ABR analysis of mice at 6 and 12 months of age.

*<p>denotes a significant p value (p<0.05).</p

Hearing analysis using ABR.

<p>Mice were subjected to ABR analysis at 6 months of age (A) and at 12 months of age (B) to determine their hearing ability. At 6 months, female mice possessed significantly better hearing than males at 8, 16 and 32 KHz. However, at 12 months of age, the hearing of the females had worsened compared to males. Error bars equal the SEM.</p

Analysis of cochlea bone.

<p>Bone mineral density of the cochlea was calculated for female and male mice using microCT scanning (panel A). Female mice possessed a significantly lower BMD index than male mice (p = 0.014). Subsequent analysis revealed that the average size of the osteocyte lacunae was significantly larger in the female mice compared to the males (p<0.001). Error bars denote the SEM and * indicates a significance of p<0.05.</p

Representative histological sections of cochlea bone for female and male mice.

<p>H&E stained sections of cochlea bone revealed that the osteocyte lacunae (arrows) appeared larger in the female mice (panel A) compared to the male mice (pane B). Scale bars equal 50 µm.</p

Threshold shift in hearing ability for male and female mice.

<p>The change in hearing acuity between 6 and 12 months for female and male mice were calculated for a click response and at various pure tone frequencies. It was found that females displayed a significantly greater shift in hearing threshold compared to males at 4 KHz (p = 0.005) and 8 KHz (p = 0.007). Error bars denote the SEM and * indicates a significance of p<0.05.</p

Additional file 1: of Protein kinase C delta null mice exhibit structural alterations in articular surface, intra-articular and subchondral compartments

Safranin O staining of the growth plate in PKC-δ+/+ and PKC-δ−/− mice. (A, B) Slightly weaker staining of safranin O was found in PKC-δ−/− mice; no significant difference in the height of the growth plate was found between PKC-δ+/+ and PKC-δ−/− mice as measured by safranin O staining regions. (C) Measurement of the height of growth plates by safranin O staining regions in PKC-δ+/+ and PKC-δ−/− mice. (TIFF 1716 kb

Cytoskeletal reorganization, lysosomal acidification and MARCKS phosphorylation in PKC-δ KO osteoclasts.

<p>(A) Osteoclasts on bone slices were immunofluorescently stained with Rhodamine Phalloidin, anti-α-tubulin, and Hoechst to visualize F-actin, α-tubulin and DNA by confocal microscopy. Scale bar represents 100 μm. (B) Osteoclasts were treated with Acridine Orange, which displays green fluorescence at neutral pH. Acridine Orange green fluorescence intensity was measured in a fluorescence microplate reader. Bar charts represent mean ± standard deviation. Experiments were performed in triplicate. n.s., no significance (p-value>0.05). (C) Osteoclasts on bone slices were immunofluorescently stained pMARCKS, Rhodamine Phalloidin and DAPI to examine the phosphorylation levels of MARCKS by confocal microscopy. Scale bar represents 20 μm.</p

Inhibition of PKC-δ and knock out of PKC-δ resulted in impaired osteoclastic bone resorption in vitro.

<p>(A) Multinucleated giant cells isolated from patients presenting with Giant cell tumor (GCT) of bone were cultured on the bovine bone slices in the presence and absence of Rottlerin (Rott). Representative light images of osteoclasts derived from GCT, and scanning electron micrographs of resorptive lacunae on bone slices. Resorbed area as a percentage of total bone slice area was determined. (B) SEM micrographs of bone discs cultured with WT and PKC<b>-</b>δ KO osteoclasts. Osteoclast bone resorption pits are highlighted by white boxes. Average bone resorption area and average pit depth was measured. Total osteoclast numbers were the same on all bone discs. (C) Percentage of CTX released into culture medium by PKC-δ KO and WT osteoclasts cultured in bone. Scale bar represents 200 μm. Bar charts represent mean ± standard deviation. *, p-value <0.05, **, p-value <0.01.</p

Altered ERK and Src signaling in PKC-δ deficient osteoclasts.

<p>WT and PKC-δ KO BMMs were serum-starved overnight before stimulation with M-CSF and 100 ng/ml of RANKL at the indicated times. (A) Western blot analysis of total protein from WT and PKC-δ KO BMMs stimulated with M-CSF and 100 ng/ml of RANKL (short time-course, 0–120 min). (B) Quantitative analysis of short-term ERK phosphorylation relative to total ERK protein expression by densitometry of western blot images. (C) Western blot analysis of RANKL-induced osteoclastogenesis (long time-course, 0–6 days). (D) Quantitative analysis of Src Tyr-416 and Tyr-527 phosphorylation status relative to total Src protein expression, and long-term ERK phosphorylation relative to total ERK protein expression, as measured by densitometry of western blot images. β-actin was probed as a loading control. Statistical analysis was performed by comparing to WT in each time point. *, p-value <0.05. **, p-value<0.01.</p

PKC-δ is the predominant isoform among PKCs expressed in osteoclasts.

<p>Microarray analysis of PKC isoform gene expression during osteoclast differentiation. (A) BMM cells (pre-OC) were treated with 100 ng/ml RANKL for 5 days to differentiate into mature osteoclasts (OC). Total RNA was harvested for microarray analysis. Heatmap demonstrating the upregulation of PKC-β, PKC-δ and PKC-η during osteoclast differentiation, with osteoclast specific genes. Up-regulation and down-regulation are shown in red and green respectively. TRAP staining for osteoclasts was also included in parallel experiment. Scale bar represents 200 μm. (B) Relative expressions of PKC isoforms was presented by arbitrary readings of microarray analysis (C) Semi-quantitative RT-PCR analysis comparing the gene expression profile of PKC isoforms in BMM and RANKL-treated osteoclasts including classical PKCs (−α, −β and −γ), novel PKCs (−δ, −ε, −η and −θ) and atypical PKCs (−ι/λ and −ζ).</p