Journal: Oncogene

Article Title: Involvement of aquaporins in colorectal carcinogenesis.

doi: 10.1038/sj.onc.1206762

Figure Lengend Snippet: Figure 1 Expression of AQPs 1, 3, and 5 in seven colon and colorectal cancer cell lines. RT–PCR was performed using sense and antisense primers for AQPs 1, 2, 3, 5, and 8 for seven cancer cell lines (W489, HT20, H455, HCT116 p53 þ / þ , HCT116 p53 /, DLD1, SW480). The name of each cell line is marked on the top of each lane, and the size of each PCR product is marked with an arrow. PCR mixtures without templates were used negative controls, and GAPDH was the loading control. Each PCR product was cloned and its sequence confirmed. All seven cell lines demonstrated expression of AQPs 1, 3, and 5(a). Western blot analysis was performed using antibodies for AQPs 1, 3, and 5 for the cell lines HCT116 p53 þ / þ , HCT116 p53 /, DLD1, and SW480. The name of each cell line is marked on the top of each lane, and the size of the protein is marked with an arrow. NIH3T3 cells transfected with AQP1 and AQP3 expression constructs were used as positive controls for the Western blots of AQPs 1 and 3, respectively. 293, a kidney cell line, was used as a positive control for the Western blot of AQP5. NIH3T3 cells were used as a negative control, and b-actin was the loading control. All four cell lines demonstrated AQPs 1, 3, and 5 expression(b)

Article Snippet: After 1 h blocking with 5% nonfat dry milk in 1 PBS at room temperature, the membranes were probed with rabbit anti-AQP1 antibody (Chemicon International), goat anti-AQP3 antibody, or goat anti-AQP5 antibody (Santa Cruz Biotechnology), all at 1 : 100 dilutions in 1 PBS for 1 h at room temperature.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Clone Assay, Sequencing, Western Blot, Transfection, Construct, Positive Control, Negative Control

Journal: Oncogene

Article Title: Involvement of aquaporins in colorectal carcinogenesis.

doi: 10.1038/sj.onc.1206762

Figure Lengend Snippet: Figure 2 In situ hybridization with AQP1 and AQP5 probes in the multistep carcinogenesis model of colon cancer development. The AQP1 antisense riboprobe clearly stains not only colonic adenoma (a, arrow) but also the primary colon cancer (b, arrow) and metastatic lesions in the liver (c, arrow). Likewise, the AQP5 antisense riboprobe clearly stains early adenoma with moderately dysplastic cells (d, arrow), late adenoma with severe dysplastic cells (e, arrow), and adenocarcinoma (f, arrow). There is almost no staining in the surrounding normal colonic mucosa when probed with AQP1 and AQP5 antisense riboprobes (a and d, star). As positive controls, the germinal centers of the tonsils were stained with antisense riboprobes of AQP1 (data not shown) and AQP5 (g). Sense riboprobes of AQP1 (data not shown) and AQP5 (h) were used as negative controls. As an internal control, vascular endothelium was stained with the AQP1 antisense riboprobe (i)

Article Snippet: After 1 h blocking with 5% nonfat dry milk in 1 PBS at room temperature, the membranes were probed with rabbit anti-AQP1 antibody (Chemicon International), goat anti-AQP3 antibody, or goat anti-AQP5 antibody (Santa Cruz Biotechnology), all at 1 : 100 dilutions in 1 PBS for 1 h at room temperature.

Techniques: In Situ Hybridization, Staining, Control

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, Real-time Polymerase Chain Reaction

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: Control, Staining, Immunostaining, Expressing, 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 transcriptional changes in developmental and polycomb repressive complex 1 (PRC1) genes. ( A ) 3′ mRNA sequencing was performed on control and AMD3100-treated E14 eSMGs at 6 h. Among 8941 up-regulated and 10,300 down-regulated genes in the treated group, a total of 535 differentially expressed genes (DEGs) with p < 0.05 and |log2 fold change (FC)| ≥ 1 were found; 220 up-regulated and 315 down-regulated. ( B ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs. Significantly enriched pathways are revealed by enrichR. ( C ) DEGs in KEGG pathways cellular senescence, cell cycle, and DNA replication are shown with log2FC and p -values. ( D ) The most frequently occurring genes in significantly enriched pathways are listed with log2FC and their number of occurrences. ( E ) Read count z -scores of epithelial differentiation marker genes consistently increased in AMD3100-treated eSMGs, contrasting to the decrease in Cxcl12 . ( F ) DEGs were filtered out from the Gene Ontology (GO) gene set GO:0030855—epithelial cell differentiation—and the inclusive DEGs are listed. ( G ) Currently-known murine transcription factors (1475 total) were extracted from PantherDB to filter the DEGs. Transcription factor genes with p ≤ 0.01 and |log2FC| ≥ 0.5 are in red. ( H ) Genes comprising PRC1 complex are listed with log2FC and p -values. Names in red indicate genes with p < 0.05.

