Mechanobiology June 26th - June 2nd 2016

Mechanobiology: June 26th  - June 2nd 2016

Supplementary Material

JCS055079 Supplementary Material

Files in this Data Supplement:

  • Supplemental Figure S1 -

    Fig. S1. Characterization of NIH 3T3 and MCF-10A cells in collagen-I microgels. (A) NIH 3T3 and MCF-10A cells stained for F-actin (green) and DNA (red). Bars, 20 µm. (B) Proliferation as quantified by BrdU incorporation to measure S-phase entry of G0-synchronized MCF-10A cells following serum stimulation. Means ± s.e.m. are from at least three independent experiments. *, p<0.05, unpaired Student�s t-test relative to 2D.

  • Supplemental Figure S2 -

    Fig. S2. Transport of soluble factors through macrogels is diffusive, and severely limited in macrogels as compared to microgels. (A) Fluorescence intensity versus time 100 µm from the bottom of a 950 µm thick collagen-I gel as measured via optical sectioning (details available on request). At time 0, Alexa-Fluor-488-conjugated bovine serum albumin (BSA) was added to the top of the gel. Open boxes represent mean of three independent experiments, and error bars represent s.e.m. Solid red line represents simulated one-dimensional diffusion, with a fitted diffusion coefficient of 2.721×10-7 cm2/sec (details available on request). This diffusion coefficient was used for simulations in Fig. 1D in the main text. (B) Average fluorescence intensity profiles as measured by Hoechst 33342 nuclear incorporation over time in MDCK cells overlaid with different volumes of collagen-I gel. Representative images illustrate that dye rapidly accumulates within 3 minutes in cells in microgels (a), but is not visible in thicker gels (b). After longer times (20-25 minutes), dye begins to accumulate in 1200 µm gels (c-d), but not in 2800 µm gels (e). Means ± s.e.m. are from at least three independent experiments. Scale bars, 10 µm. A computational model of Hoechst diffusion and accumulation in cell nuclei predicted the experimental measurements under all gel thicknesses (broken lines, details available on request).

  • Supplemental Figure S3 -

    Fig. S3. Effects of diffusion barriers on cellular responses in 3D collagen-I gels. NIH 3T3 cells in 3D macrogels and microgels were stimulated with TNFα and monitored for NF-κB signaling over time. Means ± s.e.m. are from three independent experiments.

  • Supplemental Figure S4 -

    Fig. S4. HGF promotes scattering on 2D surfaces. MDCK cells were cultured for 24 h on collagen-I-coated surfaces (top) or on top of collagen-I gels for 24 h, stimulated with 25 ng/ml HGF, and scattering was assessed via representative phase-contrast images 16 h following HGF stimulation. Controls were not stimulated with HGF. Bars, 100 µm.

  • Supplemental Figure S5 -

    Fig. S5. Quantification of sprouting in collagen-I gels. Cysts composed of MDCK cells were fixed 24 hours after exposure to HGF (or no HGF as a control), stained with f-actin and Hoechst, and scored for sprouting (details available on request). Means ± s.d. are from two independent experiments.

  • Supplemental Figure S6 -

    Fig. S6. HGF induces ERK phosphorylation similarly on 2D surfaces. MDCK cells were cultured for 24 h in starvation medium, then stimulated with 25 ng/ml HGF. Cells were lysed and probed for ERK phosphorylation at the indicated times. t=0 corresponds to no HGF stimulation. Means ± s.d. are from two independent experiments.

  • Supplemental Figure S7 -

    Fig. S7. ERK phosphorylation can be partially rescued by alleviating transport limitations. MDCK cells were stimulated with HGF and assayed for ERK phosphorylation 30 minutes following stimulation. t=0 corresponds to no HGF stimulation. Means ± s.e.m. are from three independent experiments.

  • Supplemental Figure S8 -

    Fig. S8. Gene expression changes after serum stimulation of MDCK cells. Cells co-transfected with a serum response element reporter plasmid (pSRE-Luc) and a transfection control plasmid (phRG-TK) were starved overnight, then assayed for luciferase reporter activity following 2 hour serum stimulation. Means ± s.e.m. are from at least four independent experiments. *, p<0.01, paired Student�s t-test relative to 2D and microgel.

  • Supplemental Figure S9 -

    Fig. S9. MEK inhibitor U0126 blunts HGF-stimulated ERK phosphorylation. (A) Representative immunoblot from lysates of MDCK cells on 2D collagen-coated coverslips or in 3D macrogels, stimulated with HGF for 30 minutes in the presence or absence of MEK inhibitor U0126 (�U0�; 10 µM). (B) Quantification of three independent immunoblots, means ± s.e.m.

  • Supplemental Figure S10 -

    Fig. S10. U0126 inhibits sprout formation in collagen-I gels. Cysts composed of MDCK cells were fixed 24 hours after exposure to HGF, stained with f-actin and Hoechst, and scored for sprouting (details available on request). Means ± s.d. are from two independent experiments.

  • Supplemental Figure S11 -

    Fig. S11. Stimulation rate does not significantly affect MLC phosphorylation in response to HGF. Means ± s.e.m. are from at least five independent experiments. *, experimental error rate p<0.05, paired Student�s t-test with Bonferroni correction for multiple comparisons.

  • Supplemental Figure S12 -

    Fig. S12. MDCKs harboring siRNA against MYPT1 scatter in response to HGF on collagen-I 2D surfaces. Control or MYPT1 knock-down MDCK cells were cultured on collagen-I coated surfaces or collagen-I gels for 24 h, then stimulated with 25 ng/ml HGF, and scattering was assessed via representative phase-contrast images 22 h following HGF stimulation. Bar, 100 µm.

  • Supplemental Figure S13 -

    Fig. S13. MDCK cells with knocked down MYPT1 fail to sprout in collagen-I gels. Cysts composed of MDCK cells transfected with siRNA against MYPT1 to activate myosin light chain (or no transfection as a control) were fixed 24 hours after exposure to HGF, stained with F-actin and Hoechst, and scored for sprouting (details available on request). Means ± s.d. are from two independent experiments.

  • Supplemental Figure S14 -

    Fig. S14. MDCK cells fail to sprout in the presence of an MLC phosphatase inhibitor, calyculin A. Fluorescent images of MDCK cells stimulated with HGF and treated with 10 µM ML-7 (MLCK inhibitor) or 2.5 nM Calyculin A (Cal. A, myosin phosphatase inhibitor). All scale bars indicate 20 µm.