Journal: Cell Reports Medicine

Article Title: Restoring immune homeostasis in atherosclerotic plaques via inorganic violet phosphorus nano-immunotherapy

doi: 10.1016/j.xcrm.2025.102528

Figure Lengend Snippet: Preparation, characterization, ROS scavenging, and biocompatibility of VPNS@P (A) Schematic of exfoliation and PEGylation of violet phosphorus (VP) to prepare VPNS@P from bulk VP. (B) Transmission electron microscopy (TEM) image of VPNS@P. Scale bar, 100 nm. (C) Hydrodynamic size distribution of VPNS@P measured by dynamic light scattering (DLS). (D and E) Atomic force microscopy (AFM) image (D) and thickness profile (E) of VPNS@P. Scale bar, 100 nm. (F) Raman scattering spectra of VPNS@P. (G) Time-course DLS measurements of VPNS@P incubated in PBS or DMEM supplemented with 10% fetal bovine serum (FBS) over 7 days ( n = 3 independent samples). (H–J) Scavenging capability of VPNS@P ( n = 5 independent samples) toward H 2 O 2 (H), ·OH (I), and O 2 ·− (J). (K–M) Biocompatibility of VPNS@P in vitro . Cell viabilities of RAW264.7 (K), mouse aortic vascular smooth muscle cells (MOVASs) (L), and human umbilical vein endothelial cells (HUVECs) (M) were examined with a CCK-8 assay ( n = 3 biologically independent samples). Data were analyzed using one-way ANOVA with a Dunnett’s T3 post hoc test and are shown as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. ns, not significant.

Article Snippet: Human: HUVEC umbilical vein endothelial cells , ATCC , CRL-1730; RRID: CVCL_2959.

Techniques: Transmission Assay, Electron Microscopy, Microscopy, Incubation, In Vitro, CCK-8 Assay

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) Structured illumination microscopy (SIM) image of HUVEC spread overnight on planar glass showing fibronectin (green) and fibrillar adhesions identified by α5-integrin visualized by SNAKA51 antibody recognizing the open, extended conformation (magenta). Insets show single channels of zoomed regions. Scale bar, 10µm. (B) STORM images of fibrillar adhesions (α5-integrin SNAKA51, green) and F-actin (phalloidin, magenta) in HUVEC. Fibrillar adhesions are not associated with thick F-actin cables. Insets highlight fibrillar adhesions alignment with thin F-actin cables and possibly individual filament. Scale bars, 10µm (overview), 1µm (zoom). (C) Control and fibronectin1 depleted (siFN1) HUVEC on planar substrates showing tensin3 (yellow) and external fibronectin fibrils (cyan). Depletion of FN1 results in a loss of fibrillar adhesions and redistribution of tensin3 to focal adhesion sites at stress fiber termini. Corresponding individual channels displayed separately. Scale bar, 10µm. (D) FN1 knockdown cells grown on pre-formed fibrous cell-derived matrix (CDM) stained with an antibody targeting cellular fibronectin, which is specific for cellular and not plasma fibronectin (cFN present in the deposited CDM, magenta). Focal adhesions are visualized with paxillin (FA, green) and fibrillar adhesions with α5-integrin (FB, green). Insets show zoomed regions with yellow lines indicating positions for intensity line scans in E. (E) Line scan plots of fluorescence intensities for paxillin and α5-integrin (green), together with corresponding underlying cellular fibronectin (cFN marking CDM, in magenta) along indicated lines in (D). Focal adhesions avoid fibrous ECM while fibrillar adhesions fully associate with it. (F) Cells plated on planar glass under control conditions or in the presence of 30µM ROCK inhibitor Y-27632 and RNAi depletion of myosin-IIA (siMYH9) or myosin-IIB (siMYH10), showing myosin-IIA heavy chain (magenta) and α5-integrin (green). Insets show the cell images with low magnification. Formation of fibrillar adhesions is suppressed by treatment with Y-27632 or myosin-IIA knockdown. Scale bar, 1µm. (G) Quantification of the ratio of fibrillar adhesion area to total cell area (FB Area/Cell Area) under conditions specified in (F), siControl ( n =54), +Y-27632 before spreading ( n =45), siMHY9 ( n =51), siMHY10 ( n =50), N =3, box-and-whisker plots, whiskers extend from the minimum to maximum values, the box extends from the 25th to the 75th percentile and the line within the box represents the median, P -values calculated using one way ANOVA, with each mean compared to the mean of the control (****, P < 0.0001, ns = not significant). (H) HUVEC under fibronectin knockdown (siFN1) grown on pre-existing CDM with or without treatment with 30µM Y-27632 before cell spreading, showing cFN in the pre-existing CDM and α5-integrin in fibrillar adhesions. Insets are shown with high magnification in the right column display merged regions (cFN – magenta, α5-integrin – green). Note that fibrillar adhesions are assembled along CDM fibers even in a ROCK independent fashion. Scale bar, 10µm. (I) Quantification of ratio of fibrillar adhesion area to total cell area (FB Area/Cell Area) for siFN1 HUVEC without treatment ( n =63) or treated with 30µM Y-27632 before spreading ( n =61), N =3, box-and-whiskers plot (min, median, max) with all data points shown, P -values calculated using two-tailed unpaired t-test (*, P < 0.05).

