Figure 1.
MF induces increases in filopodia-like protrusions and focal adhesions inFL cells.
A: negative control (N-con); B: sham-exposed (Sham); C: treated with100 nMEGF (EGF); D: exposed to 0.4 mT MF for 30 min (MF); E: pre-treated with PD, then exposed to MF (+PD+ MF). Arrow: appearance of filopodia; arrowhead: lamellipodia. The detailed information of experimental conditions and repeating numbers of samples is seen in Table 1.
Figure 2.
MF induced stress fibers and F-actin content decreases and cell surface increases in FL cells.
A: negative control (N-con); B: sham-exposed; C: pre-treated with 1 µM PD (+PD); D: pre-treated with 100 nM EGF (EGF); E: exposed to 0.4 mT power frequency MF (MF); F: pre-treated with 1 µM PD, then exposed to MF (+PD+MF). Microfilaments above were labeled with phalloidin-TRITC and photographed with an Olympus BX51 immuno-fluorescence microscope (×400). G: Decreases in total F-actin content in EGF- and MF-treated cells, measured by flow cytometry, P<0.05 (*). H: Gray value summary of F-actin content of FL cells from confocal images by ImageJ analysis, compared with N-con and Sham, P<0.01 (#). I: Average cell surface area increased, results were analyzed by ImageJ; compared with N-con and Sham, P<0.01 (#). Arrow: newly grown filopodia, arrowhead: loss of stress fibers in central area of the cell. Bar in A–F: 10 µm. The lines on each bar in G–I stand for standard deviation (SD), and the detailed information of experimental conditions and repeating numbers of samples is seen in Table 1.
Figure 3.
Increases in vinculin-associated focal adhesions in FL cells induced by power frequency MF.
Vinculin stained: A–D; fascin stained: E–H; Arp3 stained: I–L. A: negative control (N-con); B: sham-exposed; C: exposed to 0.4 mT power frequency MF (MF); D: pre-treated with 100 nM EGF (EGF); E: negative control (N-con); F: sham-exposed; G: exposed to 0.4 mT power frequency MF (MF); H: pre-treated with 100 nM EGF (EGF); I: negative control (N-con); J: sham-exposed; K: exposed to 0.4 mT power frequency MF; L: pre-treated with 100 nM EGF (EGF)); M: summary of the gray value analysis of the proportion of vinculin-associated adhesion spots to the total cell area, analyzed by ImageJ; compared with N-con and Sham P<0.05 (*); N: summary of the gray value analysis of fascin and Arp3 by ImageJ analysis, compared with N-con and Sham P<0.05 (*). Bar in A–L: 10 µm. The detailed information of experimental conditions and repeating numbers of samples is seen in Table 1.
Figure 4.
Motility-associated protein content in FL cells affected by power frequency MF by WB assays.
A: F-actin/G-actin content in supernatant or precipitate; NC: negative control, 1 µM phalloidin was added when cracking the cells; PC: positive control, 1 µM cytochalasin-D was added when cracking the cells. B: gray value summary of actin content by software ImageJ, compared with N-con and Sham P<0.05 (*). C: focal adhesion-associated signal protein vinculin, filopodia-associated signal protein fascin, lamellipodia-associated signal protein Arp3, and stress fiber-associated signal protein MLC content. D: gray value summary of vinculin, fascin, and Arp3 by software ImageJ, compared with N-con and Sham P<0.05 (*). E: gray value summary of stress fiber-associated MLC by software ImageJ, compared with N-con and Sham P<0.05 (*). F: total content of actin and MLC in FL cells, compared with N-con and Sham. G: gray value of total content of actin and MLC protein in FL cells; compared with Sham P>0.05. The detailed information of experimental conditions and repeating numbers of samples is seen in Table 1.
Table 1.
The information of experimental conditions and repeating numbers of samples for each target.
Figure 5.
Decrease in the efficiency of F-actin assembly by power frequency MF.
