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A – C Gradient expression of <t>PMCA1</t> in IHCs. Immunofluorescence images of PMCA1 (green) and ribbon synapses (red) in the low-frequency ( A ), high-frequency ( B ) regions, with corresponding quantification of the PMCA1 fluorescence intensity ratio ( C ). D – E The expression of PMCA1 proteins from apex- and base- basilar membrane was quantified by western blot. Three samples (2 cochlear from same mouse pooled for each sample) were used for immunoblot analysis. F Strategy for conditional knockout of Pmca1 in hair cells, depicting LoxP sites inserted to flank exon 9. G Cochlear whole-mounts immunostaining with anti-Myosin VIIA antibody (red) to label hair cells and stained <t>with</t> <t>anti-PMCA1</t> antibody to detect PMCA1 protein (green). H – J ABR thresholds are elevated in Pmca1 CKO mice, compared with WT controls. Progression of hearing loss is seen in Pmca1 CKO mice from P12, P18 and P24. K , L Representative capacitance traces ( K ) and quantification of ΔC m ( L ) evoked by 20 ms and 200 ms depolarizations in IHCs from mice of different ages, showing the functional consequence of impaired calcium clearance. Statistical analysis by two-side unpaired t test or Mann-Whitney test with significance indicated and two-way ANOVA followed by the Bonferroni post hoc test with significance indicated. All data, the number of data, statistical test used and p values can be found in the source data file. N.S., not significant, * p < 0.05; ** p < 0.01; *** p < 0.001.
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A – C Gradient expression of <t>PMCA1</t> in IHCs. Immunofluorescence images of PMCA1 (green) and ribbon synapses (red) in the low-frequency ( A ), high-frequency ( B ) regions, with corresponding quantification of the PMCA1 fluorescence intensity ratio ( C ). D – E The expression of PMCA1 proteins from apex- and base- basilar membrane was quantified by western blot. Three samples (2 cochlear from same mouse pooled for each sample) were used for immunoblot analysis. F Strategy for conditional knockout of Pmca1 in hair cells, depicting LoxP sites inserted to flank exon 9. G Cochlear whole-mounts immunostaining with anti-Myosin VIIA antibody (red) to label hair cells and stained <t>with</t> <t>anti-PMCA1</t> antibody to detect PMCA1 protein (green). H – J ABR thresholds are elevated in Pmca1 CKO mice, compared with WT controls. Progression of hearing loss is seen in Pmca1 CKO mice from P12, P18 and P24. K , L Representative capacitance traces ( K ) and quantification of ΔC m ( L ) evoked by 20 ms and 200 ms depolarizations in IHCs from mice of different ages, showing the functional consequence of impaired calcium clearance. Statistical analysis by two-side unpaired t test or Mann-Whitney test with significance indicated and two-way ANOVA followed by the Bonferroni post hoc test with significance indicated. All data, the number of data, statistical test used and p values can be found in the source data file. N.S., not significant, * p < 0.05; ** p < 0.01; *** p < 0.001.
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A – C Gradient expression of <t>PMCA1</t> in IHCs. Immunofluorescence images of PMCA1 (green) and ribbon synapses (red) in the low-frequency ( A ), high-frequency ( B ) regions, with corresponding quantification of the PMCA1 fluorescence intensity ratio ( C ). D – E The expression of PMCA1 proteins from apex- and base- basilar membrane was quantified by western blot. Three samples (2 cochlear from same mouse pooled for each sample) were used for immunoblot analysis. F Strategy for conditional knockout of Pmca1 in hair cells, depicting LoxP sites inserted to flank exon 9. G Cochlear whole-mounts immunostaining with anti-Myosin VIIA antibody (red) to label hair cells and stained <t>with</t> <t>anti-PMCA1</t> antibody to detect PMCA1 protein (green). H – J ABR thresholds are elevated in Pmca1 CKO mice, compared with WT controls. Progression of hearing loss is seen in Pmca1 CKO mice from P12, P18 and P24. K , L Representative capacitance traces ( K ) and quantification of ΔC m ( L ) evoked by 20 ms and 200 ms depolarizations in IHCs from mice of different ages, showing the functional consequence of impaired calcium clearance. Statistical analysis by two-side unpaired t test or Mann-Whitney test with significance indicated and two-way ANOVA followed by the Bonferroni post hoc test with significance indicated. All data, the number of data, statistical test used and p values can be found in the source data file. N.S., not significant, * p < 0.05; ** p < 0.01; *** p < 0.001.
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A – C Gradient expression of <t>PMCA1</t> in IHCs. Immunofluorescence images of PMCA1 (green) and ribbon synapses (red) in the low-frequency ( A ), high-frequency ( B ) regions, with corresponding quantification of the PMCA1 fluorescence intensity ratio ( C ). D – E The expression of PMCA1 proteins from apex- and base- basilar membrane was quantified by western blot. Three samples (2 cochlear from same mouse pooled for each sample) were used for immunoblot analysis. F Strategy for conditional knockout of Pmca1 in hair cells, depicting LoxP sites inserted to flank exon 9. G Cochlear whole-mounts immunostaining with anti-Myosin VIIA antibody (red) to label hair cells and stained <t>with</t> <t>anti-PMCA1</t> antibody to detect PMCA1 protein (green). H – J ABR thresholds are elevated in Pmca1 CKO mice, compared with WT controls. Progression of hearing loss is seen in Pmca1 CKO mice from P12, P18 and P24. K , L Representative capacitance traces ( K ) and quantification of ΔC m ( L ) evoked by 20 ms and 200 ms depolarizations in IHCs from mice of different ages, showing the functional consequence of impaired calcium clearance. Statistical analysis by two-side unpaired t test or Mann-Whitney test with significance indicated and two-way ANOVA followed by the Bonferroni post hoc test with significance indicated. All data, the number of data, statistical test used and p values can be found in the source data file. N.S., not significant, * p < 0.05; ** p < 0.01; *** p < 0.001.
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A – C Gradient expression of PMCA1 in IHCs. Immunofluorescence images of PMCA1 (green) and ribbon synapses (red) in the low-frequency ( A ), high-frequency ( B ) regions, with corresponding quantification of the PMCA1 fluorescence intensity ratio ( C ). D – E The expression of PMCA1 proteins from apex- and base- basilar membrane was quantified by western blot. Three samples (2 cochlear from same mouse pooled for each sample) were used for immunoblot analysis. F Strategy for conditional knockout of Pmca1 in hair cells, depicting LoxP sites inserted to flank exon 9. G Cochlear whole-mounts immunostaining with anti-Myosin VIIA antibody (red) to label hair cells and stained with anti-PMCA1 antibody to detect PMCA1 protein (green). H – J ABR thresholds are elevated in Pmca1 CKO mice, compared with WT controls. Progression of hearing loss is seen in Pmca1 CKO mice from P12, P18 and P24. K , L Representative capacitance traces ( K ) and quantification of ΔC m ( L ) evoked by 20 ms and 200 ms depolarizations in IHCs from mice of different ages, showing the functional consequence of impaired calcium clearance. Statistical analysis by two-side unpaired t test or Mann-Whitney test with significance indicated and two-way ANOVA followed by the Bonferroni post hoc test with significance indicated. All data, the number of data, statistical test used and p values can be found in the source data file. N.S., not significant, * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Cell Death Discovery