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Microscopy, Control, Knockdown, Derivative Assay, Staining, Clinical Proteomics, Fluorescence, Whisker Assay, Two Tailed Test

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) Images of cells plated on planar glass stained for α5-integrin in its open conformation (SNAKA51, magenta) together with the bent conformation of β1-integrin (MAB13, green) on the left column. Other columns show co-localization of α5-integrin with other reported fibrillar adhesion components (green: tensin1, tensin3, talin1, KANK2). Greyscale images in middle and bottom rows show individual channel images of boxed regions. Scale bar, 10µm. (B) Images comparing localization of fibrillar adhesion components (tensin3 and α5-integrin/SNAKA51) along with focal adhesion components on planar glass. Columns from left to right show paxillin with tensin3, vinculin with tensin3, zyxin with α5-integrin, and αv-integrin with tensin3. Greyscale images in middle and bottom rows show individual channel images of boxed regions. Scale bar, 10µm. (C) HUVEC transfected with GFP–tensin1 and plated on planar glass coverslips. GFP-tensin1 displays two distinct localizations: elongated central fibrillar adhesions and peripheral focal adhesion plaques. Immunostaining for fibronectin reveals a co-localization with fibrillar adhesions and a lack of co-localization along focal adhesion sites (boxed). Scale bar,10 µm (D) Western blot showing FN1 knockdown efficiency in FN1 RNAi depleted cells compared to RNAi control using an anti-fibronectin antibody and GAPDH as a loading control. Respective molecular weights indicated. (E) Schematic illustration of cell derived matrix (CDM) preparation using HUVEC. Experimental setup adapted from . (F) Quantification of the mean fluorescence intensity (AU) of the underlying cellular fibronectin in the pre-existing CDMs (cFN-CDM) along specific adhesions (Fibrillar adhesions: α5-Integrin n =45, tensin3 n =42; Focal adhesions: paxillin n =44, vinculin n =41). Box-and-whiskers-plot (min, median, max) with all data points shown. (G) HUVEC plated on pre-existing CDM and stained for cellular fibronectin to visualize the CDM (cFN-CDM, magenta), F-actin (phalloidin, grey), and vinculin (green, upper row) or tensin3 (green, bottom row). Scale bar, 10 µm. Images in the right column show higher magnification of the boxed areas in the left column. The line scans along yellow lines are shown in H. (H) Line scan along the yellow lines indicated in G, plots the fluorescence intensities for vinculin and tensin3 (green), together with corresponding underlying cellular fibronectin (cFN marking CDM, in magenta). (I) Cells grown on human plasma fibronectin coated planar glass coverslips, showing F-actin (magenta), α5-integrin (SNAKA51, yellow), and tensin3 (cyan), in siControl or after FN1 knockdown (siFN1). Loss of fibronectin secretion results in re-localization of α5-integrin and tensin3 to focal adhesion sites at the termini of stress fibers. Scale bar, 10 µm. (J) Western blot showing knockdown efficiency of myosin-IIA (siMYH9) and myosin-IIB (siMYH10) in HUVEC, using antibodies against MYH9 and MHY10, with GAPDH as loading control. Respective molecular weights indicated. (K) Images of cellular fibronectin in CDMs (cFN-CDM) secreted after 24 hours in control HUVEC or under treatment with 30µM Y-27632. Images showing failure to assemble fibronectin fibrils under ROCK inhibition. Scale bar, 10 µm.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Staining, Transfection, Immunostaining, Western Blot, Knockdown, Control, Derivative Assay, Fluorescence, Clinical Proteomics, Inhibition

