Journal: Scientific Reports

Article Title: Fatty acid binding protein type 7 deficiency preserves auditory function in noise-exposed mice

doi: 10.1038/s41598-023-48702-4

Figure Lengend Snippet: Experimental design, body weights, and FABP7 expression in the cochlea. Schematic of the experiments using wild-type (WT) and Fabp7 knockout (KO) mice ( A ). The body weight (BW) of Fabp7 KO mice was not significantly different from that of WT mice ( B ). Immunohistochemistry of FABP7 in the cochlea of WT mice revealed that FABP7 was expressed throughout the spiral ganglion (SG), organ of Corti (OC), spiral limbus (SLim), and spiral ligament (SLig) ( C ). Scale bar, 200 μm ( C – D ). No expression of FABP7 was observed in the cochlea of Fabp7 KO mice ( D ). Maximum projection images of the OC showing high expression of FABP7 in Schwann cells and SOX2-positive supporting cells around the MYO7a-positive outer hair cells (OHCs) in the cochlea of WT mice ( E ). In the SG, FABP7 was expressed in satellite cells surrounding TUJ-1-positive neurons ( F ). Scale bars, 50 μm ( E – F ). Auditory brainstem response; ABR, weeks; W, post-noise exposure day; PNED, inner hair cell; IHC. Statistical significance was determined using two-way analysis of variance, followed by Šídák multiple comparison test. Error bars represent standard deviation.

Article Snippet: After rinsing with PBS, tissue sections were blocked with 3% bovine serum albumin/0.3% Triton X-100/ PBS for 30 min at room temperature, the unconjugated AffiniPure Fab fragment of anti-mouse IgG (1:10, Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature, and incubated overnight with the following primary antibodies: (1) rabbit IgG anti-FABP7 (1:1000, Merck, Darmstadt, Germany #PRS4259), (2) mouse IgG anti-TUJ-1 (1:500, Abcam, Cambridge, UK #ab78078), (3) mouse IgG2a anti-Myosin 7a (MYO7a) (1:200, Santa Cruz Biotechnology, Dallas, TX, USA #SC-74516), and (4) goat IgG anti-SOX2 (1:500, R&D Systems, Minneapolis, MN, USA #AF2018).

Techniques: Expressing, Knock-Out, Immunohistochemistry, Comparison, Standard Deviation

Journal: The Journal of Physiology

Article Title: Gata3 is required for the functional maturation of inner hair cells and their innervation in the mouse cochlea

doi: 10.1113/JP277997

Figure Lengend Snippet: A–D , maximum intensity projections of confocal z ‐stacks taken from the apical cochlear region of control ( A , gata3 fl/fl ; C , gata3 +/+ ) and either gata3 fl/fl otof‐cre +/− ( B ) or gata3 +/− ( D ) mice using antibodies against CtBP2 (ribbon synaptic marker: red) and PSD95 (postsynaptic density marker: green). Myosin 7a (Myo7a, blue) was used as a hair cell marker. Scale bars 10 μm. E and F , number of CtBP2 puncta in the gata3 fl/fl otof‐cre +/− ( E ) and gata3 +/− ( F ) mice at P48. Note that in addition to the mean values (large circles), the individual IHC counts are also shown (smaller symbols). In E , the ribbon number in gata3 fl/fl otof‐cre +/− cells (14.4 ± 2.7, n = 28 IHCs, 3 mice, P = 0.468) was no different to that in control gata3 fl/fl IHCs (13.9 ± 3.1, n = 30 IHCs, 3 mice). In IHCs from gata3 +/− mice ( F ) the number of CtBP2 puncta (11.3 ± 2.2, n = 44 IHCs, 4 mice) was significantly reduced compared to control cells (13.5 ± 2.5, n = 44 IHCs, 4 mice, P < 0.0006). Averages are means ± SEM.

Article Snippet: Primary antibodies were: mouse anti‐myosin7a (1:100, DSHB, Iowa City, IA, USA; no. 138‐1S), rabbit anti‐myosin7a (1:500, Proteus Biosciences, Nottingham, UK; no. 25‐6790), mouse anti‐CtBP2 (1:200, BD Biosciences, Berkshire, UK; no. 612044), rabbit anti‐SK2 (1:500, Sigma‐Aldrich, no. P0483), goat anti‐choline acetyltransferase (ChAT, 1:500, Millipore, Hertfordshire, UK; no. AB144P) and mouse anti‐PSD95 (1:1000, Millipore, no. MABN68).

