Preparation of of MNPs@SiO2(RITC)
MNPs@SiO2(RITC) were prepared with a ~ 9 nm cobalt ferrite core (CoFe2O3) chemically bonded to rhodamine isothiocyanate (RITC) dye and coated by a silica shell [15]. MNPs@SiO2(RITC) are 50 nm in diameter and are known to have zeta potentials between − 40 to -30 mV [15, 48].
Cell Culture
Human embryonic kidney 293 (HEK293) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) (Lee et al. 2009). Briefly, cells were cultured in Dulbecco’s high-glucose modified Eagle’s medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin, and 100 µg/mL streptomycin (Gibco) and incubated humidified chamber at 37 °C in a 5% CO2 atmosphere.
Treatment of cells with MNPs@SiO2(RITC)
The dosage used in this study was determined by treating HEK293 cells with MNPs@SiO2(RITC) at concentrations ranging from 0.01 to 2 µg/µL for 12 h and calculating their uptake efficiencies [24]. The uptake efficiency of MNPs@SiO2(RITC) was known to be plateaued at 1 µg/µL [24]. The optimal concentration of MNPs@SiO2(RITC) was 0.1 µg/µL for in vitro use and as a magnetic resonance imaging contrast [22]. Thus, MNPs@SiO2(RITC) at 0.1 µg/µL and 1 µg/µL were used in the present study.
Immunocytochemistry and cell area analysis
Cells were seeded on cover slips and incubated for 6 h at 37 °C and 5% CO2. Then, the attached cells on the coverslips were treated with 0.1 µg/µL or 1.0 µg/µL of MNPs@SiO2(RITC) for 12 h at 37 °C and 5% CO2. The cells were then fixed in Cytofix buffer (BD Bioscience, San Jose, CA, USA) for 30 min at 4 ˚C. To reduce non-specific binding, the cover slips were blocked with phosphate-buffered saline (PBS, pH 7.4) containing 2% bovine serum albumin (BSA) and 0.1% Triton-X100 (Sigma-Aldrich, St. Louis, MO, USA). For actin labeling, cells were incubated with Alexa Fluor 488-conjugated phalloidin (Molecular Probe, Eugene, OR, USA), (1:200) diluted in PBS, for 1 h at room temperature (RT). For nuclear labeling, cells were washed three times with PBS containing 0.1% Triton-X100 and incubated with PBS containing 10 µg/mL of Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at RT. After washing three times with PBS, cover slips were mounted onto slides using Prolong Gold Antifade mounting medium (Molecular Probe). Fluorescence images were acquired by confocal laser scanning microscopy (CLSM) with a slide scanner (Axioscan. Z1, Carl Zeiss Microscopy GmbH, Jena, Germany). Attached cell areas were determined using ImageJ (NIH, Bethesda, MD, USA).
Preparation of soft and rigid flat PDMS surfaces
Surfaces were prepared using a Sylgard Silicone Elastomer Kit (Dow Corning, Cortland, NY, USA). The silicone elastomer base was mixed with a curing agent, degassed for 30 min, and spin-coated on a cover glass at 2,000 rpm for 2 min to obtain a PDMS layer 35 ± 5 µm thick. Then, the elastomer was crosslinked at 70 °C for 4 h. Surfaces with Young's moduli of 5 kPa (soft) and 2 MPa (rigid) were prepared by changing the ratio of elastomer base to curing agent to 75:1 and 10:1, respectively. Coverslips with a layer of PDMS were functionalized with 20 µg/mL fibronectin (Sigma-Aldrich) at 4 °C overnight. Prior to cell seeding, coverslips with PDMS layers were washed with PBS and growth medium. Then cells were seeded and treated with MNPs@SiO2(RITC) at 0.1 and 1 µg/µL for 12 h.
Cell morphological analysis on soft and rigid flat PDMS surfaces
Untreated and MNPs@SiO2(RITC)-treated cells were incubated on soft or rigid surfaces for 12 h and fixed with 4% paraformaldehyde in PBS for 15 min to study the spreading area, aspect ratio, and filopodia structure of the cells. They were then permeabilized with 0.1% Triton X-100 in PBS for 15 min. Next, the actin structures of the cells were labeled with Alexa Flour 488 phalloidin (Thermo Fisher Scientific) (1:2500) in PBS for 30 min at RT. The cell nuclei were then labeled with 0.2 mg/mL of 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) (1:10000) in PBS for 15 min at RT. Finally, the stained cells were imaged using a DeltaVision microscope (GE Healthcare, Chicago, IL, USA) equipped with a CoolSnap HQ2 camera (Photometrics, Tucson, AZ, USA). The cell spreading area and cell aspect ratio were measured using FIJI ImageJ (NIH), and the filopodia of cells were analyzed using a FIJI ImageJ plug-in called FiloQuant [49].