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) Scanning electron micrographs of thermoplastic polyurethane (TPU) nanofibers on top of glass coverslips made by electrospinning. Scale bars indicated on images. (B) HUVEC plated on human plasma fibronectin–coated electrospun nanofibers showing F-actin (magenta), focal adhesions visualized with paxillin (FA, green, seen as white at stress fiber termini) and fibrillar adhesions visualized with α5-integrin (FB, green). DIC channel shows nanofibers spun on planar glass. Insets highlight fibrillar adhesion formation along the nanofibers while focal adhesions form on adjacent planar regions. Scale bar, 10µm. (C) Semiquantitative estimation of mean intensities of adhesion components along electrospun nanofibers: α5-integrin SNAKA51 ( n =30), β1-integrin mAb13 ( n =30), β5-integrin ( n =20), αv-integrin ( n =25), talin1 ( n =21), paxillin ( n =22), vinculin ( n =23), zyxin ( n =19), tensin1 ( n =30), tensin3 ( n =30) and KANK2 ( n =21). Box-and-whiskers plot (min, median, max) with all data points shown. (D) Quantification of fluorescence intensity recovery after photobleaching of GFP-tensin3 positive fibrillar adhesions on flat (planar) regions (orange) or along electrospun nanofiber regions (blue). Each trajectory represents the average normalized intensity change for adhesions in one cell, with mean exponential time constant, τ (s) and mobile fractions shown on graph ( n =18 cells with 2-5 individual adhesions examined for each region, N =3, Wilcoxon signed-rank test, **** P < 0.0001, ns = not significant).

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Clinical Proteomics, Fluorescence

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) SIM images of cells plated on plasma fibronectin–coated electrospun nanofibers (DIC, grey), with F-Actin (magenta) and multiple adhesion components shown in green. Images in the right column show zoomed boxed areas merged with DIC nanofiber images. Scale bar,10 µm. (B) Images of HUVEC transfected with fluorescently tagged BAR-domain proteins: I-BAR (GFP-IRSP53) and F-BAR (mCherry-CIP4, GFP-FCHo1, YFP-FCHo2) plated overnight on plasma fibronectin–coated electrospun nanofibers. No enrichment was observed along nanofibers (seen in DIC, grey). Scale bar, 10µm. (C) Table summarizing adhesion or BAR-domain protein localization along electrospun nanofibers and fibrillar adhesions on planar substrates.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Clinical Proteomics, Transfection

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: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