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: Sequencing, Control, Analysis, Marker, Cell Differentiation, Significance Assay

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, Ubiquitin Proteomics, Activity Assay, Expressing, Control, Fluorescence, Immunostaining, 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: Schematic for AMD3100-induced epigenetic modulation and precocious differentiation in eSMGs. The treatment of eSMGs with AMD3100 is followed by down-regulation of PRC1 components, leading to the absence of ubiquitin-ligating activity on H2AK119, which, in turn, deprives the DNA of its condensing function. Such epigenetic modulation confers de novo or stronger transcription of later-stage developmental genes to eSMGs, promoting precocious differentiation in acini and ducts.

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: Ubiquitin Proteomics, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: Identification of CLIP2 as a protein partner of AQP5. A , B Tandem mass spectra of CLIP2 peptide 634–645, ATLNSGPGAQQK, (+ 2 charged ion, m/z 586.4) from AQP5 IP samples in mouse parotid ( A ) and submandibular SG ( B ). Sequence specific b- and y-ions are labeled. C AQP5 immunoprecipitation (IP) performed using proteins from mouse SGs followed by WB detection using anti-CLIP2 antibodies. Input proteins from mouse submandibular glands (mSMG; lane A) and mouse parotid gland (mPG, lane B); IP performed using mSMG (lanes C, E) and mPG (lanes D, F) proteins in the presence (lanes C, D) or absence (negative controls; lanes E, F) of anti-AQP5 antibody; IP performed in the absence of any input proteins (additional negative control, lane G). D AQP5 IP performed using proteins from NS-SV-AC cells expressing AQP5 followed by WB detection using anti-CLIP2 antibodies. Input proteins from NS-SV-AC cells transfected with HA-hAQP5 (lane A) or SNAP-hAQP5 (lane B); IP performed using NS-SV-AC HA-AQP5 (lanes C, D) and NS-SV-AC SNAP-hAQP5 (lanes E, F) in the presence (lanes C, E) or in the absence (negative controls; lanes D, F) of anti-AQP5 antibody; IP performed in the absence of input proteins (additional negative control, lane G). Considering known batch-to-batch variation of commercial Sepharose-Protein A beads, whereby Protein A can shed from the beads under elution, the non-specific bands of ± 80–100 kDa ( C ) and ± 45–50 kDa ( D ) are likely non-specific bands corresponding in all likelihood to the IgG heavy chain (± 45–50 kDa) coupled or not to protein A (± 45 kDa) shed from the Sepharose beads.HA: hemagglutinin; SNAP: small protein derived from mammalian O6-alkylguanine-DNA-alkyltransferase

Article Snippet: In addition, the AQP5 fusion protein which was used in a recent publication [ ] and which contains the extracellular loop peptide amino acids 190-265 (human AQP5 Ag14514, Proteintech, Planegg-Martinsried, Germany) was bound to Luminex beads as above, and IgG antibodies against this antigen were measured in all 123 HC and in 223 patients with SjD.

Techniques: Sequencing, Labeling, Immunoprecipitation, Negative Control, Expressing, Transfection, Derivative Assay

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: Interaction between AQP5 and CLIP2 at the molecular level. A Binding curve from the MST-experiment showing the direct interaction between AQP5 and CLIP2. Data are expressed as the mean ± S.D. of bound fraction ( n = 3). The curve-line represents the curve fitting to a one-to-one binding model. B MST-data for the individual CLIP2-MTB domains (MTB1 and MTB2) showing the absence of interaction with AQP5

Article Snippet: In addition, the AQP5 fusion protein which was used in a recent publication [ ] and which contains the extracellular loop peptide amino acids 190-265 (human AQP5 Ag14514, Proteintech, Planegg-Martinsried, Germany) was bound to Luminex beads as above, and IgG antibodies against this antigen were measured in all 123 HC and in 223 patients with SjD.

Techniques: Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: Computer docking model of the AQP5-CLIP2 complex. A Model of the complex between the AQP5 C-terminus (orange) and the two MTB-domains of CLIP2 (light cyan and teal respectively) generated by AlphaFold Multimer. AQP5 binds primarily to MTB1 in a manner that resembles how SLAIN2 (blue) interacts with CLIP1 MTB1 (grey) and how tubulin (magenta) interacts with CLIP1 MTB2 (white). B Zoom-in on the AQP5-CLIP2 interaction site. A stretch of acidic residues (EPDED) interacts with a highly conserved basic groove on MTB1 with hydrophobic residues on the proximal (I238, Y243) and distal side (W249) interacting with hydrophobic pockets on MTB1 and MTB2 respectively. C Crystal structure of human CLIP1 MTB1 in complex with a C-terminal peptide of SLAIN2 (PDB code 3RDV) and D NMR structure of human CLIP1 MTB2 in complex with the C-terminal tail of α-tubulin (PDB code 2E4H) showing a similar mode of interaction as in the predicted AQP5-CLIP2 complex

Article Snippet: In addition, the AQP5 fusion protein which was used in a recent publication [ ] and which contains the extracellular loop peptide amino acids 190-265 (human AQP5 Ag14514, Proteintech, Planegg-Martinsried, Germany) was bound to Luminex beads as above, and IgG antibodies against this antigen were measured in all 123 HC and in 223 patients with SjD.

