primary human foreskin fibroblasts  (ATCC)

Journal: bioRxiv

Article Title: Fibrillar adhesions are the primary integrin complexes shaped by matrix topography

doi: 10.1101/2025.11.26.690075

Figure Lengend Snippet: (A) Image of HUVEC transfected with GFP–tensin3 and plated on human plasma-fibronectin coated electrospun nanofibers used for evaluation of fibrillar adhesion dynamics on flat versus nanofiber regions by FRAP. Insets show adhesions formed in flat (orange box) or nanofiber (blue box) regions at the state of pre-bleaching (−2s), photobleaching (0), and recovery (45s). Scale bar, 10µm. See also Supplemental Movie 2. (B) Graph showing time recovery constants determined by single exponential fit (tau τ, seconds) of GFP-tensin3 fibrillar adhesions on flat versus nanofiber regions, with median values indicated on the graph ( n =18, N =3, box-and-whiskers plot (min, median, max) with all data points shown, P -values calculated using Wilcoxon signed-rank test) (C) Graph showing mobile fractions of GFP-tensin3 fibrillar adhesions on flat versus nanofiber regions determined by single exponential fit, with median values indicated on the graph ( n =18, N =3, box-and-whiskers plot (min, median, max) with all data points shown, P -values calculated using Wilcoxon signed-rank test) (D) HUVEC plated on plasma fibronectin coated electrospun nanofibers under control conditions or under treatment with 30µM ROCK inhibitor Y-27632 and stained (from left to right) for α5-integrin, F-actin and myosin-IIA heavy chain. Corresponding DIC images showing nanofibers are in the right column. Scale bar, 10µm. (E) Representative images of distribution of tensin3 (green) and cellular fibronectin (magenta) together with line scan intensity profiles along electrospun nanofibers in cells plated in the presence of 30 µM Y-27632 (upper panel), and after washing out the drug for 30 minutes (middle panel) or 60 minutes (lower panel). Accumulation of fibronectin after ROCK inhibitor washout correlates with the intensity of tensin3 staining. (F) Human foreskin fibroblasts (HFF) cultured overnight on planar glass in control conditions or with 30µM Y-27632 added before spreading. Left columns show merged images of F-actin (magenta), tensin3 (cyan), and cellular fibronectin (cFN; yellow). Individual grayscale panels for tensin3 and cellular fibronectin are shown in the middle and right columns. Scale bar, 10µm. (G) Images showing tensin3, cellular fibronectin and F-actin in HFFs plated on human plasma fibronectin coated electrospun nanofibers (DIC) under control conditions or in the presence of 30 µM Y-27632. Like HUVEC, fibrillar adhesions in HFF also form along nanofibers under ROCK inhibition, however formation of fibronectin fibrils is reduced. Scale bar, 10µm.

Article Snippet: Primary human Foreskin Fibroblasts (HFF) (ATCC, SCRC-1041) were cultured in DMEM + 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate, kept at an early passage (5-15) and incubated at 5% CO 2 at 37°C.

Techniques: Transfection, Clinical Proteomics, Control, Staining, Cell Culture, Inhibition