Techniques: Marker

Journal: Genes

Article Title: Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate’s Cochlea

doi: 10.3390/genes12071082

Figure Lengend Snippet: Comparison of the expression patterns of CX26 and CX30 at E115 and P0 in lateral wall fibrocytes. ( A – C ) At E115, both CX26 and CX30 expressions were detected in the upper half of the spiral ligament fibrocytes. At this stage, a relatively high expression of CX30 was observed. On the modiolus side (stria vascularis side), CX30 expression was predominantly observed, and, in several cells, only CX30 expression was detected (arrowhead in ( C )). CX26 expression was observed more laterally, and, on the lateral side of fibrocytes, only CX26 expression was detected (asterisk in ( C )). In the central region, overlapping expression of CX26 and CX30 was observed (arrow in ( C )). ( D – E ) At P0, both CX26 and CX30 were observed in the lateral wall fibrocytes and lateral side membrane of the basal cells of stria vascularis. In contrast to E115, the expression of CX30 was observed more broadly. On the lateral side of fibrocytes (type III fibrocytes), only CX30 expression was observed (asterisk in ( E )). SLC12A2 (NKCC1) was used to label stria vascularis and lateral wall fibrocytes. Nuclei were counterstained with Hoechst stain (blue). Scale bar: 100 µm in ( A , D ), 50 µm in ( B , E ), 20 µm in ( C ). StV: stria vascularis, OC: organ of Corti. ( A – E ): basal turn.

Article Snippet: Invitrogen, Carlsbad, CA, USA, #13-8100), rabbit anti-connexin 30 antibody (Z-PP9) (#71-2200, 1:500, Invitrogen, Carlsbad, CA, USA), mouse anti-myosin7a antibody (#138-1-s, 1:30, DSHB, Iowa City, IA, USA), rabbit anti-myosin7a (#25-6790, 1:200, Proteus Biosciences, Ramona, CA, USA), goat anti-SOX2 antibody (AF2018, 1:200; R&D Systems, Minneapolis, MN, USA), and goat anti-NKCC1 (SLC12A2) antibody (sc21545, 1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Comparison, Expressing, Membrane, Staining

Journal: PLoS Biology

Article Title: Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration

doi: 10.1371/journal.pbio.3001445

Figure Lengend Snippet: (A) Experimental paradigm: DT was injected into P1 wild-type or Lgr5 DTR/+ mice. EdU was injected daily from P3 to P5, and cochleae were examined at P4 or P7. (B-D, I-K) Representative images of the apical, middle, and basal turns of DT-treated wild-type cochleae showing no EdU + or Ki67 + Sox2 + SCs at P4 or P7. IPhC region is outlined by dashed lines. (E-G) In each turn of the P4 DT-treated Lgr5 DTR/+ cochlea, EdU + and/or Ki67 + Sox2 + cells were detected in the GER. Some EdU + and/or Ki67 + Sox2 + cells were also found in the IPhC and PC/DC regions. E’-G”“represent single channel images. Insets in E and E’ are high magnification images of a cell in metaphase. G”‘ and G”“are high magnification images from G’ and G”. (H) Significantly more EdU + Sox2 + cells were found in the P4 GER regions throughout the DT-treated Lgr5 DTR/+ cochlea relative to controls. Numbers decrease in an apical–basal gradient. (L-N) In all 3 turns of the P7 DT-treated Lgr5 DTR/+ cochlea, EdU + Sox2 + cells were primarily found in IPhC region, and only few were found in the GER or PC/DC region. Ki67 + Sox2 + cells were rarely detected at this age. L’-N”“represent single channel images. N”‘ and N”“are high magnification images from N’ and N”. (O) Significantly more EdU + Sox2 + cells were detected throughout the P7 IPhC regions of DT-treated Lgr5 DTR/+ cochlea relative to controls, decreasing in an apical–basal gradient. Data represent mean ± SD. ** p < 0.01, *** p < 0.001. Two-way ANOVA with Tukey’s multiple comparisons test. n = 4–7. See for H, O. DC, Deiters’ cell; DT, diphtheria toxin; GER, greater epithelial ridge; IPhC, inner phalangeal cell; PC, pillar cell; SC, supporting cell.

