Fig. 4: ATF4-dependent Col1a1 expression and multistep regulation of the collagen biosynthesis pathway contribute to fibroblast functionality.
a, Volcano plot from the genome-wide gene expression microarray on LFBs. b, Predicted binding site of ATF4 on intron 5 of Col1a1. c, ATF4 ChIP followed by RT–qPCR at the Col1a1 locus and Eif4ebp1 (positive control) (representative from two biologically independent replicates; n = 3–4 technical replicates). NEG, PCR amplification of a site with no predicted ATF4 binding sites, located at intron 6 of Col1a1 . d, Box and whisker plot of RT–qPCR of Atf4, Psat1, Shmt1 and Shmt2 (left) and Atf4, Aldh18a1 and Pycr1 (right) in LFBs (n = 5–6 biologically independent samples per group). e, Box and whisker plot of the NMR spectrometry analysis of intracellular glycine and proline levels (μM per cell) in LFBWT/WT and LFBΔ/Δ cells (n = 4 biologically independent samples per group). f, LC–ESI-MS/MS analysis to measure the metabolic flux from serine to glycine and glutamine to proline in LFBWT/WT and LFBΔ/Δ cells (n = 3 biologically independent samples per group). Values represent the mean ± s.e.m. The letters indicate a significant change from the LFBWT/WT at each isotopologue: aP < 0.01, bP < 0.001. g, Proteins were detected by immunoblotting in untreated LFBs. β-actin was used as a loading control. h, Representative images of collagen deposition from LFBWT/WT and LFBΔ/Δ using second harmonic generation (SHG) microscopy. Magnification, ×10. Scale bar, 100 μm. i, Box and whisker plot of the fluorescent signal from h. Each dot represents quantitative value from a ×10 field. j, Re-expression of a mouse ATF4 homologue from an adenoviral vector (AdmATF4) in LFBΔ/Δ cells restores collagen I levels. Proteins were detected by immunoblotting. β-actin was used as a loading control. k, LFBWT/WT were treated with TGF-β1 for 6 h and proteins were detected by immunoblotting. β-actin was used as a loading control. siNT, small interfering non-targeting RNA. Numbers below blots represent relative band intensities, normalized to T-eIF2a and β-actin. Unpaired two-sample t-test in all box and whisker plots.
