Cell-based assay for the detection of chemically induced cellular stress by immortalized untransformed transgenic hepatocytes-1

<p><b>Copyright information:</b></p><p>Taken from "Cell-based assay for the detection of chemically induced cellular stress by immortalized untransformed transgenic hepatocytes"</p><p>BMC Biotechnology 2004;4():5-5.</p><p>Published online 19 Mar 2004</p><p>PMCID:PMC406386.</p><p>Copyright © 2004 Sacco et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p>ations of NaAsO(As1: 10M, As2: 5 × 10M, As3: 10M) and CdCl(Cd1: 10M, Cd2: 5 × 10M, Cd3: 10M). . Mean ± SE of viability of PHGH and MMH-GH5 at the indicated treatment conditions as determined by the Trypan blue dye exclusion method. These results were obtained from 6 independent experiments with treatments carried out in triplicate

Prevention of the osteopetrotic phenotype in MuTuDC-treated oc/oc mice.

<p>A) Bone mineral density analyses of femur using CT Analyzer of normal littermates (+/+), MuTuDC-treated <i>oc/oc</i> mice as well as untreated oc/oc mice. The latter was smaller since they did not survive longer than 15–20 days. B) Bone morphometric analysis on control littermates, MuTuDC-treated <i>oc/oc</i> mice and untreated oc/oc mice. Four mice per group, data are from a single experiment representative of two independent experiments.</p

Bone lesions are independent of both T and B cells.

<p>Rag KO and CD3ε KO mice were used as recipients of MuTuDCs. Representative of stereomicroscopic images of skulls from wild type, Rag KO and CD3ε KO mice injected subcutaneously with MuTuDCs on the calvaria. Data are from a single experiment representative of two independent experiments. Five mice per group in each experiment.</p

Mice are protected from lytic bone lesions using bisphosphonates and osteoprotegerin (m OPG).

<p>A) Representative CT Scan images of skulls of MuTuDC injected mice (subcutaneously on the calvaria, close to the left parietal bone). Mice were treated with either PBS (control) or the biphosphonate Aclasta B) Representative stereomicroscopic images of skull bones of mice injected as above with PBS (control) or mOPG one week following adoptive transfer of MuTuDCs. Images in A and B show the absence of bone lesions after Aclasta or mOPG treatments. Arrow indicates the site of MuTuDC injections. Data are from a single experiment representative of two independent experiments. Five mice per group in each experiment. Similar results were obtained after i.v. injection of MuTuDCs (data not shown).</p

MuTuDCs are recruited to the bone marrow of Mushi mice.

<p>A) Flow cytometric analyses of bone marrow from 3 to 4 months old healthy Mushi Tg mice or sick Mushi Tg mice. Representative FACS plots of DC markers showing an extensive cellular infiltrate of GFP positive DCs in the bone marrow of the mice. Data are representative of at least three independent experiments of five mice each. B) Upper panels: H&E staining of paraffin embedded sections of bone marrow from Tg healthy or Tg sick mice (40X). Lower panels: Higher magnification image (100X) shows the presence of classical osteoclast morphology in sick Mushi Tg mice. One representative image from three independent experiments is shown.</p

Differentiation of MuTuDCs into functional osteoclasts in vitro.

<p>A) Morphology of osteoclasts induced from DCs. MuTuDCs were cultured for 12 days either with or without M-CSF and RANK-L. B) Immunofluorescence analyses for the evaluation of osteoclats differentiation. TRAP-positive cells are stained in red. Nuclei are stained with DAPI (blue). C) Resorption activity was assessed on dentine slices following culture of MuTuDCs with M-CSF and RANK-L. Blue spots indicate the presence of resorption pits for the cytokine-treated cells. All results are representative of two independent experiments performed in three biological replicates. (A-C) One representative image from three independent experiments is shown. D) mRNA relative expression levels of osteoclast specific genes. TRAP, cFos, cathepsin and NFATC1 were measured by qRT-PCR, normalized to β-actin and analyzed with qBasePlus software. The results are representative of 3 independent experiments with three biological replicates per experiment.</p

Transfer of MuTuDCs in vivo induces osteolytic lesions.

<p>A) Representative stereo microscopic images of skulls from control mice or MuTuDC injected subcutaneously on the calvaria. 4 weeks post-injection, mice that received MuTuDCs had significant osteolytic lesions compared to control mice. The bones became very fragile and disintegrated after isolation. Five mice per group, data are from a single experiment representative of three independent experiments. B) Morphometric analysis of PBS and MuTuDC injected mouse femurs using CT Analyzer one month after intravenous MuTuDC adoptive transfer. The metaphyseal trabecular bone region in the distal femur was examined. Data are representative of two independent experiments of 3–4 mice per group. C) Representative image of axial views of femoral metaphyseal region of control and injected mice using micro-CT scan. Three to four mice were used for each experimental condition.</p