Techniques: Generated

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: Deletion of hCLIP2 compromises stimulated dependent shuttling/translocation of AQP5 to the plasma membrane. A SNAP-hAQP5 localization in hCLIP2 WT and hCLIP2 KO NS-SV-AC cells treated for 8 h with FK (10 µM), TH (0.1 µM) and FK + TH. Cells were counterstained with DAPI (blue, nuclear labeling). B Violin plots (with median and interquartile range of 25–75% percentile) of the membrane upper quartile (UpQ) intensity. Cells were pretreated for 24 h with 50µM indomethacin prior to treatment for 8 h without (CTRL) or with 10 µM FK, 0.1 µM TH, or both (FK + TH), in 3 independent experiments. Statistical significance evaluated using Kruskal-Wallis test with post-hoc Dunn’s tests is indicated as follows: ***: p ≤ 0.001, ****: p ≤ 0.0001.In hCLIP2 WT, the median with interquartile range of 25–75% (IQR) are: 1 with IQR 0.716–1.288, n = 461 cells for CTRL; 1.454 with IQR 0.999–1.918, 443 for FK; 1.188 with IQR 0.813–1.544, n = 256 cells for TH; 1.294 with IQR 0.897–1.754, n = 346 cells for FK + TH. In hCLIP2 KO, the median with IQR are: 1.157 with IQR 0.922–1.406, n = 425 cells for CTRL, 0.934 with IQR 0.692–1.256, n = 525 cells for FK, 0.985 with IQR 0.717–1.337, n = 546 cells for TH and 0.982 with IQR 0.692–1.318, n = 476 for FK + TH. SNAP-hAQP5 labeling is shown with AF594 (red). Images are representative of 3 independent experiments. Scale bars are 30 μm. B: Quantification of SNAP-hAQP5 localization and intensity based on “Upper Quartile Intensity”. Data are expressed as the median with interquartile range of SNAP-hAQP5 membrane upper quartile intensity ( n = 3). CTRL: control, unstimulated; FK: forskolin; TH: thapsigargin KO: knockout; SNAP: small protein derived from mammalian O6-alkylguanine-DNA-alkyltransferase; UpQ: Upper Quartile; WT: wild type

Article Snippet: In addition, the AQP5 fusion protein which was used in a recent publication [ ] and which contains the extracellular loop peptide amino acids 190-265 (human AQP5 Ag14514, Proteintech, Planegg-Martinsried, Germany) was bound to Luminex beads as above, and IgG antibodies against this antigen were measured in all 123 HC and in 223 patients with SjD.

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Labeling, Control, Knock-Out, Derivative Assay

Journal: Cell Communication and Signaling : CCS

Article Title: CLIP2: a novel functional player in AQP5 trafficking dynamics and implications for Sjögren’s disease

doi: 10.1186/s12964-025-02476-6

Figure Lengend Snippet: AQP5-CLIP2 interaction and co-localization. A-B PLA showing AQP5-CLIP2 complexes in NS-SV-AC cells ( A ) expressing SNAP-AQP5 and in hMSGB from SICCA-NS and SICCA-SD patients ( B ). Arrows indicate the localization of spots. Upper-right corner inserts show representative images used for the signal quantification (scale bar:30 μm). C Quantification of AQP5-CLIP2 complexes in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the mean ± S.D. of AQP5-CLIP2 spots per cell ( n = 5). Data were analyzed using one-tailed Student’s t-test with Welch correction. D Localization of AQP5 and CLIP2 in hMSGB from SICCA-NS and SICCA-SD patients. AQP5 (AF594, red), CLIP2 (AF488, green) (scale bar: 25 μm). E Semi-quantification of AQP5 and CLIP2 localization in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the median with the interquartile range of the labelled area for each protein relative to the entire hMSGB area ( n = 3). Data were analyzed using the one-tailed Mann-Whitney U test. F Co-localization of AQP5 and CLIP2 in SICCA-NS and SICCA-SD hMSGB. Arrows indicate the AQP5-CLIP2 co-localization (yellow area). Images are shown in their original version and modified forms (used for quantification) (scale bar: 50 μm). Negative control (NEG CTRL) was conducted in the absence of primary antibodies. G Semi-quantification of AQP5 and CLIP2 co-localization in SICCA-NS and SICCA-SD hMSGB. Results are expressed as the median with the interquartile range of the merged labelled area for both proteins relative to the entire hMSGB area ( n = 3). Data were analyzed using one-tailed Mann-Whitney U-test. One-tailed statistical tests were performed as values for SICCA-NS were not expected to be below SICCA-SD due to previously reported decreased expression of AQP5 in SICCA-SD hMSGB . C , E , G Statistical significance is indicated as *: p ≤ 0.05

Article Snippet: In addition, the AQP5 fusion protein which was used in a recent publication [ ] and which contains the extracellular loop peptide amino acids 190-265 (human AQP5 Ag14514, Proteintech, Planegg-Martinsried, Germany) was bound to Luminex beads as above, and IgG antibodies against this antigen were measured in all 123 HC and in 223 patients with SjD.