Article Title: Folic acid prevents inner hair cell degeneration via genomic stability

doi: 10.1038/s41420-025-02880-4

Figure Lengend Snippet: A – C Gradient expression of PMCA1 in IHCs. Immunofluorescence images of PMCA1 (green) and ribbon synapses (red) in the low-frequency ( A ), high-frequency ( B ) regions, with corresponding quantification of the PMCA1 fluorescence intensity ratio ( C ). D – E The expression of PMCA1 proteins from apex- and base- basilar membrane was quantified by western blot. Three samples (2 cochlear from same mouse pooled for each sample) were used for immunoblot analysis. F Strategy for conditional knockout of Pmca1 in hair cells, depicting LoxP sites inserted to flank exon 9. G Cochlear whole-mounts immunostaining with anti-Myosin VIIA antibody (red) to label hair cells and stained with anti-PMCA1 antibody to detect PMCA1 protein (green). H – J ABR thresholds are elevated in Pmca1 CKO mice, compared with WT controls. Progression of hearing loss is seen in Pmca1 CKO mice from P12, P18 and P24. K , L Representative capacitance traces ( K ) and quantification of ΔC m ( L ) evoked by 20 ms and 200 ms depolarizations in IHCs from mice of different ages, showing the functional consequence of impaired calcium clearance. Statistical analysis by two-side unpaired t test or Mann-Whitney test with significance indicated and two-way ANOVA followed by the Bonferroni post hoc test with significance indicated. All data, the number of data, statistical test used and p values can be found in the source data file. N.S., not significant, * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Thereafter, the basilar membrane was dissected then permeabilized and blocked for 60 min in 0.5% (v/v) Triton X-100 and 4% (w/v) BSA/PBS at room temperature before incubation with the following primary antibodies: rabbit polyclonal anti-Myosin VIIa (Proteus BioSciences, USA, 138-1), mouse anti-CtBP2 IgG1 (BD Biosciences, USA, 612044), mouse anti-GluR2 IgG2 (Merck-Millipore, Germany, MAB397), rabbit polyclonal anti-PMCA1 (Alomone labs, Israel, ACP-005), rabbit polyclonal anti-SERCA3 (Alomone labs, Israel, ACP-014), rabbit polyclonal anti-NCX (Alomone labs, Israel, ANX-011), rabbit polyclonal anti-MCU (Cell Signaling Technology, USA, 14997).

Techniques: Expressing, Immunofluorescence, Fluorescence, Membrane, Western Blot, Knock-Out, Immunostaining, Staining, Functional Assay, MANN-WHITNEY