Progressive degeneration of hair bundles in <i>Tur/Tur</i> cochleae.

<p>(A,B) Immunofluorescence of anti-myosin VI on cochlear sections of wild-type (A) and <i>Tur/Tur</i> mutant at P0 (from middle turn). Arrow points to smaller tunnel of Corti in the mutant. (C-D) Phalloidin staining of whole-mount cochlear sensory epithelium (middle turn) from P0 (C,D), P14 (E,F) and P21 (G,H) with phalloidin (red). <i>Myo6</i>^{<i>Tur/Tur</i>} cochleae show irregular shapes of hair bundles compared to wild-type cochleae throughout all regions of the cochlea. Asterisks in H point to degeneration or elongation of stereocilial bundles in the mutant at P21. IHC, inner hair cell; OHC, outer hair cell. Scale bar: 25 μm.</p

Disorganized vestibular hair bundles in <i>Tur/Tur</i> mutant.

<p>(A-F) Phalloidin staining showing stereocilia in maculae in the utricle and saccule at P14. Hair bundles of <i>Tur/Tur</i> mice are irregular with no pattern of organization. Arrows in F point to elongation of stereocilia in the mutant when compared to that in normal mice (arrows in E). Panel E,F are higher magnification of boxed area in C,D. (G,H) Anti-myosin VI immunostaining on utricle sections. Scale bars: E,F, 13 μm and all other panels, 20 μm.</p

The <i>Turner</i> mutation in myosin VI.

<p>(A) Pattern program output of the results of 11 markers located between 63.3798 Mb and 96.3284 Mb, tested on DNA derived from 11 affected (9 were G3 offspring and 2 were the N2 breeding pair), that showed recombination between three markers (gnf09.071.906, 09.081.324 and rs13480312). The <i>Tur</i> mutation was determined to lie between marker rs6292345 (75.3386 Mb) and rs3698443 (87.1572 Mb). The black box (linked) indicates mice heterozygous for the C3H and the C57; the white box (unlinked) indicates mice homozygous for the C3H (patterns 1 and 2) or the C57 (pattern 4). (B) Sequence analysis of the <i>Turner</i> genomic DNA revealing a 820A>T transversion in exon 8 (NM_001039546). Control DNA shows a single A peak and heterozygous DNA (<i>Tur/+</i>) shows both A and T peaks, but homozygous DNA (<i>Tur/Tur</i>) shows a single T peak. (C) The p. N200I single amino acid substitution. Alignment of a portion of myosin VI protein from various species revealing conservation of the N200 residue. The switch I loop consensus sequence motif within the motor domain is indicated.</p

A variety of structural defects in <i>Tur</i> mutant cochlear hair bundles at P21.

<p>Higher magnifications of SEM images showing stereocilia bundles of outer hair cells in wild-type (A), <i>Tur/Tur</i> (B-E) and <i>Tur/+</i> (F-J) mice. <i>Myo6</i>^{<i>Tur/Tur</i>} cochleae show formation of multiple clumps of stereocilia within the same cell (split bundles) (B-D), stereocilial fusion bundles (arrow, D), folded stereocilia (E) or other abnormal shapes (generally deformed bundles) (C,E). In <i>Myo6</i>^<i>Tur/+</i> cochleae (F-J), the stereocilia also appear to be misshapen in the basal turn and loss of hair cells was occasionally observed (arrow in F). However, hair bundle split or degeneration was not apparent. Scale bars: A, 1 μm; B-E,G-J, 2 μm; F, 3 μm.</p

Degeneration of vestibular hair cells in <i>Tur/Tur</i> mutant.

<p>H&E staining of inner ear tissue at 4 weeks. (A-H) Posterior (A,B) and anterior (C,D) crista ampullaris, utricular macula (E,F) and Organ of Corti (G,H) in wild-type (A,C,E,G) and <i>Tur/Tur</i> mutant (B,D,F,H). Significant reduction of hair cells in the vestibular sensory organs (arrows in B,D,F) and collapse of the organ of Corti (arrow, H) are evident in the inner ear of <i>Tur/Tur</i> mice. Panels G,H are images showing the organ of Corti in the basal turn. Abb.: hc, hair cell; ihc, inner hair cell; ohc, outer hair cell; sc, supporting cell; tc, tunnel of Corti. Scale bar: A-F, 50 μm and G,H, 25 μm.</p

ABR threshold measurements of mice at 4 (unbroken lines) and 10 (broken lines) weeks of age.