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

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) HUVEC fixed 30 minutes after plating on either planar glass or electrospun nanofibers, showing F-actin (magenta), tensin3 (cyan), and cellular fibronectin (yellow). Tensin3 rapidly clusters along nanofibers (but not on planar glass) prior to endogenous fibronectin secretion. Greyscale images show the boxed areas with higher magnification. Scale bar, 10µm. (B) Images of control and FN1 knockdown cells plated overnight on electrospun nanofibers, showing for F-actin (magenta), tensin3 (cyan) and cellular fibronectin (yellow). Tensin3 clusters along electrospun nanofibers (but not adjacent planar regions) even in the absence of fibronectin secretion. Greyscale images show the boxed areas with higher magnification. Scale bar, 10µm. (C) Quantification of mean intensity of tensin3 along nanofibers in control ( n =49) or fibronectin knockdown cells (siFN1) ( n =49), N =3, box-and-whiskers (min, median, max) graph with all data points shown, P -values calculated using two-tailed unpaired t-test, ns = not significant). (D) Images of cells plated overnight on electrospun nanofibers under control conditions (upper row), in the presence of 30µM Y-27632 (middle row), and after washing out the inhibitor for 1 hour. Cells were stained for F-actin (magenta), tensin3 (green), and cellular fibronectin (grey). Tensin3 association with nanofibers persists even under treatment with Y-27632 (although not on adjacent planar regions), while fibronectin deposition is largely inhibited. Cellular fibronectin fibrils reappear along pre-existing tensin3 adhesions on nanofibers after washout of Y-27632. DIC shows nanofibers. Scale bar, 10µm. (E and F) Quantification of mean intensities of tensin3 (E) or cellular fibronectin (F) along nanofibers in control, in the presence of 30µM Y-27632, or after washout of Y-27632 for 1 hour ( n =62 for each condition, N =3, box-and-whiskers plot (min, median, max) with all data points shown, one way ANOVA with each mean compared to the mean of the control, * P < 0.05, ** P < 0.01, **** P < 0.0001).

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Control, Knockdown, Two Tailed Test, Staining

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) Images of HUVEC plated overnight on electrospun nanofibers (upper row) and similar cell after extraction with 1,6-Hexanediol (0.05 M, 5 min). α5-Integrin (SNAKA51) and tensin1 visualized, and DIC showing nanofibers. Scale bar, 10 µm. (B) Quantification of mean intensity of α5-integrin and tensin1 in fibrillar adhesions formed along electrospun nanofibers under control conditions (n =69) and after 5 min extraction with 0.05 M 1,6-Hexanediol ( n =73). Amount of tensin1 is significantly reduced while α5-integrin along nanofibers is preserved ( N =3, box-and-whiskers plot (min, median, max) with all data points shown, two-tailed unpaired t-test comparing means of α5-integrin or tensin1 average intensity in control or 1,6-Hexanediol treated cells, ns = not significant, **** P < 0.0001). (C) Western blot showing protein expression of tensin1 (TNS1), tensin3 (TNS3), and α5-integrin (ITGA5) in siControl, siTNS1&3, and siITGA5 cells. Alpha-tubulin serves as a loading control. Respective molecular weights indicated. (D) Control, tensin1 and 3 double knockdown (siTNS1&3), and α5-integrin knockdown (siITGA5) cells plated overnight on electrospun nanofibers. Images show α5-integrin and tensin3, with DIC to visualize nanofibers. Integrin-α5 localization along nanofibers is not perturbed after depletion of tensin1 and 3, but no localization of tensin3 is observed after depletion of α5-integrin. Scale bar, 10 µm. (E) Quantification of mean intensity of α5-integrin and tensin3 on electrospun nanofibers in RNAi control ( n =57), siTNS1&3 (n =56), and siITGA5 ( n =56) HUVEC. Box-and-whiskers plot (min, median, max) with all data points shown, one way ANOVA comparing means of α5-integrin or tensin1 mean intensity in siTNS1&3 or siITGA5 cells with those of control, ns = not significant, **** P < 0.0001. (F) Quantification of mean intensity of tensin3 clustered along electrospun nanofibers in cells fixed 30 minutes after plating. The electrospun nanofibers were coated with either 10 µg/mL human plasma fibronectin ( n =55) or 20 µg/mL vitronectin ( n =51) or 10 µg/mL laminin ( n =46) or 0.1% gelatin ( n =42). Only human plasma ficu1ebronectin coating promotes localization of tensin3 along nanofibers. N =3, box-and-whiskers plot (min, median, max) with all data points shown, one way ANOVA, **** P < 0.0001.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Extraction, Control, Two Tailed Test, Western Blot, Expressing, Knockdown, Clinical Proteomics

