Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Expression of MYO7A from single AAV2 and AAV5 vectors in cultured cells. ( a ) Diagram of the viral vector encoding human MYO7A cDNA. ( b ) Western blot of WT eyecup (lane 1), primary RPE cultures derived from Myo7a -null mice and infected with AAV2- MYO7A (lane 2) or AAV5- MYO7A (lane 3), or not infected (lane 4), and primary RPE cultures derived from Myo7a +/− mice (lane 5). All lanes were immunolabeled with antibodies against actin (as a loading indicator of relative protein loading) and MYO7A. ( c-f ) Immunofluorescence images of primary RPE cell cultures. Cells derived from Myo7a -null mice that were not infected (c), from Myo7a +/− mice (d), or from Myo7a -null mice infected with 1x AAV2-MYO7A (e) or 1x AAV5-MYO7A (f). Scale = 10 μm.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Expressing, Cell Culture, Plasmid Preparation, Western Blot, Derivative Assay, Infection, Immunolabeling, Immunofluorescence

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Expression of MYO7A from single AAV2 and AAV5 vectors in vivo . ( a-e ) EM images of MYO7A immunogold labelling of the connecting cilium and pericilium from rod photoreceptors in a Myo7a -null retina. ( a ) Longitudinal section from an untreated Myo7a -null retina (background label only). ( b, c ) Longitudinal sections from Myo7a -null retinas treated with 1x AAV2- MYO7A ( b ) or AAV5- MYO7A ( c ). Scale = 200 nm. ( d, e ) Transverse sections of connecting cilia from rod photoreceptors in Myo7a -null retinas treated with 1x AAV2- MYO7A ( d ) or AAV5- MYO7A ( e ). Scale = 100 nm. ( f-g ) EM images of RPE cells from Myo7a -null retinas treated with 1x AAV2- MYO7A ( f ) or AAV5- MYO7A ( g ). Scale = 2 μm. BM = Bruch’s Membrane, AP = Apical Processes. Areas indicated by rectangles are enlarged in f’ and g’, in order to show MYO7A immunogold labeling (indicated by circles). Scale = 500 nm. ( h, i ) EM image of a longitudinal section of the connecting cilium and pericilium from a rod (h) and a cone (i) photoreceptor in a Myo7a -null retina, treated with 1x AAV2- MYO7A . The section was double-labeled with MYO7A (12 nm gold) and rod opsin (15 nm gold) antibodies. Rod outer segments were labeled with the opsin antibody, while cones were identified by lack of rod opsin labeling in their outer segments. The sections show just the base of the outer segments. Nearly all the label in the connecting cilium is MYO7A, even in the rod. Scale = 200 nm. ( i-l ) Bar graphs indicating MYO7A immunogold particle density in the rod photoreceptor cilium and pericilium ( j, k ) and in the RPE ( l, m ), following treatment with AAV2- MYO7A ( j, l ) or AAV5- MYO7A ( k, m ) of different concentrations. n=3 animals per condition. Bars indicate SEM.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Expressing, In Vivo, Membrane, Labeling

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Correction of mutant phenotypes, following subretinal injections with AAV2- MYO7A or AAV5- MYO7A . ( a-e ) Correction of melanosome localization. Light micrographs showing the presence of melanosomes in the apical processes of the RPE in a WT retina ( a ) and retinas injected with 1x AAV2- MYO7A ( b ) or AAV5- MYO7A ( c ). Further away from the injection site ( d ), melanosomes are present in the apical processes of some RPE cells, but not in others (arrows indicate apical melanosomes; white lines indicate regions where melanosomes are absent from the apical processes). ( e ) Region distant from injection site, where all RPE cells lack melanosomes in their apical processes. Brackets on left side indicate RPE apical processes. Scale = 8 μm. ( f ) Diagram of a dorso-ventral section through an eyecup, indicating the relative locations of the images shown in a-e. Arrow indicates the site of injection, ONH indicates the optic nerve head. ( g ) Correction of abnormal levels of opsin in the connecting cilium and pericilium of rod photoreceptors. Bar graph showing opsin immunogold gold particle density, along the length of the connecting cilium. Ultrathin sections of retinas from Myo7a -null and WT mice were stained with rod opsin antibody. The Myo7a -null retinas had been either not treated or treated with 1x or 1:100 AAV2- MYO7A or AAV5- MYO7A . N = 3 animals per condition. Bars indicate SEM.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Mutagenesis, Injection, Staining

