Collagen production by intervertebral disc (IVD) cells in monolayer and alginate cultures

Immunocytochemistry of cytospins was used to examine collagen production at day 7; representative images are shown. In monolayer control cultures (left panels), collagen type I-immunopositive IVD cells were markedly more prevalent than collagen type II-immunopositive cells, indicative of a dedifferentiated phenotype [22]. This pattern of immunopositivity was reversed in alginate control cultures (right panels), suggesting that the cells in alginate had redifferentiated toward a chondrocytic phenotype [22]. Immunopositivity for both types of collagen was markedly decreased in glucose-deprived cultures, both in monolayer (left panels) and alginate (right panels) culture. Phalloidin-fluorescein isothiocyanate staining of IVD cells following monolayer (left panel) and alginate (right panel) culture in conditions of glucose-deprivation at day 7, demonstrating that the presence of filamentous actin (F-actin) was not markedly different in either condition (original magnification ×200).<p><b>Copyright information:</b></p><p>Taken from "The influence of serum, glucose and oxygen on intervertebral disc cell growth : implications for degenerative disc disease"</p><p>http://arthritis-research.com/content/10/2/R46</p><p>Arthritis Research & Therapy 2008;10(2):R46-R46.</p><p>Published online 23 Apr 2008</p><p>PMCID:PMC2453766.</p><p></p

Loss of hDlg1 enhances resistance to anoikis.

<p>Panel A. Cells were plated on tissue culture dishes that were either untreated, or coated with poly-HEMA to block cell attachment. After 24 h the cells were harvested and stained with Propidium Iodide, and the cell cycle distribution of a representative clone was ascertained by flow cytometry. Panel B shows the mean percentage of cells entering apoptosis (as determined by sub-G1 DNA content) from at least three separate experiments with three independent cell lines together with the standard deviations. Panel C. Cells were processed as in Panel A, but were stained with Trypan Blue. The results show the mean percentage of dead and dying cells, as determined by Trypan Blue uptake from at least three independent experiments on at least three independent cell lines together with the standard deviations. Panel D. Cells were treated with etoposide or exposed to UV C. After 12 h incubation the cells were stained with Propidium Iodide and the sub-genomic DNA content determined by flow cytometry, with the results shown being from one representative clone of each line.</p

hDlg1 depleted cells show reduced levels of E-cadherin expression but increased levels of β-catenin expression.

<p>Cells were grown to 80% confluency and then extracted into cytosolic (F1), membrane (F2), nuclear (F3) and cytoskeletal (F4) fractions. These were then analysed for the distribution of β-catenin and E-cadherin by western blotting. Controls for the integrity of the fractions were vimentin for the cytoskeletal fraction, p84 for the nuclear fraction, α-tubulin for the cytosolic fraction and transferrin receptor for the membrane fraction.</p

hScrib-depleted cells show reduced TJ and AJ formation.

<p>Control, hDlg1 and hScrib-depleted cells were grown for 24 h then switched into low calcium medium for 1 h. High calcium containing medium was then added for 6 h. The cells were fixed and stained for the TJ-associated marker ZO-1 (Panel A with insets showing junctional detail) and the AJ-associated marker E-cadherin (Panel B with insets showing junctional detail). The results show the staining under normal growth conditions, after calcium starvation and after 6 h calcium addition in representative clones; identical results were obtained with at least two additional clones of control, shRNA Dlg1 and shRNA Scrib cells.</p

Comparison of the colony-forming capacity of the different cell lines.

<p>1×10³ cells were plated in each well and after 10 days the dishes were fixed and stained with Giemsa. Panel A shows a representative assay with the indicated clones. Panels B–D show the collated results from a minimum of 3 assays with at least two different clones. Panel B shows the total colony numbers, with this separated into the number of small (Panel C) and large colonies (Panel D) based on the pixel cell density (where small colonies are <0.012 mm² and large >0.012 mm²) of the image using a BioRAD Gel Doc xR. Students T Test on Panel C: TR2 vs Dlg p = 0.0171, TR2 vs Scrib p = 0.0201 and on Panel D: TR2 vs Dlg p = 0.0569, TR2 vs Scrib p = 0.0381. Panel E shows the growth rates of representative clones of the TR2, shScrib and shDlg1 cell lines over a period of 6 days.</p

Generation of HaCaT cell lines stably depleted of hDlg1 or hScrib expression. Panel A.

<p>HaCaT cells were transfected with control plasmid (TR2) or plasmids with targeting sequences specific for either hDlg1 or hScrib. After selection, colonies were picked and levels of hDlg1 and hScrib expression were ascertained by western blotting. A selection of clones is shown at different passages, with the levels of tubulin expression shown for comparison. Panel B. Morphological appearance of the control (TR2 series) and representative hDlg1 and hScrib-depleted cell lines at low and high density. Note the typical HaCaT cobblestone morphology of the control cells and hDlg1-depleted cells, and the more fibroblastic appearance of cells in the hScrib-depleted clones at low cell densities. At higher cell density all three lines exhibit similar cobblestone morphology (right hand panels).</p

Loss of hScrib enhances cell invasion.

<p>Panel A. The cell lines were incubated in the upper compartment of matrigel chambers in serum-free medium. After 22 h, the cells that had migrated into the lower serum-containing compartment were fixed and stained with Crystal Violet. The panels show the staining of the cells on the lower surface of the collagen matrix, with the TR2 control cell line and two examples of hDlg1 depleted cells and two examples of the hScrib-depleted cells shown. Panel B shows the quantifications and standard deviations of the numbers of invading cells obtained from at least three independent assays with at least three different control, hScrib and hDlg1 knockdown cell lines.</p

Macroscopic appearance of dissected motion segments after incubation for 1 week with 20 mg/ml of trypsin, papain or no enzyme (control)

<p><b>Copyright information:</b></p><p>Taken from "Bovine explant model of degeneration of the intervertebral disc"</p><p>http://www.biomedcentral.com/1471-2474/9/24</p><p>BMC Musculoskeletal Disorders 2008;9():24-24.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2266744.</p><p></p

(a) H&E stained sections at 3 weeks post-incubation with 20 mg trypsin, 20 mg papain or no enzyme (control)

(b) Toluidine blue stained sections demonstrating severe loss of metachromasia from all of the disc in enzyme treated discs compared to non-enzymatically digested, where loss is diminished and only in the outer annulus.<p><b>Copyright information:</b></p><p>Taken from "Bovine explant model of degeneration of the intervertebral disc"</p><p>http://www.biomedcentral.com/1471-2474/9/24</p><p>BMC Musculoskeletal Disorders 2008;9():24-24.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2266744.</p><p></p

The profile from outer annulus to the central nucleus pulposus of (a) water and (b) GAG contents of fresh normal, bovine disc (i

E. not injected with enzyme or cultured).<p><b>Copyright information:</b></p><p>Taken from "Bovine explant model of degeneration of the intervertebral disc"</p><p>http://www.biomedcentral.com/1471-2474/9/24</p><p>BMC Musculoskeletal Disorders 2008;9():24-24.</p><p>Published online 25 Feb 2008</p><p>PMCID:PMC2266744.</p><p></p