Journal: PLoS ONE

Article Title: Effects of Biliverdin Administration on Acute Lung Injury Induced by Hemorrhagic Shock and Resuscitation in Rats

doi: 10.1371/journal.pone.0063606

Figure Lengend Snippet: Sections of lungs obtained 3 h after resuscitation were subjected to fluorescent immunohistochemical analysis for 8-OHdG (green) and aquaporin-5 (red). Above row: 8-OHdG-positive cells in the vehicle/sham (A), BV/sham (B), vehicle/HSR (C) and BV/HSR groups (D). Middle row: aquaporin-5-positive cells in the vehicle/sham (E), BV/HSR (F), vehicle/HSR (G), and BV/HSR groups (H). Bottom row: merged images of 8-OHdG and aquaporin-5 in the vehicle/sham (I), BV/sham (J), vehicle/HSR (K), and BV/HSR groups (L). Vehicle/sham, vehicle-administered animals subjected to sham surgery; BV/Sham, BV-administered animals subjected to sham surgery; vehicle/HSR, vehicle-administered animals subjected to HSR; BV/HSR, BV-administered animals subjected to HSR. Scale bar = 50 µm. All images shown were visualized with a 20×0.5 objective lens.

Article Snippet: Slides were incubated overnight at 4°C with a mouse anti-8-OHdG monoclonal antibody (Japan Institute for the Control of Aging) and a rabbit anti-aquaporin 5 polyclonal antibody (Alomone Labs, Ltd., Jerusalem, Israel) at a dilution of 1∶100 in 0.01 M phosphate-buffered saline containing 0.3% Triton X-100.

Techniques: Immunohistochemical staining

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: BioMed Research International

Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

doi: 10.1155/2015/460598

Figure Lengend Snippet: Analysis of AQP5 expression in nondividing EpH4 cells treated with Matrigel. (a) Shown is the average concentration of trypsinized cells [cells/mL] counted with a Coulter Counter from 4 different experiments. Averages are represented as crosses with confidence intervals; n = 3; alpha = 0.05. Treatments were performed such that the age of the cells from seeding to the time of extraction was the same within one experiment. (b) Levels of AQP5 mRNA or protein measured after shorter or longer Dex treatment times. Cells were treated with Dex for 2 h, 6 h, 12 h, 24 h, and 72 h. Numbers inside bars represent experimental replicates; mRNA was normalized to GAPDH; protein was normalized to TUBB and shown relative to untreated samples. Bars represent averages and confidence intervals; n = 3–15; alpha = 0.05. Significance was tested between controls (−Dex) and different Dex treatments, with asterisks denoting P < 0.05.

Article Snippet: A primary rabbit anti-AQP5 antibody (Alomone labs; dilution 1 : 500) and a secondary anti-rabbit HRP-conjugated antibody (Amersham GE Healthcare; dilution 1 : 1000) was used to measure levels of AQP5 protein.

Techniques: Expressing, Concentration Assay

Journal: BioMed Research International

Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

doi: 10.1155/2015/460598

Figure Lengend Snippet: Cell growth and AQP5 expression at different cell culturing stages. (a) Cell culturing and treatment times at which cells were harvested (indicated with arrows). (b) Numbers of cultured cells were counted with a Coulter Counter. Shown are averages (dots) and confidence intervals; n = 3; alpha = 0.05. (c) Total mRNA was extracted from cells used for seeding, 12 h after seeding, 26 h after seeding (with or without 2 h Dex treatment), 36 h after seeding with 12 h Matrigel treatment, and 50 h after seeding with 26 h Matrigel treatment (with or without 2 h Dex treatment). AQP5 expression is normalized to GAPDH and shown relative to the 0 time point treatment. Bars represent averages and confidence intervals; n = 3; alpha = 0.05. Significance was tested between the 26 h treatments (−Dex, −Matrigel, 26 h and 2 h Dex, −Matrigel, 26 h) and between the 50 h treatments (−Dex, 26 h Matrigel, 50 h and 2 h Dex, 26 h Matrigel, 50 h).

