Journal: International Journal of Molecular Sciences

Article Title: AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions

doi: 10.3390/ijms22084287

Figure Lengend Snippet: Fold changes in top-10 upregulated and downregulated genes.

Article Snippet: The samples were then separated with SDS-PAGE gel and immunoblotted with the antibody against AQP3 (Alomone Labs Ltd., Jerusalem, Israel, 1/200), GAPDH (BioLegend, San Diego, CA, USA), or β-actin (Santa Cruz Biotechnology) or α-tubulin (Santa Cruz Biotechnology). β-actin, GAPDH, and α-tubulin were used as loading controls.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions

doi: 10.3390/ijms22084287

Figure Lengend Snippet: The effect of AQP3 on spatiotemporal dynamics of protrusions. ( A ) Polymerase chain reaction and ( B ) Quantitative real-time reverse transcription-polymerase chain reaction of the transcript levels of the organic hydroxyl transport genes. The data shown here represent three independent experiments, and the values represent the mean ± SEM of triplicate samples. The level of each mRNA was normalized to that of the GAPDH mRNA in the same sample and presented as the fold-change over that of the 2D culture control cells. The differences in expression levels were evaluated for significance using two-tailed t-tests with unequal variance. * p < 0.05; ** p < 0.01; and *** p < 0.001. ( C ) The Western blot of AQP3 levels in 2D and 3D culture cells. The A549 cells were harvested following twenty-four h seeding to confirm AQP3 protein levels. α-tubulin was used as an internal control. ( D ) The downregulation of AQP3 following transfection of AQP3 siRNA. The A549 cells were pre-transfected for twenty-four h, and further incubated for twenty-four h to confirm the knockdown of AQP3 mRNA levels. The level of each mRNA was normalized to that of the GAPDH mRNA in the same sample and presented as the fold-change over that of the each of control groups. ( E ) The Western blot of AQP3 in 3D culture cells following transfection of AQP3 siRNA. Twenty-four h following siRNA transfection, the A549 cells were harvested to confirm the knockdown of AQP3 by evaluating the AQP3 protein levels with Western blotting. α-tubulin was used as an internal control. ( F ) The phase-contrast micrograph showing the morphologies of the A549 cells grown in 2D (left) and 3D (right) cultures. The micrograph ( F ) and representative live-cell three-dimensional holotomography ( G ) of the A549 cells showing the effect of AQP3 knockdown on the growth behavior of the A549 cells in 2D (left) and 3D (right) cultures. Twenty-four h following siRNA transfection in the 2D culture, the cells were further incubated in the 2D or 3D culture condition. Additionally, see .

Article Snippet: The samples were then separated with SDS-PAGE gel and immunoblotted with the antibody against AQP3 (Alomone Labs Ltd., Jerusalem, Israel, 1/200), GAPDH (BioLegend, San Diego, CA, USA), or β-actin (Santa Cruz Biotechnology) or α-tubulin (Santa Cruz Biotechnology). β-actin, GAPDH, and α-tubulin were used as loading controls.

Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Two Tailed Test, Western Blot, Transfection, Incubation

Journal: International Journal of Molecular Sciences

Article Title: AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions

doi: 10.3390/ijms22084287

Figure Lengend Snippet: A549 cell aggregation is an active process. ( A ) The effect of ROCK on A549 cell aggregation. The phase-contrast micrographs show the morphologies of the A549 cells following treatment with a ROCK inhibitor, Y-27632, and the Rho activator Ⅱ. ( B ) The effects of AQP3 knockdown with siRNA on actomyosin cytoskeleton remodeling. A549 cells were stained with anti-AQP3 antibody, followed by Fluorescein-conjugated antibody (green). The actin microfilaments were stained with rhodamine-conjugated phalloidin (red), and the nuclei were stained with DAPI (blue). ( C ) The effects of AQP3 knockdown with siRNA on the apoptosis signaling pathway. Twenty-four h following siRNA transfection in 2D culture condition, the cells were further incubated in the 2D or 3D culture condition. Then, cells were harvested to confirm the effect of AQP3 knockdown on the apoptotic molecular signatures using Western blotting. α-tubulin was used as an internal control.

Article Snippet: The samples were then separated with SDS-PAGE gel and immunoblotted with the antibody against AQP3 (Alomone Labs Ltd., Jerusalem, Israel, 1/200), GAPDH (BioLegend, San Diego, CA, USA), or β-actin (Santa Cruz Biotechnology) or α-tubulin (Santa Cruz Biotechnology). β-actin, GAPDH, and α-tubulin were used as loading controls.

Techniques: Staining, Transfection, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: AQP3 Increases Intercellular Cohesion in NSCLC A549 Cell Spheroids through Exploratory Cell Protrusions

doi: 10.3390/ijms22084287

Figure Lengend Snippet: Protrusion controls migration and invasion of A549 cancer cells. ( A ) The phase-contrast micrograph of the migration of NSCLC A549 cells (left) and quantification (right) in the Transwell assay. The quantification of migrated cells treated with jasplakinolide represents three independent experiments, and the values represent the mean ± SEM of triplicate samples. The differences in expression levels were evaluated for significance using unpaired two-tailed t-test. * p < 0.05; ** p < 0.01; and *** p < 0.001. ( B ) Representative images (left) and quantification (right) of A549 spheroid transfected with siRNA AQP3 . Twenty-four h following siRNA transfection in 2D culture condition, the cells were further incubated in the 2D or 3D culture condition. The migration capacity of the A549 spheroids with a knocked down level of AQP3 was much lower than that of the negative control cells. The differences in expression levels were evaluated for significance using one-way ANOVA followed by Tukey’s post-hoc tests. * p < 0.05; ** p < 0.01; and *** p < 0.001.

Article Snippet: The samples were then separated with SDS-PAGE gel and immunoblotted with the antibody against AQP3 (Alomone Labs Ltd., Jerusalem, Israel, 1/200), GAPDH (BioLegend, San Diego, CA, USA), or β-actin (Santa Cruz Biotechnology) or α-tubulin (Santa Cruz Biotechnology). β-actin, GAPDH, and α-tubulin were used as loading controls.

Techniques: Migration, Transwell Assay, Expressing, Two Tailed Test, Transfection, Incubation, Negative Control