Article Snippet: The following primary antibodies were used: rat anti-CD44 (1:200; BD Biosciences), goat anti-CtBP2 (1:500; Santa Cruz Biotechnology), chicken anti-GFP (1:1,000; Aves Labs), rabbit anti-GLAST (1:200; Abcam), mouse anti-GluR2 (1:1,000; Millipore), rabbit anti-Ki67 (1:1,000; Abcam), rat anti-Ki67 (1:400; ThermoFisher Scientific), rabbit anti-Myosin7a (1:1,000; Proteus Bioscience), mouse anti-Myosin7a (1:1,000; Developmental Studies Hybridoma Bank), mouse anti-Na + /K + ATPase α-1, α6F (1:500; Developmental Studies Hybridoma Bank), goat anti-Sox2 (1:200; Santa Cruz Biotechnology or R&D Systems), goat anti-Sparcl1 (1:500; R&D Systems), mouse anti-Tuj1 (1:1,000; Neuromics), rabbit anti-Fabp7 (1:200; Abcam), and rabbit anti-Vglut3 (1:1,000; Synaptic Systems).

Techniques: Injection

Journal: Frontiers in Cellular Neuroscience

Article Title: LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

doi: 10.3389/fncel.2016.00186

Figure Lengend Snippet: Proliferation in the cochleae of E14.5 Lgr5-eGFP embryos. (A–B′′) SOX2-positive pro-sensory cells (green, arrow) did not incorporate EdU (red). Cell nuclei were counterstained with DAPI (blue), scale bars indicate 25 μm.

Article Snippet: The following primary antibodies were used: anti-myosin7a (1:700; rabbit, Proteus Bioscience, 25-6790), anti-myosin7a (1:200; mouse, Developmental Studies Hybridoma Bank, 138-1-c), anti-SOX2 (1:200; goat, Santa Cruz Biotechnology, s-17320), anti-SOX2 (1:50, mouse, Millipore, MAB4343), anti-SOX2 (1:250, BD Pharmingen, 561469) anti-acetylated tubulin (ACTBA) (1:50; mouse, Sigma–Aldrich, T7451), anti-prestin (1:700; goat, Santa Cruz Biotechnology, N-20, sc-22692), anti-LGR4 (1:200; rabbit, Sigma–Aldrich, HPA030267, test of the antibody specificity shown in Supplementary Figure ), anti-jagged1 (JAG1) (1:50; goat, Santa Cruz Biotechnology, C-20, sc-6011), anti-cyclinD1 (CCND1) (1:100; rabbit, Thermo Fisher Scientific, SP4, MA5-14512), anti-β-tubulin class III (TUJ1, 1:200; mouse, Covance, MMS-435P), anti-p75 NGF receptor (1:400; rabbit, Sigma–Aldrich, N3908), anti-MYO6 (1:300, Abcam, ab11096).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

doi: 10.3389/fncel.2016.00186

Figure Lengend Snippet: The distribution of LGR4 in the developing mouse cochlea. (A) At E14.5, strong LGR4 staining was detected in the cochlear duct and spiral ganglion cells (white arrowhead). LGR4 was detected in the pro-sensory domain, where it overlapped with SOX2 (white arrow), and non-sensory domains lateral and medial to the pro-sensory domain. (B) At E17, LGR4 (green) was detected in IHCs and OHCs (asterisks) and their surrounding supporting cells. Hair cells were labeled with phalloidin (F-actin) (red). (C) At P1, LGR4 expression was observed in the sensory epithelium (arrowhead) and spiral ganglion cells (arrow), from which projected TUJ1-positive nerves (red). (D) At P7, LGR4 was observed in hair cells (asterisks) that co-expressed Prestin (PRES) and in the surrounding supporting cells: Deiters’ cells (arrowheads) and IPCs and OPCs. (E–G) At P21, LGR4 was down-regulated in the OHCs and pillar cells, but was still expressed in the IHCs (arrowhead), Deiters’ cells (arrows), which were also positive for acetylated tubulin (ACTBA), and phalangeal processes of Deiters’ cells (arrowheads). LGR4 signals were also observed in the spiral ganglion cells. (H) At P42, LGR4 was detected in Deiters’ cells and their phalangeal processes. Cell nuclei were counterstained with DAPI (blue), asterisks indicate hair cells, scale bars indicate 20 μm in the picture (B,D,E,G,H) and 50 μm in (A,C,F) . (I,J) Cochlear cryosections from Lgr4-LacZ mice at the age of P21 were stained for β-galactosidase activity. Expression was seen in Deiters’ cells (black arrow) and their phalangeal processes (gray arrow), IHCs (black arrowhead), and spiral ganglion cells.