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) Images of HUVEC plated on human plasma fibronectin-coated planar glass coverslips, stained for F-actin (magenta), α5-integrin (yellow), and tensin3 (cyan). Left column shows merged images representing cells under control conditions (siControl), upon double knockdown of tensin1 and tensin3 (siTNS1&3), and knockdown of α5-integrin (siITGA5). Single channel greyscale images of the same cells are shown in in columns in the middle and right. Both knockdowns result in disappearance of fibrillar adhesions and re-localization of remaining adhesion components to focal adhesion sites at the termini of F-Actin stress fibers. (The adhesion of these cells to nanofibers is shown in ). Scale bar, 10 µm. (B) Table listing the integrin receptors associated with different ECM ligands: fibronectin, vitronectin, laminin and collagen (adapted from ). (C) Images of cells fixed 30 minutes after plating on electrospun nanofibers coated with either: 10 µg/mL human plasma fibronectin, 20 µg/mL vitronectin, 10 µg/mL laminin or 0.1% gelatin. Immunofluorescent staining for tensin3, cellular fibronectin and phalloidin staining of F-actin along with DIC images of electrospun nanofibers are shown. Only coating of the substrate with human plasma fibronectin enables the formation of tensin3 positive fibrillar adhesions along electrospun nanofibers. Scale bar, 10 µm.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Clinical Proteomics, Staining, Control, Knockdown

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) HUVECs grown on plasma fibronectin-coated flexible PDMS in a stretching device, subjected to 0% stretch or a rapid 16% stretch and further incubation for 10 min. Cells visualized for F-actin (magenta) and α5-integrin (fibrillar adhesions, green, left) or vinculin (focal adhesions, green, right). Boxed areas shown with high magnification in a greyscale. Fibrillar adhesions decrease after substrate stretching while focal adhesions remain intact. Scale bar, 10µm. (B) Quantification of ratio of fibrillar adhesion area to total cell area (FB Area/Cell Area) for HUVEC under 0% stretch ( n =46) or 16% stretch ( n =47), N =3, box-and-whiskers plot (min, median, max) with all data points shown, P -values calculated using unpaired t-test, **** P < 0.0001. (C) Scatterplot showing pixel intensity correlations between myosin-IIA heavy chain (red) and α5-integrin (green), showing a strong relative mutual exclusivity ( n =10 regions of interest from 10 cells). Representative images of myosin-IIA (red) and α5-integrin (green) in a region of interest of a HUVEC cell are shown. (D) HUVECs plated overnight on planar glass coverslips and stained for myosin-IIA heavy chain (magenta) and α5-integrin (green) under control conditions or after treatment with 5µg/ml Rho activator CNO3 for 3 hours, 1nM of MLCP inhibitor calyculin A (15 min) or 1 µM nocodazole (which increases Rho activity due to release of GEF-H1 upon microtubule disruption) for 1 hour. Note that activation of myosin filament formation results in disruption of fibrillar adhesions. Pre-treatment of cells with 30 µM Y-27632 for 15 min before co-addition of 1 µM nocodazole mostly preserves fibrillar adhesion formation. Scale bar, 10 µm. (E) Quantification of ratio of fibrillar adhesion area to total cell area (FB Area/Cell Area) for control HUVEC ( n =58), cells treated with 5µg/ml CNO3 for 3 hours ( n =51), 1nM calyculin A for 15 min ( n =42), 10 µM nocodazole for 1 hour ( n =59), or 30 µM Y-27632 added for 15 min before co-addition of 1 µM nocodazole for an additional hour (nocodazole and Y-37632) ( n =54), N =3, box-and-whiskers plot (min, median, max) with all data points shown, one way ANOVA comparing means of + CNO3, + Calyculin A and + Nocodazole to control, as well as + Y-27 then Nocodazole to Nocodazole, **** P < 0.0001). (F) Mean intensity of α5-integrin clustering along electrospun nanofibers under control conditions ( n =57) or after incubation with 5µg/ml CNO3 for 3 hours ( n =56) or 1nM calyculin A for 15 min ( n =60), or 10 µM nocodazole for 1 hour ( n =57). ( N =3, Box and whiskers (min, median, max) with all data points shown, P -values calculated using one way ANOVA, **** P < 0.0001. See for representative images. (G) Graph showing measurements of cortical stiffness (Pa) in HUVEC by atomic force microscopy (AFM) in control ( n =33 cells) and cells pre-treated with 30µM Y-27632 for 1 hour ( n =34 cells), N =3 independent experiments, box-and-whiskers plot (min, median, max) with all data points shown, two-tailed unpaired t-test, **** P < 0.0001. (H) Graph showing measurements of cortical stiffness (Pa) of HUVEC by AFM in control ( n =31 cells) and cells pre-treated with 1µM nocodazole for 1 hour ( n =25 cells) N =3 independent experiments, box-and-whiskers plot (min, median, max) with all data points shown, two-tailed unpaired t-test, **** P < 0.0001. (I) Matched cortical stiffness (Pa) AFM measurements in HUVEC before and after treatment with 1 nM calyculin A for 10–20 min (pooled data on n =20 cells, in N =2 independent experiments, paired t-test, ** P < 0.01).

