Media 1: Membrane ripples of a living cell measured by non-interferometric widefield optical profilometry

Originally published in Optics Express on 26 December 2005 (oe-13-26-10665

Methodik zur lebenszyklusweiten, umweltgerechten Produkt- und Produktionsoptimierung

Originally published in Biomedical Optics Express on 01 July 2015 (boe-6-7-2624

Additional file 2 of Effects of the media conditioned by various macrophage subtypes derived from THP-1 cells on tunneling nanotube formation in pancreatic cancer cells

Additional file 2: Fig. S1. The mRNA levels of markers for M1 macrophage (a) IL-1β and (b) TLR-2, and (c) the marker for M2 macrophage CCL22, measured in the macrophages after the differentiation according to the protocols described in the Materials and Methods section. The data are from three independent experiments. ***, P < 0.005; *, P < 0.05 in comparison with those in THP-1 cells (post hoc Tukey’s test). Fig. S2. The ELISA results of EGF in (a) the conditioned media (CMs) of the THP-1 cells and macrophages, and (b) the CM of PANC-1 cells cultured in the macrophage CMs for 48 hours. The data are from three independent experiments. (c) The calibration curve of the optical density (OD) vs. the EGF concentrations. From this calibration curve, we learned that the EGF concentrations in panels (a) and (b) are all below the detection limit of ELISA. The ELISA kit was DY 236, DuoSet ELISA (R&D Systems, Minneapolis, MN, USA). The absorbance of the analytes were measured with a plate reader (Synergy 2, BioTek Instruments). Fig. S3. Formation of TNTs between two PANC-1 cells originally in contact (indicated by an arrow in the image at 0 min) in the M0 CM. This process is consistent with the “cell dislodgement” TNT formation mechanism. Fig. S4. Co-localization of kinesin (red) with the mitochondria (green) within a TNT. Most of the bright mitochondria were co-localized with the kinesin signal. The mitochondria were fused with green fluorescence protein in a stable cloned PANC-1 cell line. The kinesin was labeled with rabbit antibody (ab5629, abcam) then probed with DyLight 650-conjugated secondary antibody (ab96886, abcam)

Probability distribution of the filopodial extension and retraction rates.

<p>The data were assorted into a series of rate ranges that were equally separated by 0.02 μm⋅s⁻¹. The solid, dashed, and dotted lines represent normal distribution fits to the data of glass, PVC 3∶1, and PVC 1∶1 respectively. The coefficients of determination (i.e., <i>R-</i>square) for the three fittings are >0.9.</p

Effects of substrate stiffness on the (A) filopodial density and (B) averaged filopodial length for individual cancer cells.

<p>The filopodial density was defined as the number of filopodia per unit length of the cellular perimeter. The filopodial length was averaged from all visible filopodia of the cell. The bars represent the means of the variables and the errors denote the standard deviations calculated from 33, 18, and 43 cells for the PVC 1∶1, PVC 3∶1, and glass substrates respectively. Significant differences were found between the data of PVC 1∶1 and 3∶1, and PVC 1∶1 and glass with that ** indicates <i>p</i><0.01.</p

Effects of blebbistatin treatment on the (A) filopodial density and (B) averaged filopodial length for CL1–5 cells cultured on glass substrates.

<p>The bars represent the means of the variables and the errors denote the standard deviations calculated from 43, 24, and 31 cells treated with 0, 10, and 30 μM blebbistatin respectively. The mark * indicates <i>p</i><0.05.</p

Representative SINAP images for CL1–5 cells cultured on (A) PVC 1∶1, (B) PVC 3∶1, and (C) glass substrates.

<p>Scale bar = 5 μm.</p

Viability of CL1–5 cancer cells with and without treatment of blebbistatin of various concentrations.

<p>The cells were cultured on glass substrates for 24μM of DMSO or blebbistatin for 1 hour (<i>n</i> = 5 for each concentration). The optical densities of cells without application of the reagents were referred to as control (<i>n</i> = 8). The bars represent the means of the variables and the errors denote the standard deviations.</p

The Western blotting result of CL1 cells under different conditions.

<p>(A) Under different period of dcEF stimulation. Note that CL 1-5 cells have high EGFR expression in comparison to CL1-0 cells but neither cells showed EGFR phosphorylation at Tyr1068 under dcEF stimulation. (B) CL1-0 cells and CL 1-5 cells stimulated with 20 ng/mL EGF and CL 1-5 cells stimulated under dcEF in serum-containing medium. Note that even in serum-containing medium, CL 1-5 cells show no EGFR Tyr1068 phosphorylation under dcEF stimulation, contrary to EGF-stimulated cells. The numbers below each protein band indicate the relative densitometry intensity of the protein in different conditions compared to that in the CL 1-5 cells in the control condition.</p

Phase contrast images of CL1–5 cancer cells grown on the surfaces of commonly used compliant substrates and glass coveslips.

<p>The PVC 1∶1, 2∶1, and 3∶1 refer to the PVC composites with ratios of PVC to the plasticizer being 1∶1, 2∶1, and 3∶1 respectively; PA and PDMS are abbreviated for polyacrylamide and poly(dimethyl) siloxane respectively. Scale bar  = 100 µm.</p