Article Snippet: A primary rabbit anti-AQP5 antibody (Alomone labs; dilution 1 : 500) and a secondary anti-rabbit HRP-conjugated antibody (Amersham GE Healthcare; dilution 1 : 1000) was used to measure levels of AQP5 protein.

Techniques: Expressing, Cell Culture

Journal: BioMed Research International

Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

doi: 10.1155/2015/460598

Figure Lengend Snippet: Regulation of AQP5 expression by Dex in different cell culturing systems.

Article Snippet: A primary rabbit anti-AQP5 antibody (Alomone labs; dilution 1 : 500) and a secondary anti-rabbit HRP-conjugated antibody (Amersham GE Healthcare; dilution 1 : 1000) was used to measure levels of AQP5 protein.

Techniques: Expressing, Cell Culture

Journal: BioMed Research International

Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

doi: 10.1155/2015/460598

Figure Lengend Snippet: Analysis of AQP5 promoter methylation. (a) DNA methylation of 49 CpG sites found in the 289 base pair region located ~70 bases from the start codon of the AQP5 promoter, measured by bisulfite sequencing of individual clones (total methylation percentage shown in black). A single CpG site (CpG#24) was measured in tens of thousands of molecules with bead-emulsion amplification (methylation percentage shown in green). Nondividing cells without Dex and with Dex treatments for 2 h and 4 h were analyzed. (b) Changes in AQP5 mRNA expression analyzed with one-step qPCR. Total mRNA was extracted from Matrigel-treated cells without Dex and with Dex treatments for 2 h and 4 h. AQP5 expression is normalized to GAPDH and shown relative to no Dex treatment. Bars represent averages and confidence intervals; n = 3; alpha = 0.05. Significance was tested between control (−Dex) and differently treated samples. (c) Summary of AQP5 mRNA expression and promoter methylation.

Article Snippet: A primary rabbit anti-AQP5 antibody (Alomone labs; dilution 1 : 500) and a secondary anti-rabbit HRP-conjugated antibody (Amersham GE Healthcare; dilution 1 : 1000) was used to measure levels of AQP5 protein.

Techniques: Methylation, DNA Methylation Assay, Methylation Sequencing, Clone Assay, Amplification, Expressing

Journal: BioMed Research International

Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

doi: 10.1155/2015/460598

Figure Lengend Snippet: Changes in DNA methylation with the demethylating agent 5-Aza in actively dividing cells. (a) Changes in DNA methylation monitored by bacterial bisulfite sequencing of individual clones. Cells were treated with 5-Aza for 48 h while still dividing. Data for the control (no Aza) are combined from two experiments, one treated with Matrigel (also shown in Supplementary Figure 9, treatment 1) and one without Matrigel. (b) Expression of AQP5 mRNA analyzed with qPCR. AQP5 expression is normalized to GAPDH and shown relative to the no-5-Aza treatment. Bars represent averages and confidence intervals; n = 6; alpha = 0.05. Significance was tested between control (5-Aza) and the 5-Aza treated sample.

Article Snippet: A primary rabbit anti-AQP5 antibody (Alomone labs; dilution 1 : 500) and a secondary anti-rabbit HRP-conjugated antibody (Amersham GE Healthcare; dilution 1 : 1000) was used to measure levels of AQP5 protein.

Techniques: DNA Methylation Assay, Methylation Sequencing, Clone Assay, Expressing

Journal: BioMed Research International

Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

doi: 10.1155/2015/460598

Figure Lengend Snippet: nGRE motifs in the AQP5 promoter region. The ~7 kb region of the AQP5 promoter and 4.5 kb of the AQP5 gene were analyzed for nGRE motifs with 0, 1, or 2 nucleotides ( N ) as a spacer with and without mismatches. Sequence positions are calculated relative to the translation initiation site. For the motifs containing a mismatch, the mismatch was classified as tolerable (t) or intolerable (i) for glucocorticoid binding according to Surjit et al. . Mismatches are indicated bold and underlined.

