Fig 2: Drugs targeting mitochondrial could rescue the cilia beating and related phenotypes.A Expression analysis of DNAH9, CCDC114, LLRC6, and GAS8 in ciliated epithelium digested from WT and Dnah9 KD mouse airway organoid. Green, ace-tubulin; red, DNAH9, LRRC6, CCDC114, and GAS8; blue, DAPI. Scale bar = 10 µm. B The quantified DNAH9, LRRC6, CCDC114, and GAS8 fluorescent intensities in cilia analyzed by ImageJ in (A). *P < 0.05; ns no significance, n = 3 per group. C The expression of immune factors in mouse airway organoid after 4-day poly (I:C) treatment. *P < 0.05; n = 3 per group. D Stepwise protocol for the organoid drug screen. E CBF of Dnah9 KD mouse airway organoid after 16-day drug treatment. *P < 0.05, n = 10 per group. F Western blot of TOMM20 and COX-IV in KD mouse airway organoid treated with ATP (10 µM, 50 µM) and NADH (10 µM, 25 µM, 50 µM) and WT organoid.

Fig 3: Ciliated cells from Dnah9 KD mice show reduced expression of interactors CCDC114 and GAS8 protein.A Immunostaining analysis of DNAH9, CCDC114, LLRC6, and GAS8 in ciliated cells from Dnah9 KD nasal epithelium. Green, ace-tubulin; red, DNAH9, LRRC6, CCDC114, and GAS8; blue, DAPI; Scale bar = 10 µm. B Results of immunofluorescence images quantification corresponding to ciliated cells in (A). *P < 0.05; ns no significance, n = 3 per group. C Western blot analysis showing the protein expression of CCDC114, GAS8, and LRRC6 in lung tissues of WT and Dnah9 KD mice. GAPDH is the endogenous control. The right pattern is the statistical analysis of relative protein intensities revealed by western blot. *P < 0.05; ns no significance, n = 3 per group. D Co-IP results demonstrating interactions between DNAH9 and CCDC114/GAS8.

Fig 4: Flagellar ultrastructure is disorganized, and axonemal proteins express abnormally in Dnah2-null sperm. (A,B) Scanning electron microscopy images of Dnah2+/? (A) and Dnah2?/? (B) sperm. Dnah2?/? sperm display absence of (left) or an irregular (right) mitochondrial helical sheath in the midpiece of the sperm tail, along with abnormal head shapes. Arrowheads indicate annulus. H, head; MP, midpiece; and PP, principal piece. (C,D) Transmission electron microscopy images of Dnah2+/? (C) and Dnah2?/? (D) sperm. Representative cross-section images of TEM reveal conformational defects in the midpiece of Dnah2?/? sperm. Incomplete 9+2 axonemal structure (left), delocalized outer dense fibers (ODF, arrow) and microtubule doublet (middle, arrowhead), and lack of the axonemal components (right) were observed in the Dnah2?/? sperm. Inner (yellow asterisk) and outer (white asterisk) dynein arm structure were observed from microtubule doublet occasionally (left, inset; scale bar = 50 nm). Microtubule doublets are numbered with the absence of the corresponding microtubule doublet in red. M, mitochondria. (E) Confocal fluorescence images of axonemal proteins in WT and Dnah2?/? sperm. Dnah2?/? sperm do not express another IDA component (DNAH1, magenta) and a radial spoke protein (RSPH3, green) but an ODA protein (DNAH9, yellow). Flagella microtubules (AcTub, white) and sperm heads (Hoechst, blue) serve as controls. The corresponding DIC images are shown together for Dnah2?/? sperm.

Fig 5: Dnah9 KD mouse organoid shows reduced ciliary beating and increased secretion of inflammatory factors.A Bright-field images depicting mouse airway organoid phenotypes. Scale bar = 100 µm. B RT-PCR shows low DNAH9 expression in Dnah9 KD mouse airway organoid relative to WT. *P < 0.05; n = 3 per group. C CBF of Dnah9 KD group was significantly lower than WT group. *P < 0.05. n = 10 per group. D Whole-mount mouse airway organoid immunofluorescence shows markers for basal cells (KRT5, red), ciliated cells (acetylated a-tubulin, green), club cells (CC-10, green) and goblet cells (MUC5AC, green). Scale bar = 10 µm. E Confocal microscopy images of mouse airway organoid with a pH probe show lower pH value in the Dnah9 KD group than WT. The stronger red color indicates lower pH value in the epithelial cell of the Dnah9 KD group. Scale bar = 10 µm.