Techniques: Expressing, One-tailed Test, MANN-WHITNEY, Modification, Negative Control

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Effective-components combination improves airway remodeling in COPD rats by suppressing M2 macrophage polarization via the inhibition of mTORC2 activity.

doi: 10.1016/j.phymed.2021.153759

Figure Lengend Snippet: Fig. 2. Effective-components combination (ECC) alleviated airway remodeling on COPD. (a): Masson staining (magnification, 200 ×) and collagen volume fraction. (b): Expression level of α-Smooth muscle actin (α-SMA) (200 ×) and the integral optical density (IOD) of α-SMA. (c): AB-PAS staining imagines of the airway (200 ×, 400 ×). (d), (e): Level of Collagen 3 (COL-3) and Collagen 1 (COL-1) in the bronchoalveolar lavage fluid (BALF). (f), (g): Expression levels of mucin 5AC (MUC5AC) and recombinant aquaporin 5 (AQP5) in the BALF. (h), (i), (j): Levels of MMP-9, TIMP-1 and MMP-12 in the serum. All data showed as the mean ± SEM (n = 6). # p < 0.05, ## p < 0.01, vs. Normal group; * p < 0.05, ** p < 0.01 vs. Model group.

Article Snippet: Finally, rat AQP5 and MUC5AC were detected in the BALF using ELISA kits (Cusabio Biotech Co., Ltd., Wuhan, China).

Techniques: Staining, Expressing, Recombinant

Journal: The Journal of investigative dermatology

Article Title: Mutation in AQP5, encoding aquaporin 5, causes palmoplantar keratoderma Bothnia type.

doi: 10.1038/jid.2013.302

Figure Lengend Snippet: Figure 1. Clinical features of the proband, the identified AQP5 mutation, and immunohistochemical findings of Aquaporin 5 (AQP5). (a) Pedigree of the family with palmoplantar keratoderma Bothnia type. The arrow indicates the proband. (b,c) Palmoplantar hyperkeratosis of the proband. The keratoderma was diffuse, even, yellowish with a clear demarcation. (d) The whitish, spongy appearance of the stratum corneum after exposure to water for less than 1 minute (right hand). (e) The heterozygous mutation c.367A4T identified in AQP5 in the proband. (f) Schematic structure of AQP5 protein and multiple alignment of the loop C region among several species. The mutant site located in the loop C is highlighted with the asterisk. (g) Immunohistochemical staining of AQP5 on a paraffin-embedded section taken from the dorsum of the proband’s hand. Positive staining was noted in most secretory cells of eccrine sweat glands, especially at the apical and basolateral membrane. Bar ¼ 50mm.

Article Snippet: The complementary DNA clone of the whole coding region of human AQP5 was obtained from Origene (Rockville, MD) and subcloned into the pIRES2EGFP vector (Clontech, Mountain View, CA).

Techniques: Mutagenesis, Immunohistochemical staining, Staining, Membrane

Journal: Immune Network

Article Title: Induction of Anti-Aquaporin 5 Autoantibody Production by Immunization with a Peptide Derived from the Aquaporin of Prevotella melaninogenica Leads to Reduced Salivary Flow in Mice

doi: 10.4110/in.2021.21.e34

Figure Lengend Snippet: Compared with mouse Aqp5, Pm AQP and peptide Ags had highly conserved structures at epitope E. (A) The structures of the mouse Aqp5 and PmAqp were constructed on the SWISS-MODEL server using the structure of human AQP5 (PDB 3D9S) as a template. Blue highlighting depicts AQP5E and PmE in mouse Aqp5 and PmAqp, respectively. (B) The structures of the peptide Ags were predicted on the PEP-FOLD3 server.

Article Snippet: After Ag retrieval by incubation in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH=6) at 105°C for 20 min, the sections were blocked with 5% BSA plus goat serum and then incubated with rabbit anti-AQP5 polyclonal Abs (1:100; ATLAS Antibodies, Bromma, Sweden) and mouse sera (1:10) overnight.

