PC1 assembles into biomolecular condensates. (A) Western blot showing levels of secreted Col1A1 (medium) from 24-h-old media and intracellular expression levels of Col1A1, calnexin, and ⍺-SMA (lysate) in LX-2 cells grown with or without 10 ng/ml TGF-β for 48 h. (B) Expression levels of ⍺-SMA normalized to calnexin levels in cells grown in the absence or presence of TGF-β from three independent experiments (***P = 0.0048). (C) Levels of secreted Col1A1 normalized to intracellular calnexin levels from the 24-h-old culture medium from cells grown in the absence or presence of TGF-β from three independent experiments (**P = 0.0003). (D) Dot blot probed with anti-Col1A1 antibody showing levels of secreted Col1A1 from 4-h-old medium of cells grown with or without TGF-β. (E) Levels of secreted Col1A1 in 4-h-old culture medium of cells grown in the absence or presence of TGF-β from five independent experiments (****P < 0.0001). (F) Fluorescence images of LX-2 cells, fixed but not permeabilized and stained with anti-Col1A1 showing increased extracellular fibril formation due to increased secretion of Col1A1 upon TGF-β treatment (scale bar: 20 µm). (G) Fluorescence image of LX-2 cells grown with TGF-β (left), fixed and permeabilized, and stained with anti-Col1A1 showing the intracellular distribution of endogenous Col1A1 in distinct condensate-like structures (white arrowheads), and zoomed inset (right) highlighting their discrete spherical nature. Extracellular Col1A1 fibrils are also evident in the image since they are resistant to the permeabilization process (scale bars: 10 µm; inset: 5 µm). An expanded version of this image is presented in Fig. S1 D. (H) Fluorescence image of fixed and permeabilized LX-2 cells stably expressing Col1A1-mNG under a doxycycline-inducible promoter grown in the absence of TGF-β, 24 h after the addition of 1 µg/ml of doxycycline. White arrowheads show cells with distinct Col1A1 condensates (scale bar: 10 µm). (I) Percentage of LX-2 cells with Col1A1 condensates when grown without TGF-β, with TGF-β for 48 h or with doxycycline for 24 h but without TGF-β. A total of 229, 292, and 256 cells for the three conditions, respectively, from three independent experiments were analyzed (*P = 0.0124, ****P < 0.0001). (J) Histogram depicting the number of Col1A1 condensates per cell. 30 cells from three independent experiments were analyzed with condensate numbers ranging from 53 to 613 condensates per cell. (K) Histogram depicting condensate areas. A total of 1,934 condensates from 19 cells from three independent experiments were analyzed with a mode in the 0.50–0.55 µm² range and a mean condensate area of 0.628 µm². (L) Time-lapse images from Video 1 of LX-2 cells stably expressing Col1A1-mNG. The arrowhead indicates a fusion event between two condensates (scale bar: 2 µm). (M) Time-lapse images from Video 1 of LX-2 cells stably expressing Col1A1-mNG. The arrowhead indicates a fission event (scale bar: 2 µm). (N) Time-lapse images from Video 2 depicting FRAP of a Col1A1-mNG–expressing LX-2 cell. The arrowhead indicates condensate that was bleached and its subsequent recovery (scale bar: 2 µm). (O) Fluorescence recovery profile from two independent FRAP experiments. The data represent the mean and SD from the profiles. Source data are available for this figure: SourceData F1.