Figure 8

ERp44 KD induces intracellular accumulation of ProCol1A1, as well as defective extracellular collagen deposition in human fibroblasts (BJ cells)

BJ cells were subjected to silencing of ERp44, or treated with control duplexes (si44 and siC, respectively). About 72 h after silencing, protein extracts were collected.

(A) Representative immunoblot images of Col1A1 and ERp44 proteins with the corresponding Ponceau-stained membrane; samples were separated on a 4%–12% pre-cast SDS-PAGE gel under reducing conditions in MOPS (3-(N-morpholino)propanesulfonic acid) buffer. Membrane were probed with anti-Col1A1 antibody (Ab138492). Note the absence of intracellular mature Col1A1 signal in the protein extracts of BJ cells. Optical density quantification of total Col1A1 protein shows a 5-fold increment in si44 BJ cells with respect to controls (siC) (n = 10, Student’s t test, ∗∗p < 0.01).

(B) Protein extracts of ERp44 silenced (si44) and control (siC) BJ cells were separated on a 4%–12% precast SDS-PAGE gel under non-reducing conditions in MOPS buffer. Membranes were acquired at two different laser intensity (low and high exposition). Under high exposition, si44 sample (mean of ERp44 downregulation), reveals the accumulation of collagen high molecular weight (HMW) complexes, as well as collagen covalent assembly intermediates.

(C) Immunofluorescence images of siC (top) or si44 (bottom) BJ cells. Left: cells were fixed in methanol and then stained, without additional permeabilization, with anti-Col1A1 antibody (Ab138492, green) and DAPI (nuclei in blue); images of Col1A1 fibrils from the same samples are shown at higher magnification in middle column (black and white images). Cells in the right were fixed with PFA, permeabilized and then stained with antibodies against ProCol1A1 (yellow) and DAPI (nuclei, in blue). Intracellular collagen accumulation and thinner extracellular collagen fibers are evident in ERp44 KD (si44) cells. Scale bars, 40 and 20 μm.

(D) Transmission electron microscopy images of siC (left) or si44 (right). BJ extracellular collagen fibers: long and straight collagen fibers are deposited extracellularly in siC cells in contrast to thinner and disorganized collagen fibers in ERp44 KD cells (si44). Scale bar 500 nm.