Techniques: Construct

Journal: Immune Network

Article Title: Induction of Anti-Aquaporin 5 Autoantibody Production by Immunization with a Peptide Derived from the Aquaporin of Prevotella melaninogenica Leads to Reduced Salivary Flow in Mice

doi: 10.4110/in.2021.21.e34

Figure Lengend Snippet: Ag specificity of anti-AQP5E autoantibodies. (A) Sections of the mouse submandibular glands were stained with rabbit anti-Aqp5 Abs and sera from either the sham or PmE-L-immunized mice followed by Alexa Fluor 488–conjugated donkey anti-rabbit IgG and Alexa Fluor 594–conjugated goat anti-mouse IgG. Arrows indicate stained Aqp5. (B) Pm lysates (Lane 1: 5 µg, Lane 2: 15 µg) and 1 µg of PmE-L peptide (Lane 3) were separated on an acrylamide gel and immunoblotted with sera from sham or PmE-L-immunized mice. The red and green squares indicate PmAqp and PmE-Lm, respectively. Representative images of two experiments with similar results are shown.

Article Snippet: After Ag retrieval by incubation in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH=6) at 105°C for 20 min, the sections were blocked with 5% BSA plus goat serum and then incubated with rabbit anti-AQP5 polyclonal Abs (1:100; ATLAS Antibodies, Bromma, Sweden) and mouse sera (1:10) overnight.

Techniques: Staining, Acrylamide Gel Assay

Journal: Immune Network

Article Title: Induction of Anti-Aquaporin 5 Autoantibody Production by Immunization with a Peptide Derived from the Aquaporin of Prevotella melaninogenica Leads to Reduced Salivary Flow in Mice

doi: 10.4110/in.2021.21.e34

Figure Lengend Snippet: AQP5E-specific Ab clones were identified. Six AQP5E-specific phage clones were identified through biopanning phage Ab libraries. The amino acid sequences of the V H (A) and V L (B) were compared with those of germline gene segments, and three CDRs were annotated. (C) Recombinant IgG produced using the variable genes of clone 44 was subjected to ELISA to confirm specific binding to PmE and AQP5E. (D) The effects of clone 44 recombinant IgG and neutralizing peptides on P f T in MDCK-AQP5 cells were determined 20 min after osmotic challenge and compared with that of control recombinant IgG by t-test. Each column presents the mean±SEM of 2 experiments performed in triplicate.

Article Snippet: After Ag retrieval by incubation in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH=6) at 105°C for 20 min, the sections were blocked with 5% BSA plus goat serum and then incubated with rabbit anti-AQP5 polyclonal Abs (1:100; ATLAS Antibodies, Bromma, Sweden) and mouse sera (1:10) overnight.

Techniques: Clone Assay, Recombinant, Produced, Enzyme-linked Immunosorbent Assay, Binding Assay, Control

Journal: Cell death discovery

Article Title: Role of Sox9 in BPD and its effects on the Wnt/β-catenin pathway and AEC-II differentiation.

doi: 10.1038/s41420-023-01795-2

Figure Lengend Snippet: Fig. 1 In BPD, alveolation is impaired, and Sox9 expression level is significantly changed. A Representative images of H&E staining of lung tissue. Morphology was examined using light microscopy (×200). Scale bar: 50 μm. B Comparison of RAC values in lung tissues. C Relative levels of SPC, AQP5, Sox9 mRNAs in rat lung tissue (n = 6). D Relative levels of SPC, AQP5, Sox9 protein in rat lung tissue (n = 6). E Target band intensity was normalized to β-actin. F Paraffin-embedded tissue sections were double stained with Sox9 (red) + AQP5 (green) immunofluorescence (×400). G Relative levels of SPC, AQP5, Sox9 mRNAs, and protein in the in vitro cell model. Target band intensity was normalized to β-actin. (**P < 0.01, ***P < 0.005, and ****P < 0.001 compared with the control group).

Article Snippet: Flow cytometry detect AEC-II cell differentiation The primary cells were sealed with 5% bovine serum albumin solution at room temperature for 1 h. The AEC-I cells were fluorescently labeled using the AQP5 antibody (Biorbyt, Cambridge, England), and the AEC-II cells were fluorescently labeled with anti-rabbit IgG (H+ L) F(AB’)2 Fragment (PE Conjugate) (CST, KS, USA) and Furin AB (Novus Biologicals, CO, USA).

Techniques: Expressing, Staining, Light Microscopy, Comparison, In Vitro, Control

Journal: Cell death discovery

Article Title: Role of Sox9 in BPD and its effects on the Wnt/β-catenin pathway and AEC-II differentiation.

doi: 10.1038/s41420-023-01795-2

Figure Lengend Snippet: Fig. 2 Changes in the expression of Sox9 during primary AEC-II culture and differentiation (×400). A Sox9 and SPC immunofluorescence double staining. B Sox9 and AQP5 immunofluorescence double staining. C Changes in Sox9 protein expression in an in vitro cell model. D Total protein and plasma protein target bands were normalized to β-actin, and nucleoprotein target band intensity was normalized to PCNA. (This blot is the cropped image)(****P < 0.001 compared with the control group).

