Fig 1.
Insect behavioral and cellular response to PEMF.
(A). Distribution of wild-type Canton S larvae following 96-hour exposure in blue light (60 μmolm−2s−1) to PEMF (10 Hz) to the indicated corner. (B) Response to PEMF expressed as percentage of larvae in the petri plate corners: PEMF exposed corner (black bars), mean of 3 nonexposed corners (white bars). Data represent averages from 8 to 10 independent biological experiments (n = 8–10) (see S1 Data). Strains used are Canton S (WTS), Oregon (WTO), cry-deficient mutants (cry02 and cryb). Gal4 and UAS are nonexpressing parental strains for the cross (tim-gal4;cry02 × UAS-Hscry1;cry02) (HsCry1) that expresses the HsCry1 protein as described in ref. [27]. ***p < 0.001 (see Materials and methods for details of statistical treatment). Error bars are SEM. (C) SF21 insect cells overexpressing DmCry exposed to PEMF. Dark grown Sf21 insect cell cultures expressing high concentrations of DmCry as described [28] were illuminated for 15 minutes at 80 μmolm−2s−1 blue light in the presence (+) or absence (−) of PEMF and were viewed by confocal microscopy as described in Materials and methods. n = 5 biological repeats. Scale bar 100 μm. Data for Fig 1B is in S1 Data. DmCry, Drosophila cryptochrome; Gal4, tim-gal4;cry02; HsCry1, human cryptochrome-1; PEMF, pulsed electromagnetic field; Sf21,; UAS, UAS-Hscry1;cry02.
Fig 2.
PEMF response of HEK293 cell cultures.
(A) Cells of WT or HSCRY1 and HSCRY2 double KD (HsCry) were seeded in 24-well cell culture dishes and grown for 48 hours in the presence (PEMF, black bars) or absence (Sham, white bars) of applied PEMF as described (Materials and methods). The concentration of H202 in the culture media was determined using the Amplex Red fluorescent detection system (Materials and methods) and normalized to the number of cells. The graph presents the relative concentration of H202 from PEMF-treated cells (PEMF, black bars) compared to the control (Sham, white bars) untreated sample. Error bar represents SD of 12 independent measurements. (B) Relative number of cells per well in PEMF-treated cells (PEMF, black bars) compared to the control (Sham, white bars) untreated sample. n = 12 biological repeats. Error bar represents SD. ***p < 0.001 (see Materials and methods). Underlying data is included in S1 Data. HEK293, human embryonic kidney cells; HsCry, human cryptochrome; KD, knockdown; PEMF, pulsed electromagnetic field; WT, wild-type.
Fig 3.
Production and subcellular localization of ROS by mammalian cells exposed to PEMF.
Living HEK293, MEF, or MEF CRY1CRY2 cryptochrome-deficient double mutant knockout cell lines were exposed to PEMF (+PEMF) for 15 minutes in darkness while simultaneously treated with DCFH-DA, then viewed by an inverted Leica TCS SP5 microscope. Control cell cultures not exposed to the magnetic field (−PEMF) were treated in an identical manner. Images show a single confocal z section that crosses the nucleus. Diffuse fluorescent ROS staining can be seen in the nucleus and cytoplasm. Punctate and intense fluorescent ROS staining colocalizes with ER and nucleoli, as observed around and inside the nucleus, respectively. Qualitatively similar results were obtained for 5 independent experiments (n = 5); quantitation of representative images is presented in S9 Fig. Scale bar is 40 μm. DCFH-DA, {5-(and-6)-chloromethyl-2’,7’-dichlorofluorecein diacetate}; ER, endoplasmic reticulum; HEK293, human embryonic kidney 293; MEF, mouse embryonic fibroblast; PEMF, pulsed electromagnetic field; ROS, reactive oxygen species.