Bioluminescence.

Bioluminescence is visualized at the distal femoral metaphysis following tumor cell inoculation.</p

Representative fluorescent micrograph.

<p>Image illustrates single (line with one arrowhead) and double-labeled surfaces (line with two arrowheads) in trabecular bone.</p

Micro-CT.

<p>3-D reconstructions of normal (top) and tumor-burdened (bottom) trabecular and cortical architecture in the distal metaphysis.</p

Radiograph severity scoring on a scale from 0–5.

<p>A. 0 –No evidence of tumor-associated osteolysis. B. 1 –Mild trabecular lysis apparent (≤ 50% of the diameter of the femur affected. C. 2 –Moderate trabecular lysis apparent (> 50% of the femur’s diameter affected). D. 3 –Severe osteolysis evident with 1 cortex involved. E. 4 –Severe osteolysis evident with both cortices affected. F. 5 –Fracture.</p

Evidence of previous osteosarcoma.

<p><b>(H&E)</b> a) Distal metaphysis exhibiting trabecular destruction, necrosis, and replacement fibrosis throughout regions of previous tumor burden. The growth plate (asterisk), metaphysis (dagger), and epiphysis (double dagger) are shown for reference. (20x) Note the metaphyseal tumor does not cross the growth plate to the epiphysis, where normal trabecular architecture is evident. b) Loosely organized fibrous connective tissue and necrosis in tumor-burdened areas. (100x).</p

Active Osteosarcoma at the study endpoint.

<p>a) Rat. Distal Metaphysis of Femur. (H&E. 200x). Replacing normal trabecular architecture are poorly differentiated neoplastic cells that are polygonal to spindle shaped with lightly eosinophilic cytoplasm. b) Rat. Distal Metaphysis of Femur. (H&E. 400x). Neoplastic cells are irregularly polygonal with indistinct cell borders and moderate amounts of cytoplasm. Nuclei contain multiple, distinct nucleoli and a mitotic figure is present (asterisk).</p

Radiograph severity score over time for each treatment group.

<p>There were no significant differences between treatment groups on any given day of imaging. There was a significant difference over all groups from Day 10 to Day 24 (p ≤ 0.001). Score shown as mean +/- SE.</p

Fluorescent micrograph.

<p>Areas of dystrophic mineralization confounded quantification of single-labeled surfaces. As an alternative variable, digital images of the region of interest were used to quantify the total number of positive florescent pixels. There were no significant differences between treatment groups.</p

Video_1_Complementary Use of Super-Resolution Imaging Modalities to Study the Nanoscale Architecture of Inhibitory Synapses.MP4

The nanoscale architecture of synapses has been investigated using multiple super-resolution methods, revealing a common modular structure for scaffolds, neurotransmitter receptors, and presynaptic proteins. This fundamental organization of proteins into subsynaptic domains (SSDs) is thought to be important for synaptic function and plasticity and common to many types of synapses. Using 3D super-resolution Structured Illumination Microscopy (3D-SIM), we recently showed that GABAergic inhibitory synapses exhibit this nanoscale organizational principle and are composed of SSDs of GABAA receptors (GABAARs), the inhibitory scaffold gephyrin, and the presynaptic active zone protein, RIM. Here, we have investigated the use of 3D-SIM and dSTORM to analyze the nanoscale architecture of the inhibitory synaptic adhesion molecule, neuroligin-2 (NL2). NL2 is a crucial mediator of inhibitory synapse formation and organization, associating with both GABAARs and gephyrin. However, the nanoscale sub-synaptic distribution NL2 remains unknown. We found that 3D-SIM and dSTORM provide complementary information regarding the distribution of NL2 at the inhibitory synapse, with NL2 forming nanoscale structures that have many similarities to gephyrin nanoscale architecture.</p

Assessment of bone resorption over the region of interest.

<p>(a) There was no difference (p = 0.0627) between groups in osteoclast surface. (b) There was no difference (p = 0.1108) between groups in osteoclast number. (c) TRAP surface showed a statistically significant difference (p = 0.0152) between groups, however, pairwise comparisons (Dunn’s test) was not able to detect which groups.</p