Article Snippet: A primary rabbit anti-AQP5 antibody (Alomone labs; dilution 1 : 500) and a secondary anti-rabbit HRP-conjugated antibody (Amersham GE Healthcare; dilution 1 : 1000) was used to measure levels of AQP5 protein.

Techniques: Sequencing, Binding Assay

Journal: BioMed Research International

Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

doi: 10.1155/2015/460598

Figure Lengend Snippet: Effect of Dex on the AQP5 promoter. EpH4 cells were treated with Dex, 4 h prior ( n = 4) or during transfection ( n = 5) of the luciferase expression pGL3-basic vector including the AQP5 promoter (FF) and the pRL-TK transfection control plasmid (R). Negative controls included the pGL3-basic vector without promoter (cFF) ( n = 2) and the peGFP-N1 vector (GFP), ( n = 1). (a) Levels of AQP5 mRNA normalized to GAPDH are shown for the different transfected EpH4 cells with or without Dex treatment. Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between samples without Dex and with Dex treatment of the same transfection type. (b) Levels of Firefly enzyme activity normalized to Renilla enzyme activity measured with the luciferase reporter assay for the same transfected cells as in panel (a). Bars represent averages and confidence intervals; alpha = 0.05. Significance was tested between controls (−Dex) and treatments (+Dex), with one or two asterisks denoting P < 0.05 or P < 0.005, respectively.

Article Snippet: A primary rabbit anti-AQP5 antibody (Alomone labs; dilution 1 : 500) and a secondary anti-rabbit HRP-conjugated antibody (Amersham GE Healthcare; dilution 1 : 1000) was used to measure levels of AQP5 protein.

Techniques: Transfection, Luciferase, Expressing, Plasmid Preparation, Activity Assay, Reporter Assay

Journal: The Journal of Clinical Investigation

Article Title: Lysosomal exocytosis of HSP70 stimulates monocytic BMP6 expression in Sjögren’s syndrome

doi: 10.1172/JCI152780

Figure Lengend Snippet: ( A ) Submandibular glands of C57BL/6 mice were instilled with an AAV2 vectors encoding LAMP3 (AAV2-LAMP3) or GFP (AAV2-GFP), and the tissues and saliva flow rate were evaluated 6 months later. Mice were given i.p. injections of TAK242 or vehicle for the last 10 days. ( B ) Representative images of dual ISH for Bmp6 (white) and IF for CD68 (magenta) and nucleus (DAPI, blue) (upper panel), and images of IF for BMP6 (green) and nuclei (DAPI, blue) (lower panel) in submandibular gland sections. Scale bars: 20 μm. Enlargement original magnification, 100×. Quantification of ( C ) Bmp6 + dots per mm 2 ( n = 5 each) and ( D ) BMP6 expression area ( n = 3 each). ( E ) Representative IF images for BMP6 (green, upper panel), AQP5 (red, lower panel), and nuclei (DAPI, blue) in submandibular gland sections. Scale bars: 20 μm. Quantification of ( F ) BMP6 and ( G ) AQP5 expression area. Values shown are the mean ± SD. ** P < 0.01, by Student’s t test ( C , D , F , and G ). ( H ) Pilocarpine-stimulated salivary flow per body weight over 20 minutes in TAK242-treated mice ( n = 4) and vehicle-treated mice ( n = 3). Values shown indicate the median and the range. † P < 0.05, by Wilcoxon test.

