Figure 7

ERp44 KD induces intracellular procollagen 1A1 (ProCol1A1) and mature collagen 1A1 (Col1A1) accumulation in SAOS-2 cells

SAOS-2 cells were subjected to silencing of ERp44, or treated with control duplexes (si44 and siC, respectively). After 72 h of silencing, protein extracts were collected.

(A) Representative immunoblot images of ProCol1A1, mature 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 buffer. Membranes were probed with anti-Col1A1 antibody (Ab138492) that recognized both forms. Optical density quantification of ProCol1A1 and Col1A is shown in the graphs. The graph on the left shows the quantification of total intracellular collagen (ProCol1A1+Col1A1); the graph on the right shows the percentage of intracellular ProCol1A1 or mature Col1A1 expression on total intracellular Col1A1 signal. The downregulation of ERp44 (si44) induces a 3-fold increment of intracellular total collagen with respect to control (siC), as well as the increase of the percentage of intracellular procollagen species (n = 13, n = 7, respectively). The graphs show the results of the single biological replicates and the averages ± SD Student’s t test (∗∗∗p < 0.001, ∗p < 0.05).

(B) Immunofluorescence images of methanol fixed control (siC) or ERp44 silenced (si44) SAOS-2 cells stained with anti-Col1A1 antibody (top). Under ERp44, downregulation cells show thinner and shorter extracellular fibers deposition (see the boxed area and the higher magnification, middle column). At the bottom, SAOS-2 immunofluorescence images of control (siC) or silenced (si44) cells fixed with PFA, permeabilized and then stained with anti-ProCol1A1 antibody. ProCol1A1 accumulates in the enlarged perinuclear structures (see the boxed area and at higher magnification) in si44 condition. Scale bars, 20 μm.