Ultrastructural Analysis Using Electron Microscopy Tibias from 7-day-old mice were ﬁxed overnight and processed as previously described [Pirog-Garcia et al., 2007]. Seventy nanometer sections were cut and stained with 0.3% (w/v) lead citrate and images were collected on a FEI Tecnai 12 Biotwin electron microscope (FEI UK Limited, Cambridge, UK). Cell Proliferation and Apoptosis Assays Mice were injected intraperitoneally with BrdU labeling solution (GE Healthcare Life Sciences, Amersham, UK) and sacriﬁced after 2 hr. Knee joints were dissected and processed as previously described [Pirog-Garcia et al., 2007]. IHC was performed using an anti-BrdU antibody (1:100; Abcam, Cambridge, UK) with a biotinylated sec- ondary rabbit anti-rat IgG, (1:200; Dako cytomation, Cambridge, UK). Images of the growth plate were collected using an Axiovision microscope software (Carl Zeiss Ltd., Welwyn Garden City, UK) and the proportion of BrdU positive cells (brown stain) compared to the total number of cells (Dapi stain) in the proliferative zone was determined for each genotype. The results were quantiﬁed using one-way ANOVA. Apoptosis was visualized using the DeadEnd TM ﬂuorometric TUNEL assay (Promega UK, Southampton, UK) with citrate buffer used for unmasking [Pirog-Garcia et al., 2007]. Images were col- lected using an Axiovision microscope software (Carl Zeiss) and statistic analysis performed by independent sample t-test. Real Time qRT-PCR Cartilage was dissected from 5-day-old mice (rib) or 3-week- old mice (xiphoid) of pooled mixed gender litters and snap frozen in liquid nitrogen. The tissue samples were then homogenized in TRIzol (Invitrogen, UK) using a micro-dismembranator (2,000 rpm for 2 min) and the RNA was isolated as per the man- ufacturer’s protocol. RNA concentration was determined by a Nanodrop ultra-low-volume spectrophotometer. RNA samples of 0.5 μg were subsequently used to synthesize ﬁrst strand cDNA using superscript III (Invitrogen) and real-time analysis of wild- type and mutant mRNA levels was performed using a SYBR R Green Kit on a ABIPrismTM 7000 sequence detector system (Applied Biosystems, Warrington, UK) as described previously [Leighton et al., 2007; Nundlall et al., 2010; Pirog-Garcia et al., 2007]. Primer sequences were: Grp78/BiP: 5 -GGCACCTTCG- ATGTGTCTCTTC-3 and rev: 5 -TCCATGACCCGCTGATCAA- 3 ; Grp94: 5 -TAAGCTGTATGTACGCCGCGT-3 and rev: 5 - GGAGATCATCGGAATCCACAAC-3 ; Cnx: 5 -TGA TTT CCT CTC CCT CCC CTT-3 and rev: 5 -CAC TGG AAC CTG TTG ATG GTG A-3 ; Crt: 5 -GCT ACG TGA AGC TGT TTC CGA-3 and rev: 5 - ACA TGA ACC TTC TTG GTG CCA G-3 ; Erp72: 5 -AGT ATG AGC CCA GGT TCC ACG T-3 and rev: 5 -AGA AGT CTT ACG ATG GCC CAC C-3 . Each experiment included “no template” controls, was run in duplicate, and had an 18S RNA control. Each experiment was repeated three times for statistical relevance and the results an- alyzed by independent samples t-test. SDS-PAGE and Western Blotting Rib cartilage from 5-day-old mice or xiphoid cartilage from 3- week-old mice was homogenized in 100 μl dH2O per 5 mg tissue, boiled in SDS loading buffer containing dithiothreito (DTT) and 40 μl of total protein was loaded on an SDS-PAGE gel. The gel was 220 HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 electroblotted onto a nitrocellulose membrane, which was blocked overnight with 2% skimmed milk powder in phosphate buffered saline-triton (PBS-T). The COMP