Article Snippet: For IF, slides were blocked with 2% BSA (MilliporeSigma) at 25°C for 1 hour and then incubated at 4°C overnight with a primary antibody solution consisting of mouse anti-BMP6 (Abcam, ab15640); mouse anti-TLR4 (Abcam, ab22048); mouse anti-CD14 (Abcam, ab182032); rabbit anti-CD16 (Abcam, ab203883); rabbit anti-CD68 (Abcam, ab213363 and ab125212); rabbit anti-CD19 (Abcam, ab227019); mouse anti-CD56 (Abcam, ab200698); rabbit anti-CD138 (Abcam, ab128936); rat anti-CD3 (LifeSpan BioSciences, LS-B8765-50); rabbit anti-LAMP3 (Proteintech, 12632-1-AP); and/or rabbit anti-AQP5 (Alomone Labs, AQP-005) antibodies.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Ezrin Regulates Ca 2+ Ionophore-Induced Plasma Membrane Translocation of Aquaporin-5

doi: 10.3390/ijms222413505

Figure Lengend Snippet: Ezrin-DN inhibits stimulation-dependent plasma membrane translocation of AQP5. MLE-12 cells were transfected with wildtype or dominant negative (lacking actin-binding domain) ezrin. Subcellular localization of AQP5 was analyzed by immunofluorescence ( A ), and the amount of cell surface AQP5 was assessed by Western blotting ( B ). Cells were treated with ionomycin (1 µM) for 15 min, after which cellular localization of AQP5 was analyzed by immunofluorescence ( C ) and the amount of cell surface AQP5 was assessed by cell surface biotinylation and Western blotting ( D ). HSG cells were transfected with wildtype or dominant negative ezrin, after which cells were treated with ionomycin (1 µM) for 15 min. Subcellular localization of AQP5 was analyzed by immunofluorescence ( E ). Blue staining in merge image indicates DAPI (nuclei). Data represent the mean ± S.E. ( n = 3–6). * p < 0.05 and ** p < 0.01.

Article Snippet: A rabbit anti-AQP5 antibody (AQP005) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Translocation Assay, Transfection, Dominant Negative Mutation, Binding Assay, Immunofluorescence, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Ezrin Regulates Ca 2+ Ionophore-Induced Plasma Membrane Translocation of Aquaporin-5

doi: 10.3390/ijms222413505

Figure Lengend Snippet: Ezrin inhibition decreases stimulation-dependent membrane trafficking. MLE-12 cells were treated with ionomycin (1 µM) and NSC305787 (10 µM) for 15 min. Subcellular localization of AQP5 was analyzed by immunofluorescence ( A ). The amount of cell surface AQP5 was assessed by cell surface biotinylation and Western blotting ( B ). MLE-12 cells were pretreated with E64d (10 µM) for 30 min and then treated with ionomycin (1 µM) for 15 min, after which subcellular localization of AQP5 ( C ) and the amount of cell surface AQP5 ( D ) were determined. MLE-12 cells were transfected with wildtype, S66A, or S66D (uncleavable mutant) ezrin and then stimulated with ionomycin (1 µM) for 15 min, after which subcellular localization of AQP5 ( E ) and the amount of cell surface AQP5 ( F ) were assessed. Data represent the mean ± S.E. ( n = 3–4). * p < 0.05.

Article Snippet: A rabbit anti-AQP5 antibody (AQP005) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Inhibition, Immunofluorescence, Western Blot, Transfection, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: Ezrin Regulates Ca 2+ Ionophore-Induced Plasma Membrane Translocation of Aquaporin-5

doi: 10.3390/ijms222413505

Figure Lengend Snippet: Cytoskeleton is required for ezrin-mediated membrane trafficking of AQP5 in MLE-12 cells. MLE-12 cells were pretreated with cytochalasin D (20 µM) or vincristine (10 µM) for 2 h and then treated with ionomycin (1 µM) for 15 min. Cellular localization of AQP5 ( A ) and the amount of cell surface AQP5 were assessed ( B , C ). Data represent the mean ± S.E. ( n = 3–4). * p < 0.05 and ** p < 0.01.

Article Snippet: A rabbit anti-AQP5 antibody (AQP005) was purchased from Alomone Labs (Jerusalem, Israel).

Techniques:

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