Fig. 3

Mutation in TXNDC15 causes ciliogenesis defect in patient cells and aberrant localization of TMEM67 using TXNDC15 RNAi. a and b Immunofluorescence images of serum-starved fibroblasts from the affected individual in family 3 (II:1) and control fibroblasts (a) and TXNDC15 siRNA in human hTERT-RPE1 cells (b) stained for the ciliary marker ARL13B (green), gamma tubulin (red), and DNA (blue). Compared to controls, fibroblasts from II:1 in family 3 and TXNDC15 siRNA knockdown showed a marked ciliogenesis defect. c Bar graph showing the significant reduction in the number of ciliated fibroblast cells derived from individual II:1 from family 3. d Bar graph showing a significant reduction in the number of ciliated TXNDC15 siRNA cells compared to negative control scrambled siRNA. e Bar graph showing the efficiency of TXNDC15 siRNA compared to negative control scrambled siRNA (siScr) as quantified by qRT-PCR for the TXNDC15 transcript. f Bar graph showing the average increase in the cilium length following TXNDC15 siRNA knockdown compared to negative control scrambled siRNA (siScr). g Immunofluorescence microscopy images of serum-starved hTERT-RPE1 cells following TXNDC15 siRNA knockdown stained for the ciliary marker ARL13B (red), the transition zone marker TMEM67 (green), and DNA (blue) showing the incorrect localization of the TMEM67 ciliary receptor to the transition zone compared to negative control scrambled siRNA (siScr). h Bar graph showing an elongation in the total cilia length and elongation of the transition zone due to mislocalization of TMEM67 (arrowheads in g). Statistical significance of pair-wise comparisons are indicated by * p < 0.05, ** p < 0.01, and **** p < 0.0001 (panels c–f, Student’s paired t test for n = 3 biological replicates; and panel h, Pearson’s chi-squared test for n = 3 biological replicates)