Fig. 5

siRNA knockdown of essential ciliary genes IFT88 and IFT172 leads to axon outgrowth and branching defects. A Gene-specific siRNA significantly downregulates the expression of IFT88 and IFT172 as compared to control samples treated with non-specific scrambled siRNA or when untreated. B Knockdown (KD) of IFT88 and IFT172 expression results in fewer ciliated LUHMES neurons than in the control (scrambled siRNA n = 271; siRNA IFT88 n = 314; siRNA IFT172 n = 405). C Differently from the WT vs RFX2 -/- backgrounds (cf. Fig. 3C), a significantly higher percentage of cilia in IFT88 KD (n = 66) and IFT172 KD (n = 72) appears to be vertically oriented as compared to the control (n = 260), strongly suggesting an increased total amount of severely truncated/shorter cilia in horizontal or tilted orientation indistinguishable from the vertical orientation. D Cilia length is quantified by the PyT method and confirms that cilia of IFT88 KD (n = 66) and IFT172 KD (n = 72) neurons are shorter than the control ones (n = 260). E–G Immunocytochemistry detection of nuclei (Hoechst staining), centrosomes/basal bodies (PCNT marker) and cilia (ARL13B marker) displays the distribution of vertical (and truncated) cilia in IFT88 KD and IFT172 KD neurons. Arrowheads exemplify centrosomes of non-ciliated neurons. H Reduced expression of IFT88 and IFT172 leads to axon outgrowth defects of ciliated LUHMES neurons (scrambled siRNA n = 217; siRNA IFT88 n = 148; siRNA IFT172 n = 218), similar to non-ciliated neurons (scrambled siRNA n = 54; siRNA IFT88 n = 166; siRNA IFT172 n = 187). I Axons of IFT88 KD (n = 29) and IFT172 KD (n = 43) ciliated neurons are significantly less branched than the axons of control neurons (n = 55), similar to the axons of non-ciliated neurons (scrambled siRNA n = 22; siRNA IFT88 n = 30; siRNA IFT172 n = 33). L-N Representative images of immunocytochemistry of axon branching defects in IFT88 KD and IFT172 KD ciliated neurons (circles localize primary cilia; arrowheads point to axon branches); Hoechst staining – nucleus, ARL13B – ciliary marker, Phalloidin – cytoskeleton/F-actin marker. Mean values are shown ± s.d. (I) and ± s.e.m. (A-D, H). The results are from two independent experiments with a minimum of three technical replicates each. We conducted regular one-way ANOVA (A-B, D) and two-way ANOVA analyses (C, H-I) (not repeated measures) with multiple comparisons (Bonferroni’s test) between groups. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001