Introduction Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian computer virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ). Results The AF-specific expression signature included and was largely maintained in immortal AF cell lines. Remarkably TGFβ induced rapid 3D sheet formation in a subgroup of Azithromycin (Zithromax) AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (examination (written informed consent was obtained from the donor’s relatives and approval for the study was granted by the local ethics committee: North West Research Ethics Committee). Representative tissue biopsies were processed to paraffin wax and immunohistochemical staining performed on 5 μm sections as previously described . Briefly sections were deparafinized POLD4 rehydrated and heat-mediated antigen retrieval performed using 10 mM Tris/1mM EDTA pH9 at 95°C Azithromycin (Zithromax) for 10 minutes in a steamer. Endogenous peroxidase was blocked using 3% hydrogen peroxide in TBS for 1 hr and non-specific binding sites blocked with 25% normal goat serum in TBS for 45 minutes. Sections were incubated overnight at 4°C with rabbit polyclonal primary antibody for P4HA3 (1:100 in 1% BSA in TBS; Sigma HPA007897). Biotinylated goat anti-rabbit secondary antibody was used and staining was disclosed using Vectastain Elite ABC Reagent and a diaminobenzidine chromogen. The unfavorable control used the appropriate IgG (Dako) in place of the primary antibody at equal protein concentration. Stained sections were viewed under light microscopy and images were acquired using an InfinityX camera with DeltaPix software. Alternatively sections was scanned using the Pannoramic 250 Flash II digital slide scanner (3DHistech?) and visualised using the Pannoramic Viewer software (3DHistech?). RNA isolation and quantitative real time PCR To isolate RNA cells were disrupted in Trizol (Invitrogen). RNA isolation RNA quantification (UV)-spectrometry (Nanodrop Thermo Scientific) and cDNA synthesis were performed as described before . Real-time quantitative PCR (RT-qPCR) was performed using Mesagreen qPCR master-mix plus for SYBR? Green (Eurogentec). Validated primer sets used are depicted in Table 2. An Applied Azithromycin (Zithromax) Biosystems ABI PRISM 7700 Sequence Detection System was used for amplification: initial denaturation 95°C for 10 min followed by 40 cycles of DNA amplification. Data were analyzed using the standard curve method and normalized to assessments. To test for normal distribution of input data D’Agostino-Pearson omnibus normality assessments were performed. All quantitative data sets presented exceeded the normality assessments. In Figs ?Figs11 and ?and22 a Azithromycin (Zithromax) two-tailed student test was used and in Figs ?Figs3 3 ? 44 and ?and55 a one-tailed student test was used as only a positive difference was expected. Gene expression analyses show mean and standard deviation. Fig 1 Confirmation of AF cell phenotype morphology and gene expression phenotypes. Expression of most previously reported AF marker genes [27 30  and  was at least 2 fold higher in primary AF cultures of two impartial donors as compared to matched NP cultures at passage 5 (P5) (Fig 1D). The putative AF marker  was exclusively expressed in primary AF cells (Fig 1D). In addition we found differential expression of levels (Fig 1D). NP-specific marker expression analysis was published elsewhere . This initial data thus confirms unique tissue of origin of primary AF and NP cells. Cell line generation and characterization of AF cell clones A total of 70 cell clones (Table 1) were obtained from immortalized P5 cells that displayed a comparable fibroblastic morphology (Fig 2A) in agreement with published reports [31 32 Eleven randomly chosen clones showed nearly comparable proliferation rates with an average populace doubling time (PDL) of 50.71 hours in the exponential phase (Fig 2B). To evaluate whether cell clones retained an AF-specific marker expression profile we measured expression of genes associated with a chondrocyte-like phenotype: and . In addition we.