antibody (GeneTex Inc/Patricell Ltd., Nottingham, UK) was used at a dilution of 1:100 and antibod- ies to chaperones associated with the UPR were used at a dilution of 1:500 (Grp78, Grp94, Erp72; all from Santa Cruz Biotechnol- ogy, Inc., Santa Cruz, CA, USA). The Western blots were scanned and analyzed by densitometry using the AIDA32 software and then normalized to Ponceau staining for total protein (n ≥ 3 litters per genotype [∼5–8 mice per litter of mixed gender] and ≥3 separate experiments were performed in duplicate). Adherent HT1080 cells transfected with GFP vector alone, GFP- COMP wild-type or GFP-COMP mutant were cultured in T75 ﬂasks in DMEM4 (Sigma-Aldrich Ltd., Haverhill, UK) supplemented with 10% FBS (Fisher Scientiﬁc UK Ltd., Loughborough, UK), 1% Penicillin/Streptomycin 100× solution (Sigma-Aldrich), 1% of 200 mM L-Glutamine solution (Sigma-Aldrich), 1% nonessential amino acids 100× solution (Sigma-Aldrich), and 360 mg/L G418, changing medium every second day. After they reached conﬂuence, cells were cultured in the same medium for 48 or 72 hr, washed three times in ice-cold PBS and lysed in 1 ml of RIPA buffer (150 mM NaCl, 1% Na Deoxycholate, 0.1% SDS, and 10 mM Tris-HCl, pH 7.4) on ice for 15 min. Cell lysates were centrifuged at 13,000 g for 10 min at 4◦C and the supernatants were collected. An aliquot of the supernatant was used for protein concentration evaluation. Cell lysates were then boiled for 5 min in SDS-PAGE loading buffer containing DTT. Twenty micrograms of proteins were loaded on an SDS-PAGE 4–12% precast gel (Invitrogen) and electroblotted on a nitrocellulose membrane, which was blocked overnight with 2% skimmed milk powder in PBS-T. The monoclonal COMP anti- body (clone 12C4) was used at a dilution of 1:1000; the Polyclonal PRDX2 antibody (ProteinTech Europe, Manchester, UK) and the monoclonal β actin antibody (Abcam, Cambridge, UK) were used at a dilution of 1:500. Microarray Hybridization and Statistical Analysis For the microarray analysis, RNA was isolated using TRIzol (In- vitrogen, UK) from the rib chondrocytes of newborn and 5-day-old mice [Nundlall et al., 2010], and the cell culture model at 48 hr after conﬂuence. In order to analyze gene expression in mice that were homozygous for Comp D469del, hybridizations were performed on three separate RNA samples (3 × mutant and 3 × wild-type) isolated from chondrocytes pooled from ﬁve littermates of mixed gender. Only one RNA sample (one × mutant litter and one × wild-type litter of mixed gender) was examined from mice heterozygous for Comp D469del or homozygous for Matn3 V194D [Nundlall et al., 2010]. RNA quality was assessed using the RNA 6000 Nano Assay and analyzed on an Agilent 2100 Bioanalyser (Agilent Technologies UK Ltd., Edinburgh, UK). RNA concentration was quantiﬁed using a Nanodrop ultra-low-volume spectrophotometer (Nanodrop Tech- nologies, Wilmington, DE, USA). The hybridization cocktail was hybridized to either the Mouse 430_2 oligonucleotide array or Hu- man HG-U133_plus_2 array (Affymetrix UK Ltd., High Wycombe, UK) according to the manufacturers’ instructions. Arrays were read using an Agilent GeneArray scanner 3000 7G and processed us- ing Affymetrix GCOS (V1.4) software. Statistical analysis was per- formed using Robust Multi-array Average (RMA), which is an al- gorithm used to generate expression proﬁles from Affymetrix data [Irizarry et al., 2003].