Article Snippet: Flow cytometry detect AEC-II cell differentiation The primary cells were sealed with 5% bovine serum albumin solution at room temperature for 1 h. The AEC-I cells were fluorescently labeled using the AQP5 antibody (Biorbyt, Cambridge, England), and the AEC-II cells were fluorescently labeled with anti-rabbit IgG (H+ L) F(AB’)2 Fragment (PE Conjugate) (CST, KS, USA) and Furin AB (Novus Biologicals, CO, USA).

Techniques: Expressing, Double Staining, In Vitro, Clinical Proteomics, Control

Journal: Cell death discovery

Article Title: Role of Sox9 in BPD and its effects on the Wnt/β-catenin pathway and AEC-II differentiation.

doi: 10.1038/s41420-023-01795-2

Figure Lengend Snippet: Fig. 3 Overexpression of Sox9 in BPD rat models can promote the differentiation of AEC II. A Sox9-overexpression improved alveolar development. Scale bar: 50 μm. B Comparison of RAC values in lung tissues (****P < 0.001). RAC increased in the overexpression group compared with that in the model group (n = 6). C Paraffin-embedded tissue sections with Sox9 (red) + SPC (green) immunofluorescence double staining. D Paraffin-embedded tissue sections were double stained with Sox9 (red) + AQP5 (green) immunofluorescence.

Article Snippet: Flow cytometry detect AEC-II cell differentiation The primary cells were sealed with 5% bovine serum albumin solution at room temperature for 1 h. The AEC-I cells were fluorescently labeled using the AQP5 antibody (Biorbyt, Cambridge, England), and the AEC-II cells were fluorescently labeled with anti-rabbit IgG (H+ L) F(AB’)2 Fragment (PE Conjugate) (CST, KS, USA) and Furin AB (Novus Biologicals, CO, USA).

Techniques: Over Expression, Comparison, Double Staining, Staining

Journal: Cell death discovery

Article Title: Role of Sox9 in BPD and its effects on the Wnt/β-catenin pathway and AEC-II differentiation.

doi: 10.1038/s41420-023-01795-2

Figure Lengend Snippet: Fig. 4 Effect of Sox9 on the proliferation and differentiation of AEC-II cells. A Relative levels of SPC mRNAs after Sox9 intervention. B Relative levels of SPC protein after Sox9 intervention. C Target band intensity is normalized to β-actin. (This blot is the cropped image) (*P < 0.05 and ****P < 0.001 compared with the NC). D Flow cytometry analysis of primary cells. E Sox9 (red) + SPC (green) immunofluorescence double staining in the control group. F Sox9 (red) + SPC (green) immunofluorescence double staining in the model group. G Sox9 (red) + AQP5 (green) immunofluorescence double staining in the control group. H Sox9 (red) + AQP5 (green) immunofluorescence double staining in the model group.

Article Snippet: Flow cytometry detect AEC-II cell differentiation The primary cells were sealed with 5% bovine serum albumin solution at room temperature for 1 h. The AEC-I cells were fluorescently labeled using the AQP5 antibody (Biorbyt, Cambridge, England), and the AEC-II cells were fluorescently labeled with anti-rabbit IgG (H+ L) F(AB’)2 Fragment (PE Conjugate) (CST, KS, USA) and Furin AB (Novus Biologicals, CO, USA).

Techniques: Flow Cytometry, Double Staining, Control

Journal: Cell death discovery

Article Title: Role of Sox9 in BPD and its effects on the Wnt/β-catenin pathway and AEC-II differentiation.

doi: 10.1038/s41420-023-01795-2

Figure Lengend Snippet: Fig. 7 Changes in the expression of GAS5 and miR-1912-3p. A Expression level of GAS5 and miR-1912-3p in lung tissue (n = 6). B Expression level of GAS5 and miR-1912-3p in the in vitro cell model. C Relative levels of Sox9 mRNAs and protein after the GAS5 intervention.Target band intensity was normalized to β-actin (This blot is the cropped image). (***P < 0.005 and ****P < 0.001 compared with the control group). D Sox9(red)+ SPC (green) immunofluorescence double staining in primary cell model. E Sox9 (red) + AQP5 (green) immunofluorescence double staining in primary cell model. F Double luciferase report of GAS5 and miR-1912-3p. 7G: Double luciferase report of Sox9 and miR- 1912-3p.

Article Snippet: Flow cytometry detect AEC-II cell differentiation The primary cells were sealed with 5% bovine serum albumin solution at room temperature for 1 h. The AEC-I cells were fluorescently labeled using the AQP5 antibody (Biorbyt, Cambridge, England), and the AEC-II cells were fluorescently labeled with anti-rabbit IgG (H+ L) F(AB’)2 Fragment (PE Conjugate) (CST, KS, USA) and Furin AB (Novus Biologicals, CO, USA).

