Journal: Journal of cell science

Article Title: Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy.

doi: 10.1242/jcs.252015

Figure Lengend Snippet: Figure 5. The ATG5-ATG12-ATG16L1 complex is sequestered at MakA-induced

Article Snippet: Colony-forming unit (CFU) was counted after 24 h. Antibodies EEA1 (#3288, IF: 1:100), Rab7 (#9367, IF: 1:100), Rab11 (#5589, IF: 1:100), LC3B (#2775, WB: 1:1000, IF: 1:200), LAMP1 (#9091, IF: 1:200), ATG16L1 (#8089, IF: 1:100), GM130 (#12480, IF: 1:200), ATG12 (mouse specific) (#2011, WB: 1:1000, IF: 1:100), ATG12 (human specific) (#2010, WB: 1:1000, IF: 1:100), Cav1 (#3267, IF: 1:400), Galectin-3 (#87985, IF: 1:400) and Na/K-ATPase α1 (#23565, IF: 1:100) antibodies were purchased from Cell Signaling Technology.

Techniques:

Journal: Journal of cell science

Article Title: Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy.

doi: 10.1242/jcs.252015

Figure Lengend Snippet: Figure 5. The ATG5-ATG12-ATG16L1 complex is sequestered at MakA-induced

Article Snippet: Colony-forming unit (CFU) was counted after 24 h. Antibodies EEA1 (#3288, IF: 1:100), Rab7 (#9367, IF: 1:100), Rab11 (#5589, IF: 1:100), LC3B (#2775, WB: 1:1000, IF: 1:200), LAMP1 (#9091, IF: 1:200), ATG16L1 (#8089, IF: 1:100), GM130 (#12480, IF: 1:200), ATG12 (mouse specific) (#2011, WB: 1:1000, IF: 1:100), ATG12 (human specific) (#2010, WB: 1:1000, IF: 1:100), Cav1 (#3267, IF: 1:400), Galectin-3 (#87985, IF: 1:400) and Na/K-ATPase α1 (#23565, IF: 1:100) antibodies were purchased from Cell Signaling Technology.

Techniques:

Journal: Nature Communications

Article Title: Myosin-VIIa is expressed in multiple isoforms and essential for tensioning the hair cell mechanotransduction complex

doi: 10.1038/s41467-020-15936-z

Figure Lengend Snippet: a MYO7A immunoreactivity in WT and Myo7a-ΔC organ of Corti (P5), counterstained with phalloidin (F-actin). MYO7A is predominantly decreased in the IHCs (quantified in Fig. ) (scale bars: 10 μm). b MYO7A is significantly reduced in all utricle hair cells (by 63%, p < 1e−4, two-tailed, unpaired t -test). (scale bars: 100 μm left, 10 μm right). c MYO7A immuno- and phalloidin reactivity in IHCs and OHCs in WT, Myo7a-ΔC , and Myo7a full KO mice (scale bar: 10 μm). Although MYO7A levels are depleted in Myo7a-ΔC IHCs, hair bundle morphology is not affected. d Representative SEMs of IHCs of P7 WT and Myo7a-ΔC (scale bars: 1 μm). e No difference in stereocilia length in P7 IHCs between WT and Myo7a-ΔC mice by phalloidin immunofluorescence (IF) analysis (using Imaris 3D module). Length of first row stereocilia (mean per cell ± SD): WT (3.57 ± 0.18); Myo7a-ΔC (3.54 ± 0.13), p = 0.706. Analyzed numbers (stereocilia, cells, animals): WT (159; 7; 4), Myo7a-ΔC (127; 8; 3). Second row (mean per cell ± SD): WT (2.13 ± 0.13), Myo7a-ΔC (2.14 ± 0.11), p = 0.936. Analyzed numbers (stereocilia, cells, animals): WT(89; 8; 4), Myo7a-ΔC (95; 8; 3) (n = number of cells). f Quantification of stereocilia length in P7 IHCs by SEM found no significant differences between WT and Myo7a-ΔC mice. Length of first row stereocilia (mean per cell ± SD): WT (2.88 ± 0.35), Myo7a-ΔC (2.83 ± 0.45), p = 0.664. Second row: WT (1.80 ± 0.16), Myo7a-ΔC (1.84 ± 0.19), p = 0.363. Analyzed numbers (stereocilia, cells, animals): WT (141; 41; 10), Myo7a-ΔC (123; 24; 7). g Quantification of stereocilia length in P7 OHCs by SEM found no significant differences between WT and Myo7a-ΔC mice. Length of first row stereocilia (mean per cell ± SD): WT (1.57 ± 0.077), Myo7a-ΔC (1.61 ± 0.074), p = 0.23. Length of second row (mean per cell ± SD): WT (1.23 ± 0.036), Myo7a-ΔC (1.22 ± 0.052), p = 0.80. Length of third row (mean per cell ± SD): WT (0.74 ± 0.036), Myo7a-ΔC (0.73 ± 0.045), p = 0.64. Analyzed numbers (stereocilia, cells, animals): WT(73; 7; 3), Myo7a-ΔC (70; 8; 3). p values of e – g were derived from two-tailed, unpaired t -tests. Boxplots indicate medians, 25th and 75th percentiles as box limits, and minima and maxima as whiskers, respectively. N for statistical analysis in e – g was number of cells. Source data are provided in the file.