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Clinical Proteomics, Incubation, Staining, Control, Activity Assay, Disruption, Activation Assay, Microscopy, Two Tailed Test

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) HUVECs grown on planar glass coverslips. Left column shows cells stained for myosin-IIA heavy chain (magenta) and fibronectin (green) under control conditions and after treatment with 5µg/ml Rho activator CNO3 for 3 hours, 1nM of MLCP inhibitor calyculin A for 15 min and 1µM nocodazole (activating RhoA via GEF-H1 release following microtubule depolymerization) for 1 hour. The right column shows greyscale images of fibronectin fibers. Increase in myosin-IIA filaments results in disappearance of fibronectin fibrils. Scale bar, 10 µm. (B) Cells grown on electrospun nanofibers under control conditions and after treatment with 5µg/ml CNO3 for 3 hours, 1nM calyculin A for 15 min or 1µM nocodazole for 1 hour which all promote myosin filament formation. Top rows show merged images of myosin-IIA (magenta) and α5-integrin (green). Greyscale images show α5-integrin in corresponding cells (middle row) and nanofibers visualized by DIC (bottom row). Increase in myosin-IIA filaments disrupts clustering of α5-integrin along nanofibers. Scale bar, 10µm. (C) Cells plated overnight on planar glass, under control conditions (left column), treatment with 10 mM methyl-β-cyclodextrin (MβCD) for 90 mins (central column), or pre-treatment with 30 µM Y-27632 for 15 min and co-addition of 10 mM MβCD for 90 mins. Top row shows merged images of myosin-IIA (magenta) and α5-integrin (green), bottom row shows greyscale images of α5-integrin in corresponding cells. Pre-treatment of cells with ROCK inhibitor before co-addition of MβCD does not preserve fibrillar adhesions. Scale bar, 10 µm. (D) Quantification of ratio of fibrillar adhesion area to total cell area (FB Area/Cell Area) for cells under control conditions ( n =37), treated with 10 mM MβCD ( n =34), or pre-treated with 30 µM Y-27632 followed by co-addition of 10 mM MβCD ( n =25). P -values calculated using one way ANOVA, ns, not significant, **** P < 0.0001.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Staining, Control

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) Cells spread on planar glass substrates under control conditions or in the presence 10 mM methyl-β-cyclodextrin (MβCD) for 90 minutes or hypo-osmotic medium (HypoShock – 0.25X hypotonic) for 20 minutes to increase membrane tension. Images show F-actin (magenta), fibrillar adhesions visualized by tensin3 (cyan) which disassemble, and focal adhesions visualized by paxillin (yellow) which are not affected. Scale bar, 10 µm. (B) Cells plated overnight on plasma fibronectin coated electrospun nanofibers under control conditions or treated with 10 mM MβCD for 90 min as well as hypo-osmotic medium (0.25X hypotonic) for 20 min, showing tensin3 (green) containing fibrillar adhesions assembled along nanofibers and paxillin (magenta) containing focal adhesions. Electrospun nanofibers shown in DIC (greyscale) in composite. Fibrillar adhesions are disassembled but focal adhesions are not affected upon MβCD and hypotonic medium treatment. Scale bar, 10 µm. (C) Quantification of ratio of fibrillar adhesion area to total cell area (FB Area/Cell Area) for control HUVEC ( n =55), cells treated with 10 mM MβCD ( n =51), and hypo-osmotic shock (0.25X hypotonic) ( n =50), N =3, box-and-whiskers plot (min, median, max) with all data points shown, P -values calculated using one way ANOVA, **** P < 0.0001. (D) Mean intensity of fibrillar adhesions along electrospun nanofibers under control conditions ( n =70), with the addition of 10 mM MβCD ( n =71), or after treatment 0.25X hypotonic medium ( n =72) ( N =3, box-and-whiskers plot (min, median, max) with all data points shown, P -values calculated using one way ANOVA, **** P < 0.0001). (E) Still images from time-lapse imaging of HUVEC on planar glass transfected with GFP-tensin3 (green) under isotonic conditions and indicatable for 13:55 mins in 0.25x hypotonic medium. Boxed areas with peripheral focal adhesions (FAs, blue box) and central fibrillar adhesions (FB, yellow box) are shown at higher magnification in figures below. See Supplemental Movie 4. Fibrillar adhesions are disappearing while focal adhesions are not affected by hypo-osmotic shock. Scale bar, 10 µm.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Control, Membrane, Clinical Proteomics, Imaging, Transfection

