Fig 2: ER retention and activation of ER stress/UPR by mutant COMP.a Wild-type, V66E, and R718W mutant COMP were expressed in primary tendon cells or RCS cells. Cell lysates and culture medium were respectively collected. Immunoblotting results indicate poor secretion of V66E mutant COMP by primary tendon cells but not RCS cells. The secretion of R718W-COMP is impaired in both types of cells. RCS rat chondrosarcoma cells, FBN fibronectin. The secretion ratios of different COMP from three experiments are quantified and summarized in the right panel, two-tailed t test, ***p = 0.00095 (V66E) and *p = 0.0161 (R718W) for primary tendon cells, p = 0.107 (V66E) and *p = 0.0108 (R718W) for RCS cells, error bars are ± SEM. b–d Normal (n = 2) and Family 1 patients’ TCL tissues (n = 3) were analyzed. b Immunofluorescent staining indicates strong co-localization of COMP (green) and endoplasmic reticulum (ER, recognized by KDEL antibody, red) in patients’ TCL cells. DAPI stains cell nucleus (blue). c Transmission electron microscopy (TEM) shows distended and fragmented ER in CTS patients’ TCL cells (red arrows). d Immunohistochemical staining shows upregulation of BIP, ATF4, and CHOP in patients’ TCLs, indicating activation of ER stress/unfolded protein response (UPR) in CTS patients. Arrows point to the perinuclear staining of the ER-resident BIP. The boxed areas (black box) in the figures are magnified and shown in the insets (white box). Source data are provided as a Source Data file.

Fig 3: Surface translocation of glucose-regulated protein 78 (GRP78) in beta cells is an early phenomenon, taking place in the early apoptotic cell population.MIN6 cells exposed to cytokines (human interleukin-1ß (50 U/ml), mouse interferon-? (250 U/ml), and mouse tumor necrosis factor-a (1000 U/ml)) for the indicated time points (4, 8, 16, and 24 h) were stained with the late-apoptosis/necrosis marker DRAQ7-AF700, early apoptosis marker Annexin V-APC (see Results for the interpretation of these combined dyes to discriminate between living, early, and late apoptotic cells), and anti-GRP78-FITC, followed by flow cytometry. a, b Percentage of living (a) and early apoptotic (b) cell population in control (C) and cytokine (Cyt) exposed MIN6 cells. c, d Percentage of sGRP78-positive cells in the living (c) and early apoptotic (d) subpopulation. e, f Geometric mean fluorescence intensity (MFI) for sGRP78 in the living (e) and early apoptotic (f) subpopulation. Data are presented as mean ± SEM (n = 4) and statistically analyzed by a two-way analysis of variance followed by Sidak posthoc test for multiple group comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Fig 4: Pancreatic UPR becomes maladaptive in older animals. (A) CHOP expression is increased when aged mice are challenged with sleep deprivation. Young (3 months) and aged (24 months) mice were subjected to 6 h of SD. Western blots of pancreatic tissue indicate that there is a significant increase in BiP in young (*P = 0.001) and not in aged pancreas with SD and significantly more CHOP expressed in the aged mice at baseline that is further increased with SD (*P < 0.01). Gel shows BiP and CHOP protein in young control (YC) and SD (YSD) and in old control (OC) and old SD (OSD) probed on the same gel. Bar graph shows BiP and CHOP expression normalized to β-actin (average ± SEM) in young and aged mice from four gels (n = 8). (B) There is increased ER stress in the pancreas of aged mice. Phosphorylation of PERK (P-PERK), indicative of ER stress, and activation of the unfolded protein response are evident in β-cells. Representative images of P-PERK (green) in B cells (red) immunostained for insulin in young undisturbed (YC), young SD (YSD), old undisturbed (OC), and old sleep-deprived (OSD) mice.

Fig 5: The PERK-p-eIF2α signaling pathway is specifically down-regulated by the treatment of ISRIB in 13del HCs.At p10 stage, the expression of p-eIF2α in ISRIB-treated 13del HC was not changed. Higher magnification of the boxed region was shown to demonstrate the expression of p-eIF2 clearly, and the positive staining cells were indicated by arrows. (Scale Bar = 200 μm) (B) The vehicle-treated WT, vehicle- and ISRIB-treated 13del p10 HZ were equally fractionated into two chondrocyte populations (UHC and LHC), and protein from each population was isolated. The protein expression level of ATF4 and ER stress sensor BiP were examined via western blot. Beta-actin was used as internal control. (C) The WT, vehicle- and ISRIB-treated 13del p10 growth plates were fractionated into different chondrocyte populations (PC, pHC, UHC, MHC and LHC), and total RNA from each population was isolated. The expression levels of Atf4, its downstream targets (Atf3, Ddit3, and Fgf21), ER stress sensor Hspa5, total Xbp1T and its active form Xbp1S were quantified via qRT-PCR, results were presented as fold induction compared with WT proliferating chondrocytes (PC) from five independent animals of each group. Error bars shown as S.D. and significance between vehicle- and ISRIB-treated HCs was determined Two-tailed Mann-Whitney U-test. *: p-value<0.05, **: p-value<0.005.