Figure 1.
Phase contrast images of CL1–5 cancer cells grown on the surfaces of commonly used compliant substrates and glass coveslips.
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.
Figure 2.
The optical densities of formazan dye in cells grown on various substrates.
The dye has a purple color and was quantified by the optical absorbance at 570(n = 8, 5, 4, 4, 3, and 3 for the glass, PVC 3∶1, PVC 2∶1, PVC 1∶1, PA gel, and PDMS substrate respectively).
Figure 3.
Representative SINAP images for CL1–5 cells cultured on (A) PVC 1∶1, (B) PVC 3∶1, and (C) glass substrates.
Scale bar = 5 μm.
Figure 4.
Effects of substrate stiffness on the (A) filopodial density and (B) averaged filopodial length for individual cancer cells.
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 p<0.01.
Figure 5.
The length temporal profile of a filopodium measured from a series of SINAP images taken every 10 seconds.
The cell was cultured on glass substrate. The filopodial length was 0.92 μm at the start of image acquisition, continuously extended to 1.81 μm, and retracted to 1.02 μm at the end of image acquisition. The SINAP images corresponding to the length data marked by circles are shown in the right and annotated with the recording time. The tracked filopodium was highlighted by the arrow in the 1st image.
Figure 6.
Variation of filopodial extension and retraction rate at various filopodial lengths.
(A), (B), and (C) depict the relationship between the extension rates and filopodial lengths for the cells cultured on the PVC 1∶1, PVC 3∶1, and glass substrate respectively; (D), (E), and (F) show variation of the retraction rates with respect to various filopodial lengths for the cells cultured on the PVC 1∶1, PVC 3∶1, and glass substrate respectively. The bars represent the means of the rate data assorted in the length ranges and the errors denote the standard deviations. The data were assorted into a set of length ranges separated by 2 μm. The numbers of filopodia tracked for the rate measurement are 51, 59, and 34 for the PVC 1∶1, PVC 3∶1, and glass substrates respectively.
Figure 7.
Probability distribution of the filopodial extension and retraction rates.
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., R-square) for the three fittings are >0.9.
Figure 8.
Probability distribution of the estimated tendency for a filopodium to retract.
The retraction tendency was defined as the fraction of time that the filopodium spent for retraction. 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., R-square) for the three fittings are >0.9.
Figure 9.
Viability of CL1–5 cancer cells with and without treatment of blebbistatin of various concentrations.
The cells were cultured on glass substrates for 24μM of DMSO or blebbistatin for 1 hour (n = 5 for each concentration). The optical densities of cells without application of the reagents were referred to as control (n = 8). The bars represent the means of the variables and the errors denote the standard deviations.
Figure 10.
Effects of blebbistatin treatment on the (A) filopodial density and (B) averaged filopodial length for CL1–5 cells cultured on glass substrates.
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 p<0.05.
Figure 11.
Bright field and immunofluorescence images of cells cultured on the three kinds of substrates with and without blebbistatin treatment.
(A), (B), and (C) are bright field images of cells treated with regular culture medium as control; (D), (E), and (F) are their corresponding immunofluorescence images of nucleus (blue) and vinculin (green); (G)—(L) show the bright field and corresponding immunofluroresence images of cells treated with 30 µM blebbistatin for one hour to inhibit myosin II activities. Scale bar = 10 μm.
Figure 12.
Effects of blebbistatin treatment on (A) filopodial density and (B) averaged filopodial length.
The white bars represent the means of the data measured from cells without blebbistatin treatment (i.e., data in Fig. 4); the gray bars denote the means of the data measured from cells with blebbistatin treatment; and errors specify the standard deviations of the data. The numbers of cells treated with blebbistatin are 10, 13, and 31 for the PVC 1∶1, PVC 3∶1, and glass substrates respectively. # and ## denote p<0.05 and p<0.01 respectively for the difference between the data acquired from the same kind of substrates with and without blebbistatin treatment; * and ** are annotated for p<0.05 and p<0.01 respectively for the difference between the data acquired from different kind of substrates with and without blebbistatin treatment. Note that for the cells without blebbistatin treatment, significant differences were found between the data of PVC 1∶1 and PVC 3∶1, and between that of PVC 1∶1 and glass; for the cells treated with blebbistatin, significant difference was only found between the data of PVC 1∶1 and glass.