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Expression of MYO7A from the overlapping AAV2- MYO7A dual vectors. ( a ) Diagram of the the overlapping AAV2- MYO7A dual vectors. The overlapping region contains 1365 bases. ( b ) Western blot of proteins from primary RPE cultures derived from Myo7a -null mice and not infected (lane 1), or infected with AAV2- MYO7A (dual) (lane 2); and primary RPE cultures derived from Myo7a +/− mice (lane 3). All lanes were immunolabeled with anti-MYO7A and anti-actin. ( c ) Western blot of primary cultures derived from Myo7a -null mice and not infected (lane 1), or infected with AAV2- MYO7A (dual (lane 2) or with AAV5- MYO7A single vector (1x) (lane 3). Lanes were immunolabeled with anti-MYO7A and anti-actin. Densitometry of the actin labeling showed that lane 2 was loaded with 3-fold more protein than lane 3; the MYO7A to actin ratio is 7-fold greater in lane 3 compared with lane 2 in this blot. ( d-g ) Immunofluorescence of cultured RPE cells transduced with AAV2-MYO7A(dual). ( d-f ) Primary RPE cultures derived from Myo7a -null mice and ( g ) ARPE19 cells. Scale = 10 μm. ( h ) Bar graph indicating the distribution of MYO7A immunogold particle density among RPE cells from retinas of Myo7a -null mice, injected with AAV2-MYO7A(dual). N = 3 animals.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Expressing, Western Blot, Derivative Assay, Infection, Immunolabeling, Plasmid Preparation, Labeling, Immunofluorescence, Cell Culture, Transduction, Injection

Journal: Gene therapy

Article Title: Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

doi: 10.1038/gt.2013.3

Figure Lengend Snippet: Correction of mutant phenotypes, following subretinal injections with AAV2- MYO7A (dual). ( a ) Light microscopy of a semi-thin section from a treated Myo7a -null mouse retina. Region shown is near the injection site. Arrows indicate melanosomes in the apical processes. White lines indicate cells that still show the Myo7a- null phenotype, with an absence of melanosomes in the apical processes. Scale = 10 μm. ( b ) Low magnification of an immunoEM image of the RPE from a retina treated with AAV2- MYO7A (dual). As in a , the white line indicates a region that still shows the Myo7a- null phenotype. Rectangle, c, includes melanosomes in the apical region, indicating a corrected RPE cell. Scale = 10 μm. ( c-e ) Higher magnification of regions outlined by rectangles in b . MYO7A immunogold particles are indicated by circles. Scale = 1 μm. ( f ) Bar graph illustrating MYO7A immunogold particle density measured in RPE cells from Myo7a -null retinas, WT retinas, or from Myo7a -null retinas treated with AAV2- MYO7A (dual) and determined to be corrected or not corrected by the location of their apical melanosomes. N = 3 animals per condition. Bars indicate SEM. ( g ) ImmunoEM image of a rod photoreceptor cilium double-labeled with antibodies against MYO7A (small gold particles) and against rod opsin (large gold particles). MYO7A labeling is associated with the connecting cilium and periciliary membrane, indicating expression and correct localization of MYO7A, while this region is devoid of opsin labeling, which is restricted to the disk membranes, consistent with the WT phenotype, thus indicating correction of the mutant phenotype. Scale = 300 nm.

Article Snippet: After transfer, blots were blocked with 5% non-fat milk, and probed with mouse anti-MYO7A antibody, generated against residues 927-1203 of human MYO7A (Developmental Studies Hybridoma Bank, Iowa, USA) , and mouse anti-actin antibody (Sigma, MO, USA) as a loading control.