<p>(A) Average threshold ± s.e.m. for wild-type (+/+) at 4 weeks (n = 3) and 10 weeks (n = 6), <i>Tur</i> heterozygous <i>(Tur/+)</i> at 10 weeks (n = 8) and homozygous ears (<i>Tur/Tur</i>) at 4 weeks (n = 5) and 10 weeks (n = 6) of age. Averages of 90 dB SPL were used for thresholds beyond the upper limit of the sound system. The <i>Tur/+</i> mice at 10 weeks of age had mild hearing loss in both ears (threshold shifted by 10–20 dB between 16 and 45 kHz), while <i>Tur/+</i> mice at 4 weeks of age (n = 5) had relatively normal hearing. However, all homozygous mice at 4 weeks of age had severe hearing loss (threshold shift by ~20–40 dB), while homozygous mice at 10 weeks of age had >35 dB loss between 5–11 kHz and >50 dB loss between 16–45 kHz.</p

Disorganization and degeneration of hair bundles in <i>Tur/Tur</i> mutant cochleae at P0 and P21.

<p>SEM images showing stereocilial bundles of outer hair cells in the mid-basal turn in wild-type at P0 (A) and P21 (E), <i>Tur/Tur</i> at P0 (B-D) and P21 (F,G). <i>Myo6</i>^{<i>Tur/Tur</i>} cochleae show misshapen stereocilia, which appear to be smaller/narrower when compared to those in wild-type mice at P0. At P21, stereocilial degeneration is apparent in the majority of cells in the mid-basal turn (arrows). Scale bars: A-D, 2 μm; E,F, 3.0 μm; G, 2.5 μm.</p

The penetrant rate for gross and behavioral abnormalities of <i>Turner</i> mice.

<p>The penetrant rate for gross and behavioral abnormalities of <i>Turner</i> mice.</p

<i>In vivo</i> analysis of tumor-associated macrophage polarization upon anti-CD47 treatment; bioluminescence and survival analysis of treated mice.

<p>(A) Ratio of CD80 and CD206 positive cell count per total macrophage count in untreated and anti-CD47-treated mice (p = 0.0054 for CD80 and 0.164 for CD260, paired t-test). (B) Median fluorescent intensity (MFI) measurement of CD80-AF647 and CD206-PE (p = 0.0002 for CD80 and 0.423 for CD206, paired t-test). (C) Bioluminescence in vivo imaging data (left panel), photon flux values at days 21 and 50 (middle panel) and Kaplan-Meier analysis of mice grafted with GBM5 and treated with Hu5F9-G4 (right panel, 250 μg/dose, every other day, starting at week 3; n = 5 per group, p = 0.0018, log-rank analysis). Legend: blue shapes: mice engrafted with GBM4, black shapes: mice engrafted with GBM5.</p

Differential phagocytosis rate of human M1 and M2 macrophages towards various human glioma cells upon CD47-SIRPα disruption.

<p>(A) Representative flow cytometric phagocytosis assay of human M1 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD80^high live singlets was measured and compared between untreated (left column) and anti-CD47 antibody-treated (right column) co-cultures. (B) Representative flow cytometric phagocytosis assay of human M2 macrophages against CSFE-labeled GBM1 and PGBM1 cells. The percentage of CSFE+CD11b+CD163^high live singlets was measured and compared between untreated and anti-CD47 antibody-treated co-cultures. (C) Bar graph demonstrating the change in phagocytosis rates by human M1 macrophages towards individual co-incubated (GBM1-4 and PGBM1) tumor cells -/+ anti-CD47 (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests). (D) Bar graph demonstrating the change in phagocytosis rates by human M2 macrophages towards individual tumor cell (GBM1-4 and PGBM1 -/+ anti-CD47 (significant difference in means of technical triplicates indicated by * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, multiple t-tests).</p