Fig 2: H3K4me3 and H3K27me3 levels in wild-type cells compared with those in GCD2-homozygous primary corneal fibroblasts. a Heat map of ChIP-chip signal for two wild-type and two GCD2-homozygous primary corneal fibroblasts. b Heat map of the H3K4me3-enriched TGFBIp gene location in wild-type and GCD2 primary corneal fibroblasts. Each line represents the average H3K4me3 levels in each cell type. A continuous X-axis indicates the location from the promoter region to downstream of the TGFBI gene. c Heat map of methylated DNA and CpG island regions of the TGFBIp gene in primary corneal fibroblasts

Fig 3: Validation of the kinetics of classically secreted proteins by Western blotting. A, The kinetic behavior of three representative classically secreted proteins, MMP1, MMP3 and TGFBI/TGFβig-h3, following pharmacological blockade of secretion in CAMs, were validated using a standard immunoblotting approach. When translation was inhibited with cycloheximide, the accumulation in the medium of MMP1 and TGFβig-h3 was already reduced at 1 h. For these two proteins (and MMP2), secretion was clearly inhibited at 4 h. Similarly, upon perturbation of secretion using brefeldin A (BFA), the appearance of MMP1 was suppressed at 1 h and of all three proteins from 2 h. B, The kinetics of secretion of MMP1, MMP3 and TGFβig-h3 determined by SIDLS clearly match those determined by the orthogonal approach of Western blotting. C, Evidence that SIDLS can discern proteins of the regulated secretory pathway. Western blotting confirmation that secretion of secretogranin-2 (SCG2), an established marker of a minor, endocrine-like secretory phenotype known to be present in stromal myofibroblasts (CAMs), was stimulated by a short (30 min) stimulation with ionomycin. This is consistent with calcium-evoked exocytosis and the response was resistant to cycloheximide (left) and BFA (right), supporting the idea that this represents release from intracellular storage vesicles.