Techniques: Mutagenesis, Light Microscopy, Injection, Labeling, Membrane, Expressing

Journal: Cell Death and Differentiation

Article Title: Live imaging the phagocytic activity of inner ear supporting cells in response to hair cell death

doi: 10.1038/cdd.2015.48

Figure Lengend Snippet: HC corpses accumulate in cisplatin-treated utricles. (a) Utricles were cultured in either control medium (upper row), neomycin (middle row), or cisplatin (bottom row) until 50% of the HCs died (or a matched time point in control medium). Four hours later, utricles were fixed and stained for the HC marker myosin VIIA (green), which was compared with the tdTomato signal present in living HCs (red). HC corpses (cells that are myosin VIIA-positive and tdTomato-negative) are indicated by asterisks in merged images from each condition. All images were acquired at the plane of the HC nuclei, with the exception of those in the fourth column, which were taken at the level of the reticular lamina and include the f-actin signal (white) in the merged image. Scale bar=5 μm. (b) The percentage of HC corpses was significantly increased in neomycin-treated utricles over controls, while the percentage of HC corpses was highest in the cisplatin-treated utricles. Bars represent mean±S.E.M. N=358–2583 HCs across 3–4 utricles per condition. *P<0.05 versus control, #P<0.05 versus neomycin. (c) Myosin VIIA signal intensity was not reduced in neomycin-treated tissue relative to cisplatin-treated tissue. Both tdTomato+ and tdTomato− HCs were analyzed at the nuclear plane. Bars represent mean±S.E.M. N=7 HCs per condition. (d) HC volume was analyzed over time in control, neomycin, and cisplatin-treated tissue. As the HCs cultured in either neomycin or cisplatin approached HC death (indicated by loss of tdTomato signal at t=0 h), HCs from both conditions shrank in volume, particularly in the last 3–4 h before death. HCs cultured in control medium were tracked for the same amount of time as the drug-treated utricles, but they did not die and did not shrink in volume. N=1–31 HCs per time point, values represent mean±S.E.M. *P<0.0002 via t-test with Bonferroni correction, neomycin versus cisplatin

Article Snippet: Utricles were incubated overnight at 4 °C with a mouse monoclonal antibody against myosin VIIA (138-1, diluted 1 : 100 in blocking buffer, Developmental Studies Hybridoma Bank, Iowa City, IA, USA).

Techniques: Cell Culture, Control, Staining, Marker

Journal: Cells

Article Title: Heat Shock Factor 1 Prevents Age-Related Hearing Loss by Decreasing Endoplasmic Reticulum Stress

doi: 10.3390/cells10092454

Figure Lengend Snippet: Cochlea of mice with age-related hearing loss (ARHL) exhibit reduced HSF1 and heat shock protein (HSP) expression, and increased expression of endoplasmic reticulum (ER) stress and intrinsic apoptosis markers. ( A ) The auditory brainstem response (ABR) threshold was significantly higher at 8, 16, and 32 kHz in the aging group (n = 12) than in the control group (n = 12). Data are means ± standard error of the mean (SEM). ( B ) Hematoxylin and eosin (H & E)-stained sections from the middle and base turns of the cochlea. Black darts indicate inner (IHCs) and outer hair cells (OHCs) and their supporting cells (SPs) within boxes. Red asterisks indicate cell loss. Scale bars, 30 μm. ( C ) IHC, OHC, and SP counts in young and aging mice from the apex to the basal turn of the cochlea, measured on histological sections. Data are means ± SEM of eight independent sections. ( D ) Sections were immunolabeled with anti-MYO7A, anti-HSF1, anti-CHOP, anti-p-eIF2α and anti-Bax antibodies to evaluate the relationship between ER stress and apoptosis with HSF1 expression levels. DAPI was used as a counterstain. Arrow heads indicate IHCs and OHCs (yellow) and SPs (white). Cell loss is indicated by white asterisks. Scale bars, 20 μm; DAPI was used as a counterstain. Darts indicate IHCs and OHCs (yellow) and SPs. Cell loss is indicated by white asterisks. Scale bars, 20 μm. ( E ) Protein expression was measured in cochlea extracts from the young and aging groups. Relative band intensities are presented as means of the average of at least three independent experiments. Densitometry experiments were performed using ImageJ software . All experiments were performed with at least three replications for each condition and repeated at least twice ** p < 0.01; *** p < 0.001 (one-way analysis of variance (ANOVA), followed by Tukey’s honest significant difference (HSD) test).

Article Snippet: Antibody against anti-MYO7A (sc-25834) was obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Control, Staining, Immunolabeling, Software