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    This document provides supplementary information for a research article on how substrate rigidity controls activation and movement (durotaxis) of pancreatic stellate cells. Supplementary Figure 1 shows bright field images of stained pancreatic stellate cells forming clusters and networks when cultured on a soft substrate. Supplementary Figure 2 provides a schematic of the surface preparation method used to attach cells to soft and stiff substrates. The remaining supplementary figures provide additional data supporting the findings of the primary article through immunofluorescent staining and quantification of protein markers. Supplementary Table 1 lists the reagent proportions used to obtain specific substrate rigidities. Supplementary videos 1-3 show durotactic movement, random movement after FAK inhibition, and random movement with blebbistatin treatment.

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    Substrate Rigidity Controls Activation and Durotaxis in Pancreatic Stellate Cells

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    41598_2017_2689_MOESM1_ESM

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    This document provides supplementary information for a research article on how substrate rigidity controls activation and movement (durotaxis) of pancreatic stellate cells. Supplementary Figure 1 shows bright field images of stained pancreatic stellate cells forming clusters and networks when cultured on a soft substrate. Supplementary Figure 2 provides a schematic of the surface preparation method used to attach cells to soft and stiff substrates. The remaining supplementary figures provide additional data supporting the findings of the primary article through immunofluorescent staining and quantification of protein markers. Supplementary Table 1 lists the reagent proportions used to obtain specific substrate rigidities. Supplementary videos 1-3 show durotactic movement, random movement after FAK inhibition, and random movement with blebbistatin treatment.

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    11 views8 pages

    2017 2689 Moesm1 Esm

    Uploaded by

    Stephan Samir
    This document provides supplementary information for a research article on how substrate rigidity controls activation and movement (durotaxis) of pancreatic stellate cells. Supplementary Figure 1 shows bright field images of stained pancreatic stellate cells forming clusters and networks when cultured on a soft substrate. Supplementary Figure 2 provides a schematic of the surface preparation method used to attach cells to soft and stiff substrates. The remaining supplementary figures provide additional data supporting the findings of the primary article through immunofluorescent staining and quantification of protein markers. Supplementary Table 1 lists the reagent proportions used to obtain specific substrate rigidities. Supplementary videos 1-3 show durotactic movement, random movement after FAK inhibition, and random movement with blebbistatin treatment.

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    SUPPLEMENTARY INFORMATION

    Substrate Rigidity Controls Activation and Durotaxis in Pancreatic Stellate Cells

    Dariusz Lachowski1*, Ernesto Cortes1*, Daniel Pink1*, Antonios Chronopoulos1, Saadia A. Karim2,

    Jennifer Morton2, and Armando E. del Río Hernández1§

    1
    Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial

    College London, London SW7 2AZ, United Kingdom


    2
    Pancreatic Cancer Research Team, CRUK Beatson Institute Glasgow G61 1BD, United Kingdom

    *These authors contributed equally

    §Corresponding author:

    Armando E. del Río Hernández, PhD

    Cellular and Molecular Biomechanics Laboratory

    Department of Bioengineering

    Imperial College London

    London, United Kingdom

    email: a.del-rio-hernandez@imperial.ac.uk
    Supplementary Figure S1

    Supplementary Figure 1: Bright field images of Oil Red O stained PSCs cultured on
    Matrigel for 6 days and showing the clusters connected by a filamentous network
    characteristic of PSCs quiescence. Scale bar 50 µm
    Supplementary Figure S2

    Supplementary Figure 2: Schematic representation of surface


    preparation for cell attachment on soft and stiff PAA gels. Crosslinking of
    PAA hydrogels with fibronectin through the use of sulfo-SANPAH. sulfo-
    SANPAH is a heterobifunctional crosslinker containing an amine-reactive
    NHS and a photoactivatable nitrophenyl azide. Upon UV exposure, this
    nitrophenyl azide forms a nitrene group that binds to NH2 groups within
    the hydrogel, leaving a NHS group free at the gel surface to allow
    binding of fibronectin.
    Supplementary Figure S3

    Supplementary Figure 3: Immunofluorescent images of aSMA and vimentin of PSCs


    seeded on matrices represented in Fig. 1a. Scale bar 50 µm. Quantification in Fig 1e,f.
    Supplementary Figure S4

    a b
    Cell proliferation Fibronectin staining
    1.0 1.5

    Staining intensity (a.u.)


    % of Ki67 positive cells

    *** ***
    0.8 *** n.s. ***
    1.0
    0.6

    0.4 n.s.
    0.5
    0.2

    0.0 0.0
    Matrigel 1 kPa 25 kPa Matrigel 1 kPa 25 kPa

    Supplementary Figure 4: Mechanical activation of PSCs by stiff substrates increases


    cell proliferation and fibronectin production. (a, b) Quantification of ki67 positive cells
    and fibronectin immunofluorescent staining as markers of cell proliferation and ECM
    production, respectively. Histogram bars represent mean ± SEM. Representative of 3
    independent experiments. (Anova and Tukey posthoc test) *** p<0.001.
    Supplementary Figure S5

    Supplementary Figure 5: Immunofluorescent images of αSMA and vimentin of PSCs seeded


    on matrices represented in Fig. 2a. Scale bar 50 µm. Quantification in Fig 2 f,g.
    Supplementary Figure S6

    a b
    Cell proliferation Fibronectin staining
    1.5

    Staining intensity (a.u.)


    1.0 n.s. n.s.
    % of Ki67 positive cells

    0.8
    *** *** *** ***
    1.0
    0.6

    0.4
    0.5
    0.2

    0.0 0.0
    Glass 1 kPa 25 kPa Glass 1 kPa 25 kPa

    Supplementary Figure 6: Mechanical deactivation of PSCs by soft substrates decreases cell


    proliferation and fibronectin production. (a, b) Quantification of ki67 positive cells and
    fibronectin immunofluorescent staining as markers of cell proliferation and ECM
    production, respectively. Histogram bars represent mean ± SEM. Representative of 3
    independent experiments. (Anova and Tukey posthoc test) *** p<0.001.
    Supplementary Table S1: Reagent proportions required to obtain specific PAA hydrogel
    rigidities.

    Total
    Stiffness volume PBS APS TEMED acrylamide/bisacrylamide (29:1)
    (kPa) (μl) (μl) (μl) (μl) 40% vol (μl)

    1.3 500 461.6 2.5 1 34.9


    25.5 500 371.2 2.5 1 125.3

    Supplementary Video S1

    This video represents the durotactic movement (towards the stiff region) of one
    pancreatic stellate cell in the boundary region between a soft (right side) to a rigid
    substrate (left). The total video represents 6 h in real time.

    Supplementary Video S2

    This video represents the random undirected movement of one pancreatic stellate cell
    (siRNA FAK) in the boundary region between a soft (right side) to a rigid substrate (left).
    The total video represents 6 h in real time.

    Supplementary Video S3

    This video represents the random undirected movement of one pancreatic stellate cell
    (treated with blebbistatin) in the boundary region between a soft (right side) to a rigid
    substrate (left). The total video represents 6 h in real time.

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