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. 2018 Jan 15;8:773. doi: 10.1038/s41598-017-19052-9

Figure 4.

Figure 4

PERK inhibition led to accumulation of the high-molecular-weight type 2 A collagen (COL2A1), resulting in blockade of endoplasmic reticulum (ER)-to-Golgi transport. (A) Protocol for the procollagen transport assay. Mouse primary chondrocytes were treated with 2 μM PERK inhibitor for 12 h and exposed to a temperature of 40 °C for 3 h (accumulation in ER), followed by a temperature shift to 32 °C combined with exposure to 100 μg/ml cycloheximide and 50 μg/ml ascorbic acid for the indicated times (exit from ER). Procollagen transport was visualised by immunostaining. (B) Representative micrographs of immunostaining for Hoechest 33258, GM130 and COL2A1 in mouse primary chondrocytes after ER block release in the procollagen transport assay. Scale bar, 50 μm. (C) Representative micrographs of immunostaining for Hoechest 33258, GM130 and COL2A1 in mouse primary chondrocytes before and after ER block release in the procollagen transport assay. Mouse primary chondrocytes were pretreated with mock inhibitor or 2 μM PERK inhibitor for 12 h. Scale bar, 50 μm. (D) Quantification of transported COL2A1 in mock inhibitor- and PERK inhibitor-treated mouse primary chondrocytes. A scatter plot comparing mock inhibitor- and PERK inhibitor-treated mouse primary chondrocytes regarding the ratio of Golgi-localised COL2A1 to total COL2A1 is shown and each group data consisted of images of 10 cells (n = 5 technical replicates, **P < 0.01). (E) Quantification of transported COL2A1 in primary chondrocytes derived from wild-type or Perk−/− mice at 18.5 dpc and each group data consisted of images of 10 cells (n = 5 technical replicates, **P < 0.01). (F) Protocol for high-molecular-weight (HMW) detergent-resistant complex separation to measure COL2A1 folding. Mouse primary chondrocytes treated with 2 μM PERK inhibitor for the indicated times were lysed in lysis buffer, adjusted to 0.8% SDS, loaded on separation buffer and then ultracentrifuged at 100,000 × g for 45 min. The HMW detergent-resistant complex containing the collagen-specific chaperon HSP47 represents unfolded or misfolded COL2A1. (G) Representative immunoblots and quantification of HSP47 in SDS-resistant HMW complexes (PELLET) and total cell lysate (INPUT) from mouse primary chondrocytes treated with 2 μM PERK inhibitor for the indicated times. The protein levels of HSP47 in PELLET were normalised to the total HSP47 levels in INPUT and shown as the mean ± SD (n = 5 technical replicates, *P < 0.05).