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. 2021 Apr 20;22(8):4287. doi: 10.3390/ijms22084287

Figure 4.

Figure 4

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 Supplementary Movie 1 and Supplementary Movie.