Techniques: Expressing, In Vitro, Control, Double Staining, Luciferase

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: Effect of AQP5 overexpression and 24-h treatment with hydrogen peroxide on the viability of breast cancer cell lines: ( A ) MCF7 cell line; ( B ) SkBr-3 cell line; ( C ) SUM 159 cell line. Cell lines were either non-transfected (control denoted as ctrl) or transfected with plasmid encoding human AQP5 (denoted as AQP5) or empty plasmid (denoted as pCMV6). The results are expressed as means ± SDs, n = 5. Significance markers (†, ‡, §) represent differences between H 2 O 2 -treated cells and their respective controls (untreated cells) for: †—control (non-transfected) cells, ‡—pCMV6 cells, §—AQP5-overexpressing cells. (*) indicates differences between control (non-transfected) vs. pCMV6 cells for the same treatment. (#) indicates differences between pCMV6 vs. AQP5-overexpressing cells for the same treatment. (+) indicates differences between control (non-transfected) vs. AQP5-overexpressing cells for the same treatment. Significance levels: ‡, §, *, +, # for p < 0.05; §§, **, ++, ## for p < 0.01; ††††, ‡‡‡‡, §§§§, ++++ for p < 0.0001. Abbreviations: H 2 O 2 , hydrogen peroxide.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: Effect of the AQP5 overexpression on the intracellular ROS generation in the breast cancer cell lines. ( A ) Intracellular ROS levels upon a range of H 2 O 2 concentrations in MCF7, SkBr-3, and SUM 159 cells. ( B ) Intracellular ROS levels upon the addition of selected H 2 O 2 concentrations (10 and 40 µM). Cell lines were either non-transfected (control denoted as ctrl) or transfected with plasmid encoding human AQP5 (denoted as AQP5) or empty plasmid (denoted as pCMV6). The results are expressed as means ± SDs, n = 3. Significance markers (†, ‡, §) represent differences between H 2 O 2 -treated cells and their respective controls (untreated cells) for: †—control (non-transfected) cells, ‡—pCMV6 cells, §—AQP5-overexpressing cells. (#) indicates differences between pCMV6 vs. AQP5-overexpressing cells for the same treatment. (+) indicates differences between control (non-transfected) vs. AQP5-overexpressing cells for the same treatment. Significance levels: †, ‡, § for p < 0.05; ‡‡, ++, ## for p < 0.01; ††††, ‡‡‡‡, §§§§ for p < 0.0001. Abbreviations: ROS, Reactive oxygen species; H 2 O 2 , hydrogen peroxide.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques: Over Expression, Transfection, Control, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: AQP5 and AQP3 mRNA and protein expression in breast cancer cell lines: ( A ) MCF7, ( B ) SkBr-3, ( C ) SUM 159, and ( D ) representative western blots. Cell lines were either non-transfected (control denoted as ctrl, shown in shades of red with increasing intensity as H 2 O 2 concentration rises) or transfected with plasmid encoding human AQP5 (denoted as AQP5, shown in shades of blue with increasing intensity as H 2 O 2 concentration rises) or empty plasmid (denoted as pCMV6, shown in shades of green with increasing intensity as H 2 O 2 concentration rises). The results are expressed as means ± SDs, n = 3. Significance markers (§) represent differences between H 2 O 2 -treated and untreated (control) AQP5-overexpressing cells. (#) indicates differences between pCMV6 vs. AQP5-overexpressing cells for the same treatment. (+) indicates differences between control (non-transfected) vs. AQP5-overexpressing cells for the same treatment. Significance levels: §, # for p < 0.05; §§, ++ for p < 0.01; +++ for p < 0.001; §§§§, ++++, #### for p < 0.0001. Abbreviations: AQP3, aquaporin 3; AQP5, aquaporin 5; H 2 O 2, hydrogen peroxide.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Transfection, Control, Concentration Assay, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: Effects of the AQP5 and hydrogen peroxide treatment on the NRF2 signaling pathway: mRNA expression of NRF2 and protein expression of NRF2, KEAP1, AKR1B10, and HO-1 in breast cancer cell lines: ( A ) MCF7, ( B ) SkBr-3, and ( C ) SUM 159. Cell lines were either non-transfected (control denoted as ctrl, shown in shades of red with increasing intensity as H 2 O 2 concentration rises) or transfected with plasmid encoding human AQP5 (denoted as AQP5, shown in shades of blue with increasing intensity as H 2 O 2 concentration rises) or empty plasmid (denoted as pCMV6, shown in shades of green with increasing intensity as H 2 O 2 concentration rises). The results are expressed as means ± SDs, n = 3. Significance markers (†, ‡, §) represent differences between H 2 O 2 -treated cells and their respective controls (untreated cells) for: †—control (non-transfected) cells, ‡—pCMV6 cells, §—AQP5-overexpressing cells. (*) indicates differences between control (non-transfected) vs. pCMV6 cells for the same treatment. (#) indicates differences between pCMV6 vs. AQP5-overexpressing cells for the same treatment. (+) indicates differences between control (non-transfected) vs. AQP5-overexpressing cells for the same treatment. Significance levels: ‡, §, *, +, # for p < 0.05; ††, ‡‡, §§, **, ++, ## for p < 0.01; ***, ### for p < 0.001; ††††, ‡‡‡‡, ****, ++++ for p < 0.0001. Abbreviations: NRF2, nuclear factor erythroid 2–related factor 2; KEAP1, Kelch-like ECH-associated protein 1; HO-1, Heme oxygenase 1; AKR1B10, Aldo-keto reductase family 1 member B10.