: Cell-specific expression of aquaporin-5 ( Aqp5 ) in alveolar epithelium is directed by GATA6/Sp1 via histone acetylation

doi: 10.1038/s41598-017-03152-7

Figure Lengend Snippet: Knockdown of HDAC3 increases GATA6/Sp1/p300 interactions. Representative co-immunoprecipitation (co-IP) of cell lysate from MLE-15 cells transduced with Hdac3 shRNA or non-silencing shRNA (NS) demonstrates decreased HDAC3/Sp1 interaction ( A ), increased GATA6/Sp1 and GATA6/p300 ( B ), and increased p300/Sp1 ( C ) interaction following HDAC3 knockdown. n = 3. ( D ) Model of Sp1-mediated Aqp5 transcriptional regulation and involvement of HDAC3, GATA6, and p300: HDAC3 and GATA6 regulate Sp1-mediated Aqp 5 transcription via H3 deacetylation and acetylation, respectively, at the proximal promoter/enhancer. Mechanisms underlying GATA6-dependent H3 acetylation at the Aqp5 enhancer/promoter might involve competition between GATA6 and HDAC3 for binding to Sp1, as well as recruitment of histone acetylase p300 to the Sp1/GATA6 complex. In addition to effects on H3 acetylation, p300 might also modulate GATA6 and/or Sp1 activity.

Article Snippet: Primary antibodies (Abs) used included rabbit polyclonal anti-AQP5 (#AQP005, Alomone Labs, Jerusalem, Israel), -p300 (#SC584, Santa Cruz Biotechnology, Dallas, Texas), -HDAC2 (#SC7899, Santa Cruz Biotechnology) and -HDAC3 (SC11417, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transduction, shRNA, Binding Assay, Activity Assay