A: sham-exposed G-actin to power frequency MF observed by AFM. B: exposed G-actin of self-assembled microfilaments to power frequency MF observed by AFM. C: free G-actin, without self-assembly, in sham exposure observed by AFM. D: AFM image index information. Arrow: free G-actin. Bar scale at the bottom of the image: 500 nm. The detailed information of experimental conditions and repeating numbers of samples is seen in Table 1.
Figure 6.
The influence of power frequency MF was greatly reducedby 2hours after withdrawal of the field.
A: Time-dependent recovery of F-actin content after withdrawing power frequency MFs. Sham: sham-exposed to power frequency MF for 0.5 hours; 0–2 h: exposed to power frequency MF for 0.5hours,field withdrawn and then sham-exposed for 0hour to 2hours; the summary of the gray value analysis of F-actin and cell area was analyzed by software ImageJ; compared with Sham, 0–1.5 h P<0.05 (*), 2 h P>0.05. B: content of vinculin, fascin, MLC and Arp3 proteins in FL cells 2 hours after withdrawing power frequency MF; C: gray value summary of vinculin, fascin, MLC and Arp3 content in FL cells 2 hours after exposing to MFs by software ImageJ, compared with Sham, P>0.05. The detailed information of experimental conditions and repeating numbers of samples is seen in Table 1.
Figure 7.
Power frequency MF downgrades EGFR ligand release.
FL cells transfected by TGF-α were grouped into 4 conditions: sham exposure, treated with PMA as a positive control, 0.4 mT power frequency MF exposure, and treated with PMA plus power frequency MF exposure. The cells were washed, and conditional medium was added. After 30 min, the medium from each group was collected as the corresponding pre-sample. Then, medium was collected again for all groups after incubation in the indicated conditions for 30 min. OD data were measured for each sample or pre-sample. The data presented in Figure 7 are the ratio of the OD of sample (OD405S) over the OD of pre-sample (OD405P). P<0.05 (*) between each two group. The detailed information of experimental conditions and repeating numbers of samples is seen in Table 1.
Figure 8.
Influence of power frequency MF on the average migration rate of FL cells.
A–F: Sham for 0 h, 3 h, 6 h, 12 h, 18 h, and 24 h successively; G–L: MF for 0 h, 3 h, 6 h, 12 h, 18 h, and 24 h successively; M–R: EGF for 0 h, 3 h, 6 h, 12 h, 18 h, and 24 h successively; S–X: MF+EGF for 0 h, 3 h, 6 h, 12 h, 18 h, and 24 h successively; Y: Effects on FL cell migration area; the areas were calculated by software ImageJ; compared with Sham, P<0.05(*) or <0.01(#); Z: Effect on the migration rate of FL cells, compared with Sham, p<0.05 (*) or <0.01(#). Bar: 200 µm. The repeating and experimental condition information is seen in Table 1.
Figure 9.
A possible model for power frequency MF interaction with the actin cytoskeleton.
A proposed physics model of power frequency MF disrupting F-actin assembly. The actin monomers can be seen as electric dipoles, and it is necessary for the monomers to be in a proper angle and orientation to join to the F-actin string (A). It’s suggested that power frequency MF induces electrical fields that have an effect on actin monomers. In the center of the magnetic field generation device, the direction of MFs is vertical to the coil plates, and the induced electrical fields (EFs) are parallel to the plates in clockwise or counterclockwise orientation (B). The intensity of the MF is vibrated as a sine function at the frequency of 50 Hz (C). Then, the intensity of the induced EF changes as a cosine function (C). Thus, in the MF-induced EF, the orientation or/and position of the actin dipoles flips over and over, following the EF direction, as shown in B (D). While the mean time for an actin monomer binding to F-actin is approximately 0.02 seconds, the induced EF changes its direction before the other candidate monomer with a proper orientation binds to the F-actin stream. As the result, the efficiency of F-actin assembly decreases.
Table 2.
[Ca2+]i in FL cells (FI526).