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques: Expressing, Transfection, Control, Concentration Assay, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: Effects of the AQP5 and hydrogen peroxide treatment on the PI3K/AKT signaling pathway in breast cancer cell lines: ( A ) MCF7, ( B ) SkBr-3, ( C ) SUM 159, and ( D ) representative western blots. Cell lines were either non-transfected (control denoted as ctrl, shown in shades of red with increasing intensity as H 2 O 2 concentration rises) or transfected with plasmid encoding human AQP5 (denoted as AQP5, shown in shades of blue with increasing intensity as H 2 O 2 concentration rises) or empty plasmid (denoted as pCMV6, shown in shades of green with increasing intensity as H 2 O 2 concentration rises). The results are expressed as means ± SDs, n = 3. Significance markers (†, ‡, §) represent differences between H 2 O 2 -treated cells and their respective controls (untreated cells) for: †—control cells (non-transfected), ‡—pCMV6 cells, §—AQP5-overexpressing cells. (*) indicates differences between control (non-transfected) vs. pCMV6 cells for the same treatment. (#) indicates differences between pCMV6 vs. AQP5-overexpressing cells for the same treatment. (+) indicates differences between control (non-transfected) vs. AQP5-overexpressing cells for the same treatment. Significance levels: ‡, §, *, +, # for p < 0.05; ††, ‡‡, §§, **, ++, ## for p < 0.01. Abbreviations: PI3K, Phosphoinositide 3-kinases; pAKT, phosphorylated protein kinase B; AKT, protein kinase B; PTEN, Phosphatase and tensin homolog.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques: Western Blot, Transfection, Control, Concentration Assay, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: Effects of the AQP5 and hydrogen peroxide treatment on the FOXO1 and FOXO3a mRNA and protein expression in breast cancer cell lines: ( A ) MCF7, ( B ) SkBr-3, ( C ) SUM 159. Cell lines were either non-transfected (control denoted as ctrl, shown in shades of red with increasing intensity as H 2 O 2 concentration rises) or transfected with plasmid encoding human AQP5 (denoted as AQP5, shown in shades of blue with increasing intensity as H 2 O 2 concentration rises) or empty plasmid (denoted as pCMV6, shown in shades of green with increasing intensity as H 2 O 2 concentration rises). The results are expressed as means ± SDs, n = 3. Significance markers (†, ‡, §) represent differences between H 2 O 2 -treated cells and their respective controls (untreated cells) for: †—control (non-transfected) cells, ‡—pCMV6 cells, §—AQP5-overexpressing cells. (*) indicates differences between control (non-transfected) vs. pCMV6 cells for the same treatment. (#) indicates differences between pCMV6 vs. AQP5-overexpressing cells for the same treatment. (+) indicates differences between control (non-transfected) vs. AQP5-overexpressing cells for the same treatment. Significance levels: †, ‡, §, *, +, # for p < 0.05; ††, ‡‡, ** for p < 0.01; †††, ***, +++, ### for p < 0.001; ++++ for p < 0.0001. Abbreviations: FOXO1, forkhead box class O 1; FOXO3a, forkhead box class O 3a.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques: Expressing, Transfection, Control, Concentration Assay, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: Experimental design. ( A ) Establishing cell lines stably expressing AQP5 or empty vector (pCMV6); ( B ) treatment with H 2 O 2 . Abbreviations: HR+—hormone receptor positive; HER2+ HER2-overexpressing cells; TNBC—triple-negative (hormone (estrogen and progesterone) receptor-negative and HER2-negative breast cancer.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques: Stable Transfection, Expressing, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: Primer sequences used for qPCR analysis.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Influence of AQP5 on the Response to Hydrogen Peroxide in Breast Cancer Cell Lines

doi: 10.3390/ijms26073243

Figure Lengend Snippet: Primary antibodies used for western blot.

Article Snippet: Breast cancer cell lines (3 × 10 5 cells per well) were seeded in six-well plates (TPP, Trasadingen, Switzerland), allowed to attach overnight, and transfected the following day with either the AQP5 (Myc-DDK-tagged)-Human-pCMV6 vector (RC206069) or the pCMV6-Entry vector (mock control, PS100001) (Origene, Rockville, MD, USA) using Lipofectamine 3000 (L3000001, Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s protocol.

Techniques: Western Blot