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. 2022 Mar 21;12(3):502. doi: 10.3390/jpm12030502

Figure 2.

Figure 2

RNA and protein studies in RPGR c.1415 − 9A>G variant fibroblast cells. (A) Sanger sequencing of PCR-amplified cDNA across the exon 11–12 junction showed an additional eight base pairs (blue intron) interrupting canonical splicing (green exons) in the patient cells, compared to the control. (B) The expression of RPGR, determined by RT-qPCR, was decreased in patient fibroblasts compared to Control 1 cells at exons 1–3 and exons 11–12 (unpaired t-test, * p < 0.05, SEM from n = 3 independent experiments per line). Expression levels are relative to both the HPRT and POLR2A housekeeper gene expression. (C) (i) Immunofluorescence staining of the primary cilium in control fibroblast cells using acetylated α-tubulin (red) showed RPGR (green) localisation to the ciliary TZ (white arrow). (ii) The TZ localization of RPGR was lost in the patient’s fibroblast cell cilia (white arrow outline). (iii) Quantification of RPGR presence at the cilia TZ confirmed a decreased presence of RPGR at the TZ in the patient’s cells compared with the control (unpaired t-test, ** p < 0.01, SEM from n = 3 independent experiments per line, n = 159 control cilia counted, n = 219 patient cilia counted). The number of cilia with RPGR staining in the ciliary TZ was manually counted, and this was expressed as a percentage of the total number of cilia present.