Article Snippet: The following primary antibodies were used: anti-myosin7a (1:700; rabbit, Proteus Bioscience, 25-6790), anti-myosin7a (1:200; mouse, Developmental Studies Hybridoma Bank, 138-1-c), anti-SOX2 (1:200; goat, Santa Cruz Biotechnology, s-17320), anti-SOX2 (1:50, mouse, Millipore, MAB4343), anti-SOX2 (1:250, BD Pharmingen, 561469) anti-acetylated tubulin (ACTBA) (1:50; mouse, Sigma–Aldrich, T7451), anti-prestin (1:700; goat, Santa Cruz Biotechnology, N-20, sc-22692), anti-LGR4 (1:200; rabbit, Sigma–Aldrich, HPA030267, test of the antibody specificity shown in Supplementary Figure ), anti-jagged1 (JAG1) (1:50; goat, Santa Cruz Biotechnology, C-20, sc-6011), anti-cyclinD1 (CCND1) (1:100; rabbit, Thermo Fisher Scientific, SP4, MA5-14512), anti-β-tubulin class III (TUJ1, 1:200; mouse, Covance, MMS-435P), anti-p75 NGF receptor (1:400; rabbit, Sigma–Aldrich, N3908), anti-MYO6 (1:300, Abcam, ab11096).

Techniques: Staining, Labeling, Expressing, Activity Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

doi: 10.3389/fncel.2016.00186

Figure Lengend Snippet: Hair cell formation in the embryonic cochlea of Lgr4-LacZ mice. (A–D′′) The cochlear cryosections obtained from Lgr4 deficient mice and their wild type counterparts were stained for a hair cell marker (MYO7a, green) and two pro-sensory cell markers (SOX2, and JAG1, both red). Developing hair cells (asterisks) were detected only in the mid-basal turns. Cell nuclei were counterstained with DAPI (blue), scale bars indicate 20 μm.

Article Snippet: The following primary antibodies were used: anti-myosin7a (1:700; rabbit, Proteus Bioscience, 25-6790), anti-myosin7a (1:200; mouse, Developmental Studies Hybridoma Bank, 138-1-c), anti-SOX2 (1:200; goat, Santa Cruz Biotechnology, s-17320), anti-SOX2 (1:50, mouse, Millipore, MAB4343), anti-SOX2 (1:250, BD Pharmingen, 561469) anti-acetylated tubulin (ACTBA) (1:50; mouse, Sigma–Aldrich, T7451), anti-prestin (1:700; goat, Santa Cruz Biotechnology, N-20, sc-22692), anti-LGR4 (1:200; rabbit, Sigma–Aldrich, HPA030267, test of the antibody specificity shown in Supplementary Figure ), anti-jagged1 (JAG1) (1:50; goat, Santa Cruz Biotechnology, C-20, sc-6011), anti-cyclinD1 (CCND1) (1:100; rabbit, Thermo Fisher Scientific, SP4, MA5-14512), anti-β-tubulin class III (TUJ1, 1:200; mouse, Covance, MMS-435P), anti-p75 NGF receptor (1:400; rabbit, Sigma–Aldrich, N3908), anti-MYO6 (1:300, Abcam, ab11096).

Techniques: Staining, Marker

Journal: Frontiers in Cellular Neuroscience

Article Title: LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea

doi: 10.3389/fncel.2016.00186

Figure Lengend Snippet: Proliferation in the embryonic cochleae of Lgr4 deficient mice. (A–B′′) In the cochleae from heterozygous and homozygous Lgr4-LacZ mice, CCND1 (green) expression increased in the mid-basal turn, where CCND1 merged with red signal of SOX2 (arrows) in the pro-sensory domain. (C–D′′) The proliferation marker, EdU (red), was incorporated into pro-sensory cells (arrows) positive for SOX2 (green) in the mid-basal turn of the cochlea from Lgr4 deficient mice. Cell nuclei were counterstained with DAPI (blue), scale bars indicate 20 μm.

Article Snippet: The following primary antibodies were used: anti-myosin7a (1:700; rabbit, Proteus Bioscience, 25-6790), anti-myosin7a (1:200; mouse, Developmental Studies Hybridoma Bank, 138-1-c), anti-SOX2 (1:200; goat, Santa Cruz Biotechnology, s-17320), anti-SOX2 (1:50, mouse, Millipore, MAB4343), anti-SOX2 (1:250, BD Pharmingen, 561469) anti-acetylated tubulin (ACTBA) (1:50; mouse, Sigma–Aldrich, T7451), anti-prestin (1:700; goat, Santa Cruz Biotechnology, N-20, sc-22692), anti-LGR4 (1:200; rabbit, Sigma–Aldrich, HPA030267, test of the antibody specificity shown in Supplementary Figure ), anti-jagged1 (JAG1) (1:50; goat, Santa Cruz Biotechnology, C-20, sc-6011), anti-cyclinD1 (CCND1) (1:100; rabbit, Thermo Fisher Scientific, SP4, MA5-14512), anti-β-tubulin class III (TUJ1, 1:200; mouse, Covance, MMS-435P), anti-p75 NGF receptor (1:400; rabbit, Sigma–Aldrich, N3908), anti-MYO6 (1:300, Abcam, ab11096).

