Calreticulin

For qRT-PCR, the rib cages from 5-day-old mice (wild type (wt) and mutant (m/m)) were dissected and treated with collagenase (type 1A, 2 mg/ml) for 1 h at 37°C in Dulbecco’s modified Eagle’s medium (DMEM). The costal cartilage was dissected from individual ribs, and the perichondrium layer was removed. The cartilage was digested a second time with collagenase for 3 h to remove the collagen matrix and release the chondrocytes. The chondrocytes were passed through a cell strainer (70 μm) and washed with DMEM containing 10% FBS and following centrifugation washed again with PBS. The cell pellet was resuspended in 500 μl TriZol (Invitrogen), and total RNA was isolated according to the manufacturer’s instructions. First-strand cDNA was synthesised using random hexamer primers (Superscript III, Invitrogen), and qPCR was performed using the SYBR® green PCR protocol. Primer sequences were: BiP: 5′-ggcaccttcgatgtgtctcttc-3′ and rev: 5′-tccatgacccgctgatcaa-3′; Grp94: 5′-taagctgtatgtacgccgcgt-3′ and rev: 5′-ggagatcatcggaatccacaac-3′; Calnexin: 5′-tga ttt cct ctc cct ccc ctt-3′ and rev: 5′-cac tgg aac ctg ttg atg gtg a-3′; Calreticulin: 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 independent experiment was repeated three times, and the results were analysed by independent-samples t test.
For Western blot analysis, chondrocytes were isolated as above, but aliquots of 2 × 10⁵ chondrocytes were prepared and resuspended in 5× sodium dodecyl sulphate (SDS) loading buffer containing DTT. These protein aliquots were separated by 4–12% SDS-polyacrylamide gel electrophoresis (PAGE; Invitrogen) then transferred to nitrocellulose membranes for Western blot analysis. Ponceau staining was used to confirm equal loading of total protein isolates.
Antibodies to key chaperones associated with the unfolded protein response were used at a dilution of either 1:500 (BiP, Grp94, Erp72 and PDI; all from Santa Cruz) or 1:100 (ATF-6 from Imgenex and Bcl-2 from Abcam).

Mouse embryonic fibroblasts were isolated from calreticulin-deficient and wild-type embryos, immortalized, and designated K41 and K42, respectively (Nakamura et al., 2000 (link)). K42 crt^−/− cells were transfected with the pcDNA3 expression vector containing cDNA encoding rabbit calreticulin to generate crt^−/− cell lines expressing recombinant calreticulin (designated K42CRT). K42 cells were also transfected with expression vectors encoding the N + P domain or P + C domain of calreticulin to generated K42N+P and K42P+C lines, respectively. cDNA encoding the N + P domain of calreticulin (amino acid residues 1–287) was synthesized by PCR-driven reaction using the following oligodeoxynucleotides with 5′ flanking EcoR1 (primer N5′) and Kpn1 (primer P3′) restriction sites: N5′, 5′-ATATGAATTCATGCTGCTCCCTGTGCCGCT-3′ and P3′, 5′-ATATCTCGAGGTCGGGCGAGTACTCGGGGT-3′.
cDNA encoding the P + C domain of the protein (amino acid residues 172–401) was synthesized by PCR-driven reaction using the following P5′ and C3′ primers with 5′ flanking Kpn1 and Xho1, respectively: P5′, 5′-ATATGGTGACCAACAGCCAGGTGGAGTCGGG-3′ and C3′, 5′-ATATCTCGAGGCCGGCGGCCGCCTCCTCCT-3′.
cDNA for the N + P and P + C domain was preceded by cDNA encoding calreticulin signal sequence and COOH-terminal KDEL ER retrieval signal.

MX was purchased from Shanghai Macklin Biochemical Co., Ltd. (China). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-Pyrrolidinopyridine (4-ppy), PEG-NH₂ (2000), dithiodibutyric acid, N-hydroxysuccinimide (NHS), folic acid, DSPE-PEG-NH₂ (2000), trichloroacetic acid, DL-Dithiothreitol (DTT) and 4-Dimethylaminobenzaldehyde were purchased from Shanghai Aladdin Reagent Co., Ltd. (China). 1-MT-Boc was purchased from SINO High Goal Chemical Technology Co., Ltd. Dialysis bag was purchased from Yobios Science And Technology Co., Ltd. (China).
Roswell Park Memorial Institute-1640 (RPMI-1640), fetal bovine serum (FBS), trypsin solution, penicillin–streptomycin solution and phosphate buffered saline (PBS, pH 7.4) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). 4% paraformaldehyde solution, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and HMGB1 ELISA Kit were obtained from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Anti-HMGB1 Rabbit mAb and Anti-Calreticulin Rabbit pAb were obtained from ABclonal Technology Co., Ltd. (Wuhan, China). TritonX-100 and Enhanced ATP Assay Kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Recombinant mouse interferon gamma (IFN-γ) was bought from BioLegend, Inc. Kyn and Trp were obtained from Sigma Co., Ltd. BALB/c nude mice (male, 5–6 weeks) were purchased from the Beijing Huafukang Biotechnology Co., Ltd. All animal experiments were approved by the Ethical Committee on Animal Experimentation of Yantai University (Yantai, China) (Figure 1).