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

Figure 6.

Figure 6

NPHP3 regulated intraflagellar transport protein 88 (IFT88) expression. (A) Pre-designed IFT88 promoter (HPRM46777) was obtained from GeneCopoeia Inc. The mutant IFT88 promoter (pmt) was prepared by the deletion of 1396 bp (−2308~−912). (B–D) AccuTarget™ negative control siRNA or IFT88-siRNA were co-transfected with pEZX-PG02-IFT88-wildtype (WT)-pmt-Gaussia luciferase (Gluc) plasmids (C,D) or pEZX-PG02-IFT88 mutant-pmt-Gluc plasmids (D) into HeLa cells. Cells were incubated for 24 h (B–D) or 36 h (C). Cell lysates were prepared and IFT88 protein was detected by western blot analysis (B left top). Expression level of NPHP3 transcripts was measured by RT-PCR (B left bottom). The density of NPHP3 amount in the NPHP3-siRNA-transfected group was quantitated with NIH image analysis software (version 1.54h) and normalized to control. Fold changes in NPHP3 were represented with a bar graph (B right). The activity of Gluc in cultured media was measured with a luminometer using a Gluc substrate. (C,D). (E–G) pcDNA3.1 or pcDNA6-Flag-NPHP3 plasmids were co-transfected into HeLa cells with pEZX-PG02-IFT88-WT-pmt-Gluc plasmids (E,G) or pEZX-PG02-IFT88 mutant pmt-Gluc plasmids (G). Cells were incubated for 24 h (E–G) or 36 h (E). Cell lysates were prepared and flag protein was detected by western blot analysis (F, top). The expression level of NPHP3 transcripts was measured by RT-PCR (F bottom). The activity of Gluc in cultured media was measured with a luminometer using a Gluc substrate. (E,G). Data were representative of four experiments. Processing (such as changing brightness and contrast) is applied equally to control across the entire image (B left, F). Data were representative of four experiments. Data in bar graphs represents the means ± SEM (B right, C,D,E,G). * p < 0.05, ** p < 0.01; significantly different from control siRNA-transfected group (B right, C,D) or pcDNA3.1-transfected group at each time point (E,G). && p < 0.01; significantly different from WT IFT88-pmt plasmid-transfected and control siRNA-transfected (D) or pcDNA3.1 plasmid-transfected group (G). ## p < 0.01; significantly different from WT IFT88-pmt plasmid-transfected and NPHP3-siRNA-transfected (D) or pcDNA6-Flag-NPHP3 plasmid-transfected group (G).