Techniques: Expressing, Marker

Journal: Frontiers in Molecular Neuroscience

Article Title: Junctional E-cadherin/p120-catenin Is Correlated with the Absence of Supporting Cells to Hair Cells Conversion in Postnatal Mice Cochleae

doi: 10.3389/fnmol.2018.00020

Figure Lengend Snippet: Increase in junctional E-cadherin/p120ctn in the postnatal mice cochleae. (A–C) Representative images of the apex turn in cochleae from P1, P3, and P7 mice immuno-labeled for E-cadherin (green) and p120ctn (red) with the same confocal intensity. (A3–C3) Magnified images of A2-C2 showing the differences in the junctional p120ctn widths between adjacent outer hair cells in the same row. (A4–C4) Double-labeling of E-cadherin (green) and p120ctn (red). ( D) Quantification of apical junctional region (AJR) widths in the apexes of cochleae from P0, P3, and P7 mice. (E) Relative Atoh1, Hes1, Hes5, E-cadherin , and P120-catenin mRNA expression levels in cochleae from P1, P3, and P7 mice ( n = 3 for each age). The mRNA levels for each gene were plotted relative to the respective P0 mRNA levels. (F) Representative examples of Western blots showing the E-cadherin, p120ctn, and total GAPDH (internal control) protein expression levels in pure cochlear sensory epithelia harvested from P1, P3, and P7 mice. (G) Quantification of Western-blot experimental results. E-cadherin and p120ctn were normalized to the total GAPDH levels, and the values are expressed as percentages relative to the P0 levels for comparison. The average percentages relative to P0 are shown ( n = 3). OHC, Outer hair cell region; IHC, Inner hair cell region. The error bars in (D,E,G) show the SEMs. * p < 0.05, ** p < 0.01. The scale bars represent 20 μm in (A1) and 5 μm in (A3) .

Article Snippet: Explant cultures were harvested and fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 plus 10% donkey serum for 1 h. The explants were then incubated with the following primary antibodies for 24 h at 4°C: rabbit anti-myosin7A (1:100; Proteus Biosciences, Ramona, CA, USA), mouse anti-myosin7A (1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit anti-Prox1 (1:1,000; Millipore), mouse anti E-cadherin (against the C-terminus, 1:200, BD Transduction Laboratories, San Jose, CA, USA), and goat anti-p120-catenin (1:200; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Labeling, Expressing, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Junctional E-cadherin/p120-catenin Is Correlated with the Absence of Supporting Cells to Hair Cells Conversion in Postnatal Mice Cochleae

doi: 10.3389/fnmol.2018.00020

Figure Lengend Snippet: DAPT induces E-cadherin/p120ctn disorganization in the outer hair cell region of the sensory epithelium. (A–D) Images of the mid-apex of P0 cochleae treated with DMSO or DAPT for 2–4 days and immunostained for E-cadherin (blue), p120ctn (green), and Myo7a (red). In control cochleae, E-cadherin internalization and p120ctn depletion did not occur in the sensory epithelium (A,B) . In the DAPT-treated groups, internalization of E-cadherin occurred along with depletion of p120ctn in the outer hair cell region (C,D) . The short arrows in both (C1,D1) show that E-cadherin internalization occurred following the DAPT treatments. The long arrows in (C2) indicate that p120ctn was decreased in the sensory epithelium after 2 days of DAPT treatment. Following 4 days of DAPT treatment, p120ctn was depleted in the outer hair cell region (long arrows in D2 ). The short arrows in (C5) indicate SCs that internalized E-cadherin. The short arrows in (D5) represent HCs that internalized E-cadherin. (E) Relative Atoh1, Hes1, Hes5, E-cadherin , and p120ctn mRNA expression levels in P0 cochleae treated with DMSO or DAPT for 48 h ( n = 3 for each gene). The mRNA levels for each gene were plotted relative to the respective vehicle control mRNA levels. (F) Representative images of Western blots showing the E-cadherin, p120ctn, and total GAPDH (internal control) protein expression levels in cochlear sensory epithelia treated with DMSO or DAPT for 4 days. (G) Quantification of Western blot experiments. E-cadherin and p120ctn were normalized to the total GAPDH levels, and the values are expressed as percentages relative to DMSO for comparison. Average percentages relative to DMSO are shown ( n = 3). (H) 3D projections showing that Myo7a + HCs and Prox1 + SCs in the sensory region are surrounded by p120ctn and do not undergo SC-to-HC conversion in control cochleae (long arrows). HCs and SCs were surrounded by p120ctn in x-z- axial sections (H2) and in y-z- axial sections (H3) . (I) In DAPT-treated cochleae, p120ctn was largely depleted in both the HC and SC layers, whereas Myo7a + HCs were significantly increased, and Prox1 + SCs were dramatically decreased (short arrows). (I2) x-z- axial sections. (I3) y-z- axial sections. Myo7a (blue), Prox1 (green), and p120ctn (red). The error bars in (E,G) show the SEMs. ** p < 0.01. The scale bars represent 10 μm in (A–D) and 25 μm in (H,I) .