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) The adhesion receptor is depicted as two springs connected at an angle to each other. The main assumption of the theory is that free energy approaching minimum when the plane of the cell membrane from which the receptor is protruding (grey) and plane of the substrate covered with the receptor’s ligand (orange) are tilted relatively to each other. (B, C, D) Situations when the tilted configuration can be approached. Upper row: entire cell (blue) interacting with the substrate (orange); the areas where adhesions are preferentially formed are boxed (red). Lower row: boxed areas at high magnification, showing the positions of adhesion receptors (not in scale) and the angles between the local tangents to cell membrane and the substrate (red dotted lines). (B) Cell on the flat substrate forming a small protrusion/fold (this situation was analyzed in detail in ). (C) Cell spreading over the semi-cylindrical pattern (cross-section). The adhesion receptors are concentrated along the concave edges of the semi-cylinders forming grooves with high negative curvature. (D) Cell engulfing a cylindrical nanofiber with a small diameter and high positive curvature. The adhesion receptors are concentrated along the nanofiber. (E, F) Numerical calculation of the clustering pattern of adhesion receptors along the nanofiber geometry calculated based on the assumption of (E) an isotropic receptor tilt and (F) an anisotropic receptor tilt. Fraction of adherent receptors Φ( x ) represented by spectrum scale on the right. (G) (left) SIM merged image of α5-integrin (green) clustering along electrospun nanofibers (DIC, grey) in HUVEC. The boxed area is shown in individual channels in images on the right. (H-K) Numerical calculation of clustering patterns of adhesion receptors (based on the assumption of isotropic receptor tilt) on various curved geometries. (H) a semi-cylinder geometry with concave edges of high negative curvature (I) a triangle geometry with concave and convex edges, (J) a rectangle geometry with concave edges, and (K) a geometry with smooth concave edges. Calculated fraction of adherent receptors Φ( x ) is represented by spectrum scale on the right. Note that linear receptor clustering occurs preferentially along concave edges with high negative curvature. (L-O) Experimental validation of numerical calculations in (H-K). Top panels show SEM images of PDMS microfabricated substrates of (L) semi-cylindrical substrates with concave edges, (M) triangular substrates with concave and convex edges, (N) rectangular substrates with concave edges, and (O) cylindrical segments with smooth concave edges. Middle panels are 2D confocal images of HUVEC plated on corresponding plasma fibronectin-coated PDMS patterns showing α5-integrin (green) localization along the micro-ridge edges (DIC, grey). The bottom panels show confocal 3D reconstructions of α5-integrin (green) clustering along the respective micro-ridge geometries with plasma fibronectin coating labelled using fluorescent fibrinogen (magenta) to visualize the ridge surface.

Article Snippet: Human umbilical vein endothelial cells (HUVEC) (ATCC, CRL-1730) were cultured in endothelial growth medium EGM-2 Bulletkit (Lonza) including all provided supplements.

Techniques: Membrane, Biomarker Discovery, Clinical Proteomics