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. 2024 Sep 26;25(19):10369. doi: 10.3390/ijms251910369

Figure 2.

Figure 2

Decreased anticancer effect of vinblastine (VBL) on cells treated repeatedly with VBL was associated with primary cilium formation and NPHP3 expression. (A–E) Wildtype (WT) cells were treated with VBL once or repeatedly three (3×) to five (5×) times. Then each cell population was plated and treated with 1nM of VBL for 24 h. Cell viability was measured by the trypan blue exclusion assay (A). VBL-treated cells were observed under the bright-field microscope (B). The cells were fixed and stained with antibodies against Ac-tubulin (green) and Arl13b (orange-red). The nucleus was stained with DAPI (blue). The ciliated HeLa cells (n > 500 cells) were counted (C). The primary cilium was observed and photographed at 400× or 1000× magnification with 40× or 100× objectives, respectively under a fluorescence microscope. The image with primary cilia at 1000× magnification is representative of ≈30 pictures. White arrows indicated primary cilia (D). Total RNA was prepared by using NucleoZOL® and the expression level of NPHP3 was measured by RT-PCR (E left top). Cell lysates were prepared and each protein was detected by western blot analysis (E left bottom). The density of NPHP3 amount in repeatedly (3× or 5×) VBL-treated group was quantitated with NIH image analysis software (version 1.54 h) and normalized to control. Fold changes in NPHP3 were represented with a bar graph (E right). (F) WT cells and VBL-treated cells repeatedly with VBL three (3×) or five (5×) times were transfected with pEZX-PG02-NPHP3-promoter (pmt) Gaussia luciferase (Gluc) plasmid and incubated in the presence or the absence of FBS for up to 24 h. The activity of Gluc in cultured media was measured with a luminometer using a Gluc substrate. Data were representative of four experiments. Processing (such as changing brightness and contrast) is applied equally to controls across the entire image (C,E). Data were representative of four experiments. Processing (such as changing brightness and contrast) is applied equally to control across the entire image (B,D). Data in bar graphs represents the means ± SEM (A,C,E right, F). && p < 0.01; significantly different from the VBL-untreated control group (A). * p < 0.05, ** p < 0.01; significantly different from VBL-treated WT cells (A,C) or WT control cell (E right, F).