Article Snippet: Although MYO7A levels are depleted in Myo7a-ΔC IHCs, hair bundle morphology is not affected. d Representative SEMs of IHCs of P7 WT and Myo7a-ΔC (scale bars: 1 μm). e No difference in stereocilia length in P7 IHCs between WT and Myo7a-ΔC mice by phalloidin immunofluorescence (IF) analysis (using Imaris 3D module).

Techniques: Two Tailed Test, Immunofluorescence, Derivative Assay

Journal: Nature Communications

Article Title: Myosin-VIIa is expressed in multiple isoforms and essential for tensioning the hair cell mechanotransduction complex

doi: 10.1038/s41467-020-15936-z

Figure Lengend Snippet: a MYO7A and MYO6 immunoreactivity in apical and basal turns of organ of Corti of P5 Myo7a-ΔC and WT mice (scale bar: 20 μm). b MYO7A immunoreactivity was quantified in apical, middle, and basal turns by normalizing to MYO6 immunoreactivity. Compared with WT, MYO7A levels in Myo7a-ΔC mice were reduced by ~85% ( p < 1e−4) in all IHCs, and by ~30% and 52% ( p < 1e−3 and <1e−4) in middle and apical-turn OHCs. Each data point in b corresponds to a mean MYO7A/MYO6 immunofluorescence ratio (mean of 12 IHCs and 36 OHCs per organ and position). Error bars indicate SD. N (animals) = 5 in WT and 7 in Myo7a-ΔC . p values in b were derived from two-tailed, unpaired t -tests. c Design of the HA-Myo7a-C KI mouse. HA-tag was knocked-in after the ATG of Myo7a-C by CRISPR-mediated gene editing. d Organ of Corti of an HA-Myo7a-C KI mouse, stained with a HA-specific antibody. The white boxes indicate approximate positions of the apical and basal cochlear regions shown on the right. HA immunoreactivity, representing the expression of MYO7A-C, is strong in all IHCs. In OHCs, HA immunoreactivity is readily detected at the cochlear apex, but decreases toward the base (scale bars: 200 μm in whole organ of Corti image, 20 μm in magnified panels). e The genomic position from which the Myo7a promoter used in the Myo7a::Actin-GFP transgenic mouse is derived. f Actin-GFP signal and phalloidin reactivity in the organ of Corti of Myo7a ::Actin-GFP transgenic mice at P6. The Actin-GFP signal was predominantly observed in the IHCs. Actin-GFP signal was detected at low levels in the apical OHCs, and decreased tonotopically toward the basal end of the cochlea (scale bar: 10 μm). Source data are provided in the file.

Article Snippet: Although MYO7A levels are depleted in Myo7a-ΔC IHCs, hair bundle morphology is not affected. d Representative SEMs of IHCs of P7 WT and Myo7a-ΔC (scale bars: 1 μm). e No difference in stereocilia length in P7 IHCs between WT and Myo7a-ΔC mice by phalloidin immunofluorescence (IF) analysis (using Imaris 3D module).

Techniques: Immunofluorescence, Derivative Assay, Two Tailed Test, CRISPR, Staining, Expressing, Transgenic Assay

Journal: Nature Communications

Article Title: Myosin-VIIa is expressed in multiple isoforms and essential for tensioning the hair cell mechanotransduction complex

doi: 10.1038/s41467-020-15936-z

Figure Lengend Snippet: a WT and Myo7a-ΔC organ of Corti immunostained for harmonin, F-actin, and MYO7A. Harmonin staining was indistinguishable between WT and Myo7a-Δ C, while severe mislocalization (formation of aggregates in the cuticular plate) was observed in Myo7a full KO mice (scale bar: 10 μm). b , c Harmonin immunoreactivity at the UTLDs in WT and Myo7a-ΔC IHCs at P7. Compared with WT, harmonin signal is slightly increased at the UTLD of Myo7a-ΔC IHCs. For quantification, harmonin fluorescence intensities in stereocilia (UTLDs of longest stereocilia only) were measured using ImageJ, normalized to phalloidin reactivity at the same site, and a mean intensity determined for each cell. Normalized harmonin intensities (mean ± SD): WT = 0.40 ± 0.092, Myo7a-ΔC = 0.47 ± 0.093, p = 2.34e−2 (two-tailed, unpaired t -tests). N = number of cells. Analyzed numbers (UTLDs; cells; animals): WT (167; 16; 3), Myo7a-ΔC (209; 17; 4) (scale bar: 1 μm). d , e ADGRV1 immunoreactivity at the stereocilia base was not significantly changed in Myo7a-ΔC IHCs at P7 compared with WT. ADGRV1 immunofluorescence intensity (from middle cochlear region) was determined for each cell and normalized to background immunofluorescence in the cuticular plate region of the same cell. Normalized ADGRV1 intensities (mean ± SD): WT = 1.39 ± 0.49, Myo7a-ΔC = 1.32 ± 0.16, p = 0.08 (two-tailed, unpaired t -tests). N = number of cells. Analyzed numbers (cells; animals): WT (24; 7), Myo7a-ΔC (33; 7) (scale bar: 1 μm). Data are plotted as mean ± SD. Source data are provided in the file.

Article Snippet: Although MYO7A levels are depleted in Myo7a-ΔC IHCs, hair bundle morphology is not affected. d Representative SEMs of IHCs of P7 WT and Myo7a-ΔC (scale bars: 1 μm). e No difference in stereocilia length in P7 IHCs between WT and Myo7a-ΔC mice by phalloidin immunofluorescence (IF) analysis (using Imaris 3D module).

Techniques: Staining, Fluorescence, Two Tailed Test, Immunofluorescence