Figure 5.

RNAi Silencing of Paramecium DNAH9 Shows Severe Defective Motile Cilia Phenotype

(A) Quantitative RT-PCR assessment of expression level of DNAH9 orthologs after RNAi knockdown in Paramecium. The expression level is reduced by >80% in the DNAH9-RNAi cells compared to its expression in the control ND7-RNAi cells (ND7 gene not associated with cilia motility); p < 0.001, independent samples t test.

(B) Reduced cilia beat frequency after DNAH9-RNAi silencing. At 48 hr of RNAi, the ciliary beat frequency of DNAH9 knockdown cells is reduced by ∼50% compared to ND7 control knockdown cells. 7–10 paramecia evaluated per condition; p < 0.001, Mann-Whitney U test.

(C) Analysis of swimming velocity of Paramecium DNAH9 and ND7 knockdown cells shows a significant reduction (∼70%) in velocity of DNAH9 knockdown cells compared to the control ND7 knockdown cells over 72 hr of RNAi silencing. More than 150 paramecia were evaluated per condition; p < 0.001, independent samples t test.

(D) Paramecium swimming pattern visualized in dark-field microscopy, shown by the Z projection of track recordings, captured using a 10× objective after 24 and 48 hr of RNAi feeding. DNAH9 knockdown cells (right) show a severe motility phenotype compared to the control ND7 silenced cells (left).

(E) Transmission electron micrographs of Paramecium cilia in cross-section showing normal 9+2 arrangement for the control ND7 knockdown (left, outer dynein arms indicated by white arrows) and absence of the outer (gray arrows) dynein arms in the DNAH9 knockdown cells (right).

(F) Quantification of TEM dynein arms counts across >40 cross-sections per knockdown experiment showed reduced ODA and IDA in DNAH9 knockdown cells compared to ND7 control knockdown cells with ODA loss more extensive than IDA loss. p > 0.001, independent samples t test. All error bars indicate SEM.