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. Author manuscript; available in PMC: 2022 Aug 9.
Published in final edited form as: Nat Cell Biol. 2020 Jan 6;22(1):74–86. doi: 10.1038/s41556-019-0441-z

Figure 3. Circadian clock-regulated PC-I trafficking and collagen fibre assembly in culture.

Figure 3

(A) Time series immunofluorescence study using anti-collagen-I and anti-PDI-specific antibodies. Areas boxed in white are magnified in the lower panels. Representative images from each time point are shown, from three independent experiments. (B) Time-series co-localisation of collagen-I and PDI (black) and collagen fibres per cell (red), from (A) were quantified using multiple regions to assess co-localisation from 4 independent regions. Bars show mean ± SD. (C) Heat map from LC-tandem mass spectrometry analyses of protein expression showing CTSK and collagen α1(I) and collagen α2(I) oscillation over 48 hours (n = 4 biological replicates). (D) Increased hydroxyproline content of Achilles tendons dissected from mice and incubated with 1 μM odanacatib (OD) for 3 days, compared to control (n=6; ***p=0.0004, two-tailed unpaired t-test, mean and standard deviation are indicated). (E) Col1a1 mRNA expression in mouse tail tendon fibroblast incubated with OD (n=3 tendon samples, p=0.0055, two-tailed unpaired t-test). Bars show mean ± SEM. (F) Representative western blot analyses (n=2 biologically independent experiments) of CRISPR/Cas9 Sec61a2-knock out and Sec61a1/Sec61a2-double knock out MEFs (Sec61a2 KO, Sec61a1 + Sec61a2 KO). Levels of vinculin protein served as a loading control. (G) Immunofluorescence analysis of synchronised MEFs show that depletion of Sec61a1 and Sec61a2 prevent collagen-I fibre assembly in the extracellular matrix (n=6 independent tests each with similar results). (H) Representative western blot analysis of TANGO1 protein in MEFs treated with siRNA targeting Mia3. Levels of vinculin protein served as a loading control. (I) Immunofluorescence analysis of control and siMia3 cells shows collagen-I retention in the ER (PDI-staining) in cells with Mia3 knock-down (Mia3 KD). Staining of control cells show collagen-I fibres in the extracellular matrix (n=4 independent tests each with similar results). Bars 10 μm. See also Source Data for Figure 3 and Supplementary Information 2 for unprocessed western blots.