Journal: Journal of Biomedical Science
Article Title: Early downregulation of hair cell (HC)-specific genes in the vestibular sensory epithelium during chronic ototoxicity
doi: 10.1186/s12929-025-01180-4
Figure Lengend Snippet: Effect of subchronic ototoxicity on the expression of PMCA2, DNER, KCNA10, osteopontin, calretinin, and MYO7A in vestibular sensory epithelium assessed by immunofluorescent labelling and confocal microscopy. A – B Images of whole mount utricles from control and treated rats showing the effects of IDPN ( A ) or streptomycin ( B ) on PCMA2 expression in stereocilia bundles. Images are maximum intensity projections of a stack of 6 planes (2 µm total thickness). C Expression of DNER in the neck region of crista HCs in control and streptomycin rats. Images are maximum intensity projections of a stack of 4 planes (1.2 µm total thickness). D Quantitative analyses of PCMA2 and DNER expression. Bars show mean ± SEM fluorescence intensity values. Each point is from an individual animal, averaging values from > 180 hair bundles ( a and b ) or > 400 HCs (c) per animal. ***: p < 0.001, ****: p < 0.0001, Student’s t-test. E Immunolabelling of CASPR1 and KCNA10 in a control crista (upper row) and in a crista of a rat exposed to IDPN for 4 weeks (bottom row). In control tissue, both proteins are located in the calyceal junction area. After IDPN, most afferent calyces show very reduced expression of CASPR1 (arrows), while a few retain a large amount of label (asterisk). The corresponding HCs show a marked depletion of the KCNA10 label in the calyceal junction, although the non-specific label shown by supporting cells with this antibody persisted. F Quantitative analysis of the effect of chronic IDPN ( a ) and streptomycin ( b ) on KCNA10 expression in the calyceal junction area. Bars show mean ± SEM fluorescence intensity values. Each point is from an individual animal, averaging values from > 400 HCs per animal. **: p < 0.01, NS nonsignificant, Student’s t-test. G and H Ostepontin and MYO7A labels in sections of cristae from control rats and IDPN-4wk rats. I Calretinin label in HCII of the periphery of the utricles of control and IDPN-4wk rats. J Quantitative analyses of the effect of chronic IDPN on the expression of osteopontin, MYO7A, and calretinin. Bars show mean ± SEM fluorescence intensity values. Each data point is from an individual animal, averaging values of 30 HCs per animal. ****: p < 0.0001, ns: nonsignificant, Student’s t-test. Scale bars = 10 µm in ( A , B , C , E , and H ); 25 µm in ( G and I )
Article Snippet: For immunohistochemistry of the vestibular sensory epithelia, the following commercial primary antibodies were used at the indicated dilutions: mouse monoclonal anti-contactin-associated protein (CASPR1) (clone K65/35, cat.#MABN69, Millipore, RRID: AB_2083496, 1/400), mouse monoclonal anti-myosin-7A (MYO7A) (cat.# 138-1, supernatant, Developmental Studies Hybridoma Bank, RRID: AB_2282417, 1/100), rabbit anti-potassium voltage-gated channel subfamily A member 10 (KCNA10, Kv1.8)(cat.# APC-157, Alomone Labs, RRID: AB_2341039, 1/200), rabbit anti-MYO7A (cat.# 25–6790, Proteus Biosciences, RRID: AB_10015251, 1/400), rabbit anti-oncomodulin (cat.# OMG4, Swant, RRID:AB_10000346, 1/400), rabbit anti-plasma membrane calcium transporting ATPase 2 (PMCA2, encoded by the Atp2b2 gene) (cat.# PA1-915, Invitrogen, RRID: AB_2243199, 1/400), goat anti-Delta/Notch Like EGF Repeat Containing (DNER) (cat.# AF2254, R&D Systems, RRID:AB_355202, 1/200), goat anti-Osteopontin (encoded by the Spp1 gene) (cat.# AF808, R&D Systems, RRID: AB_2194992, 1/200), and guinea pig anti-calretinin (cat.# 214104, Synaptic Systems, RRID: AB_10635160, 1/500).
Techniques: Expressing, Confocal Microscopy, Control, Fluorescence
Developmental Studies Hybridoma Bank is a verified supplier 
Developmental Studies Hybridoma Bank manufactures this product 