FA size analysis on soft and rigid flat PDMS surfaces
Cells were fixed and permeabilized for FA analysis as previously described. The cells were then blocked with 1% BSA (Gibco) for 1 h. The cells were subsequently treated with anti-paxillin primary antibody (ab32084, Abcam, London, UK) 1:500 in 1% BSA for 1 h at RT, and the cell nuclei were labeled with DAPI for 15 min. The cells were then imaged with a DeltaVision microscope (GE Healthcare). Finally, the FA area of the cells was analyzed using FIJI ImageJ.
Western blotting
For analysis of cytoskeletal proteins, untreated and MNPs@SiO2(RITC)-treated cells were lysed in a protease inhibitor cocktail (Sigma-Aldrich) containing radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich), and total protein concentrations were determined by the bicinchoninic acid kit (Bio-Rad, Hercules, CA, USA). Proteins were separated using 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then washed three times with 0.1% Tween-20 (Sigma-Aldrich) in tris-tween buffered saline (T-TBS) and blocked with 5% non-fat milk/T-TBS. The membranes were incubated with mouse monoclonal antibodies against phosphorylated Src (p-Src), total Src (tSrc), phosphorylated FAK (p-FAK), total FAK (tFAK), phosphorylated MLY2 (p-MLY2), total (tMLY2), phosphorylated paxillin (p-PXN), total PXN (tPXN), β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-rabbit FSCS1 (ab126772) (1:10000; Abcam) as primary antibodies. After washing with T-TBS three times, the membranes were incubated with mouse Ig kappa binding protein conjugated with horseradish peroxidase (1:1000; Santa Cruz Biotechnology). Blots were developed using an enhanced chemiluminescence solution (Thermo Fisher Scientific), and luminescence was captured on medical blue X-ray films (Agfa, Mortsel, Belgium) in a dark room.
Pillar fabrication and pillar force analysis
Photolithography using reactive ion etching was used to fabricate a linear array of holes (0.9 µm diameter, 2 µm depth, 1.8 µm distance between holes) in a 5-inch silicon wafer. The PDMS pillar array (2 µm in height, 0.9 µm diameter, 1.8 µm center to center distance, k = 24.21 nN/µm) was made from the Sylgard Silicone Elastomer Kit. The silicone elastomer base was mixed with the curing agent in a ratio of 10:1 and then degassed for 15 min to remove air bubbles. The mixed elastomer was then spin-coated on the mold at 1,000 rpm for 1 min and degassed for 15 min. The coated mold was then cured at 80 °C for 3 h. The pillar array was removed from the silicon mold while immersed in 99.5% isopropanol. The bending stiffness k of the column was calculated using the Euler Bernoulli beam theory:
where D and L are the diameter and length of the pillar, respectively, and E is the Young’s modulus of the PDMS (2 MPa). In this study, the stiffness (k) of the pillar was 24.21 nN/µm.
$$=\frac{\sqrt{{(Ax+Bx)}^{2}+{(Ay+By)}^{2}}}{\sqrt{\left({Ax}^{2}+{Ay}^{2}\right)}+\sqrt{\left({Bx}^{2}+{By}^{2}\right)}}$$
For this purpose, cell images taken within 30 min of cell surface contact were used for γ calculation. Average γ values for each type of cell were obtained by calculating 20 frames of two different cells.
Statistical analysis and error correction
Data are represented as the mean ± standard error of the mean of the samples in a group. Student’s t-test was used to compare 0.1 or 1 µg/µL MNPs@SiO2(RITC)-treated samples with untreated samples. A p-value of less than 0.05 was considered significant (*P < 0.05, **P < 0.01, ***P < 0.001). In the experiments using pillars, errors of the pillar deflections were corrected by reducing the average pillar deflection of pillars outside the cell.