Fig 1: Rescue of hH723R-Pendrin Expression and Function by Japanese Encephalitis Virus (JEV)(A) Surface biotinylation assays were performed in PANC-1 cells expressing hH723R-pendrin. Blockade of endoplasmic reticulum (ER-to-Golgi traffic via Arf1-Q71L overexpression induced the cell-surface expression of core-glycosylated H723R-pendrin (band B). Overexpression of DNAJC14 (3× HA-tagged) alone or with Hsc70 (Myc-tagged) induced the cell-surface expression of core-glycosylated H723R-pendrin in PANC-1 cells. (B) Surface biotinylation assay in PANC-1 cells transfected with plasmids encoding wild-type (WT)- and hH723R-pendrin after treatment with JEV (106 pfu). Band B, ER core-glycosylated immature pendrin; band C, fully glycosylated mature pendrin. The first lane is the positive control cells, in which the band C form of WT-pendrin was expressed on the cell surface. The second lane is the negative control cells, in which the cell-surface band C form of hH723R-pendrin was not expressed and the band B form was weakly expressed. The last lane is cells treated with JEV (106 pfu), which induced the cell-surface expression of the band B form of hH723R-pendrin on the cell surface. (C) The surface expression ratio of pendrin (compared to total lysate pendrin) was significantly increased by JEV treatment (106 pfu) (n = 8 in each group). ∗∗∗p < 0.001. (D) Cl−/HCO3− exchange activity measured by recording the pH-sensitive fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in PANC-1 mock cells and in WT- and hH723R-pendrin stably expressing cells, as detailed in the Materials and Methods. (E) The quantitation of multiple experiments showed that the Cl−/HCO3− exchange activity was significantly increased in the cells expressing hH723R-pendrin with JEV (3 × 106 pfu) treatment (n = 6 in each group). ∗∗p < 0.01.
Fig 2: Japanese Encephalitis Virus (JEV) Restores the Expression of Cell-Surface hH723R-PendrinCell-surface pendrin expression was examined in PANC-1 cells with immunostaining. (A) The cells were stained for pendrin (anti-Flag, green) and co-stained for DNAJC14 (red). Nuclei were counterstained with DAPI. DIC images were overlapped in the second lane to clarify the boundary of cells (dashed line). Yellow arrow line indicates the axis for the quantification of fluorescence. In the lower panel, fluorescence intensities for pendrin (green) and DAPI (blue) were quantified along the long axis of each cell, where the yellow box refers to the position of the plasma membrane. Scale bar, 20 μm. (B) The plasma membrane fraction of pendrin was calculated as fluorescence signals between a distance of 5 μm from the cell membrane-to-total fluorescence signal in the cell. Compared to that in lane 2 (hH723R-pendrin), both groups treated with JEV at different titers (1 × 104 and 5 × 104 pfu) showed increased expression of pendrin in the cell membrane (n = 4). ∗∗∗p < 0.001.
Fig 3: Overexpression of DNAJC14 in hH723R-Pendrin Transgenic Mouse(A) For activation of DNAJC14 in the established hH723R-pendrin transgenic (hH723R Tg) mouse model, hH723R Tg mice were crossed with Tg mice overexpressing DNAJC14. (B) Based on western blotting of the cochlea, expression of pendrin was increased by overexpressing DNAJC14. (C) Quantification of the density of pendrin is depicted in (AU, arbitrary unit) (n = 4 in each group). ∗p < 0.05. (D) DNAJC14 expression (red) was identified in the DNAJC14 Tg(+);hH723R Tg(+);mPDS(−/−) mice, and the expression of pendrin was increased in most of the cochlear tissue (Pax2-Cre dependent), as shown by immunostaining using the Alexa 488 secondary antibody (green). DAPI (blue) was used for nuclear staining. Scale bar, 100 μm; sm, scala media; st, scala tympani; sv, scala vestibule.
Fig 4: DNAJC14 Overexpression Results in the Insufficient Rescue of Hearing Function and Vestibular Abnormal Morphologies in hH723R Transgenic Mice(A and B) Auditory brainstem responses by click and tone-burst stimuli were measured in wild-type (WT), hH723R Tg(+);mPDS−/−, and DNAJC14 Tg(+);hH723R Tg(+);mPDS−/− mice. Auditory threshold was not recovered even if DNAJC14 was overexpressed in hH723R Tg(+);mPDS−/− mice based on click (A) and tone-burst (B) stimuli (n = 8 mice in each group). (C) Immunostaining of endolymphatic duct, endolymphatic sac, and ampulla in P0 mice of each strain. hH723R-pendrin (green) was predominantly identified in the epithelial lines (endolymphatic duct, endolymphatic sac, and ampulla) as well as supporting cells, around the endolymphatic duct and common crus (possibly ectopic expression of pendrin depending on CMV promoter) of DNAJC14 Tg(+);hH723R Tg(+);mPDS−/− mice. DNAJC14, red; DAPI, blue; ed, endolymphatic duct; cc, common crus; es, endolymphatic sac; ca, crista ampullaris; cu, cupula; white arrow, ed in wild-type; scale bar, 100 μm. (D) Light microscopy and surface electron microscopy were used to observe otoconia morphology (red arrow). Giant otoconia were identified in the macula of hH723R Tg(+);mPDS−/− mice (middle), whereas normal-sized otoconia were observed in the macula of WT mice (upper). Giant otoconia were also observed in the macula of DNAJC14 Tg(+);hH723R Tg(+);mPDS−/− mice (lower). Scale bar, 100 μm.
Fig 5: Comparison of Cochlear Histology and KCNJ10 Expression in Mice with Different Genotypes(A) H&E staining of cochlea from wild-type (WT), mPDS−/−, hH723R Tg(+);mPDS−/−, and DNAJC14 Tg(+);hH723R Tg(+);mPDS−/− mice at 4–5 weeks of age was performed. (B) Cochlear hydrops in DNAJC14 Tg(+);hH723R Tg(+);mPDS−/− mice were significantly reduced (n = 6 mice in each group). **p < 0.01; WT, wild-type; KO, mPDS−/−. (C) The stria vascularis (SV) and spiral ligament (SL) depth were thicker in DNAJC14 Tg(+);hH723R Tg(+);mPDS−/− mice than in hH723R Tg(+);mPDS−/− mice. (D) SV depth was statistically compared (n = 6 mice in each group). (E) SL depth was statistically compared (n = 6 mice in each group). (F) Whole-mount of cochlear outer hair cells (OHC) at the middle turn. (G) DNAJC14 expression increased the number of OHCs (n = 6 mice in each group). WT, wild-type; KO, mPDS−/−. (H) KCNJ10 (green) immunostaining showed increased expression of KCNJ10 in the stria vascularis, implying that the endocochlear potential might have been partially rescued. DAPI (blue) was used for nuclear staining. Scale bar, 100 μm. (I) KCNJ10 fluorescence histogram of multiple area sections of the stria vascularis (n = 3). * p < 0.05.
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