Article Snippet: Explant cultures were harvested and fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 plus 10% donkey serum for 1 h. The explants were then incubated with the following primary antibodies for 24 h at 4°C: rabbit anti-myosin7A (1:100; Proteus Biosciences, Ramona, CA, USA), mouse anti-myosin7A (1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit anti-Prox1 (1:1,000; Millipore), mouse anti E-cadherin (against the C-terminus, 1:200, BD Transduction Laboratories, San Jose, CA, USA), and goat anti-p120-catenin (1:200; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Junctional E-cadherin/p120-catenin Is Correlated with the Absence of Supporting Cells to Hair Cells Conversion in Postnatal Mice Cochleae

doi: 10.3389/fnmol.2018.00020

Figure Lengend Snippet: E-cadherin/p120ctn disorganization and SC-to-HC conversion decline with age and cease by P7. (A–F) Whole mounts of apexes harvested from P0, P3, and P7 cochleae treated with DMSO and DAPT for 4 days. Myo7a (red), Prox1 (green), and p120ctn (white). p120ctn was imaged with the same confocal intensity for all samples. (A2–F2) p120ctn expression in the apex from P0 to P7. p120ctn disintegration was detected in the apexes of P0 (short arrow in B2 ) and P3 (short arrow in D2 ) mice treated with DAPT. No p120ctn disruption was observed in P7 mice or controls. (A3–F3) Higher magnification of p120ctn in the HC layers. p120ctn was depleted and HCs were regenerated in P0 (short arrows in B3 ) and P3 mice (short arrows in F3 ). (A4–F4) Higher magnification of p120ctn and Prox1 + SCs in the SC layer. After DAPT treatments, p120-depleted areas were notably smaller in the apexes of P3 mice (short arrows in F4 ) compared with those in the apexes of P0 mice (short arrows in B4 ). (G,H) Ratio of Myo7a + HCs and Prox1 + SCs following DAPT treatments compared with DMSO treatments in the apexes of P0, P3, and P7 cochleae. (G–I) Quantification of p120ctn depletion widths in the apexes of P0, P3, and P7 mice treated with DAPT for 4 days. (J) Scatter plot of the number of Myo7a + HCs/100 μm ( y- axis) and Prox1 + SCs/100 μm ( x- axis) in the apexes of P0, P3, and P7 mice. A higher number of generated Myo7a + HCs was associated with the retention of fewer Prox1 + SCs. (K,L) Scatter plot of Myo7a + HCs/100 μm ( I , y-axis), Prox1 + SCs/100 μm ( J , y-axis), and p120 depletion widths ( x -axis) in the apexes of P0, P3, and P7 mice. The data in (G–L) are presented as the means ± SEMs. ** p < 0.01. The scale bars represent 25 μm.

Article Snippet: Explant cultures were harvested and fixed with 4% paraformaldehyde for 30 min and then treated with 0.1% Triton X-100 plus 10% donkey serum for 1 h. The explants were then incubated with the following primary antibodies for 24 h at 4°C: rabbit anti-myosin7A (1:100; Proteus Biosciences, Ramona, CA, USA), mouse anti-myosin7A (1:200; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), rabbit anti-Prox1 (1:1,000; Millipore), mouse anti E-cadherin (against the C-terminus, 1:200, BD Transduction Laboratories, San Jose, CA, USA), and goat anti-p120-catenin (1:200; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing, Generated