Sensitivity to chemotherapy and Cetuximab is not influenced by BEMER therapy.

<p>(A) Flow chart of colony formation assay. Cells were plated in 3D lrECM, treated with respective agents followed by BEMER therapy 23 h later. (B) Basal surviving fraction after Cisplatin (0.1 μM; DMEM as control) treatment and BEMER therapy (~13 μT, 8 min). (C) Basal surviving fraction after Gemcitabine (10 nM; DMEM as control) treatment and BEMER therapy (~13 μT, 8 min). BEMER sham-treated (sham) cells served as control. (D) Basal surviving fraction after Cetuximab (5 μg/ml; IgG as control) treatment and BEMER therapy (~13 μT, 8 min). IgG-treated cells served as control. All results represent mean ± SD. Student's t-test. n = 3. * P < 0.05; ** P < 0.01. n.s., not significant.</p

QLT0267 treatment increases the number of radiation-induced DSBs without affecting apoptosis.

<p>(A) After treatment with 10 µM QLT0267 for 24 h, 2D or 3D lrECM grown cells remained unirradiated or received a single dose of 2 Gy. After 24 h, cells were isolated, fixed and co-stained against 53BP1 and γH2AX. Double stained foci from 150 cells were microscopically counted per experiment. Number of foci of irradiated cells was normalized to number of foci of unirradiated cells. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated or QLT0267/2Gy- vs. DMSO/2Gy-treated cells. *<i>P</i><0.05, **<i>P</i><0.01. Photographs illustrate immunofluorescence staining of 53BP1 (green) and γH2AX (red) of 3D grown cell cultures. Nuclei were stained with DAPI (blue). (B) In parallel, cells were treated as indicated, fixed and stained with DAPI to microscopically determine cells with typically apoptotic nuclear morphology. Results are means±s.d. (n = 3).</p

BEMER therapy-mediated radiosensitization remains unaltered upon chemotherapy and Cetuximab.

<p>(A) Flow chart of colony formation assay. (B) Clonogenic survival after 6-Gy irradiation combined with BEMER therapy (~13 μT, 8 min) and Cisplatin (0.1 μM; DMEM as control). (C) Clonogenic survival after 6-Gy irradiation combined with BEMER therapy (~13 μT, 8 min) and Gemcitabine (10 nM; DMEM as control). Sham-treated (sham) but irradiated cells served as control. (D) Clonogenic survival after 6-Gy irradiation combined with BEMER therapy (~13 μT, 8 min) and Cetuximab (5 μg/ml; IgG as control). IgG-treated, irradiated cells served as control. All results represent mean ± SD. Student's t-test. n = 3. * P < 0.05; ** P < 0.01. n.s., not significant.</p

QLT0267 significantly reduces basal cell survival and sensitizes hHNSCC cells to ionizing radiation.

<p>(A) For 2D or 3D clonogenic assays, single cells were plated onto lrECM or inserted into lrECM and exposed to increasing concentrations of QLT0267 (0–20 µM) for 1 h or 24 h. Colonies were counted microscopically after 8–11 days. Results are means±s.d. (n = 3). Student's t-test compared clonogenic survival of QLT0267-treated cells under 2D vs. 3D growth conditions. *<i>P</i><0.05. (B) Photographs show colonies 11 days (FaDu) or 8 days (UTSCC45) after treatment with DMSO or 10 µM QLT0267. (C) Subsequent to a 1-h or a 24-h exposure with DMSO or QLT0267, 2D and 3D cell cultures were irradiated with 2 Gy X-rays. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267-treated/irradiated vs. DMSO-treated/irradiated cells. *<i>P</i><0.05; **<i>P</i><0.01</p

QLT0267 and irradiation induce accumulation of G2 phase cells in a cell culture model-dependent manner.

<p>(A) After treatment with QLT0267 for indicated time periods, cells were incubated with BrdU and cell cycle analysis was performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006434#s4" target="_blank">Materials and Methods</a>. Results are means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated cells. *<i>P</i><0.05; **<i>P</i><0.01. (B) Cell cycle distribution was assayed in cells after a 24-h QLT0267 treatment plus 6 Gy X-rays (FaDu: 12 h post irradiation; UTSCC45: 18 h post irradiation). Results are means±s.d. (n = 3). Student's t-test compared QLT0267/irradiated vs. DMSO/irradiated cells. *<i>P</i><0.05; **<i>P</i><0.01.</p

QLT0267 fails to antagonize the radiosensitization mediated by a constitutively active form of ILK.

<p>FaDu cells stably transfected with a constitutively active form of ILK (IH43) or control vector (EV2) were used. Cells cultured in 2D or 3D were exposed to 1 µM or 10 µM QLT0267 for 24 h and irradiated with 2 Gy X-rays. Results are means±s.d. (n = 3). Student's t-test compared QLT0267/2Gy- vs. DMSO/2Gy-treated cells. *<i>P</i><0.05; **<i>P</i><0.01.</p

BEMER therapy radiosensitizes microtumors.

<p>(A) Flow chart of colony formation assay. (B) Basal surviving fraction of BEMER (~13 μT, 8 min, 1 h, 24 h) treated and BEMER sham-treated (sham) microtumors. (C) Clonogenic survival after BEMER therapy (~13 μT, 8 min, 1 h, 24 h) combined with radiotherapy (2 and 6 Gy). All results represent mean ± SD. Student's t-test compares BEMER therapy versus sham samples. n = 3. * P < 0.05; ** P < 0.01.</p

Modification of clonogenic survival and protein phosphorylation by QLT0267 are independent from ILK.

<p>Mouse <i>ILK</i>^fl/fl and <i>ILK^−/−</i> fibroblasts were cultured under 2D or 3D cell culture conditions, exposed to QLT0267 for 24 h plus/minus irradiation (2 Gy X-rays) and basal (A) and radiation cell survival (B) were measured as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006434#s4" target="_blank">Materials and Methods</a>. Results are means±s.d. (n = 3). Student's t-test compared QLT0267-treated <i>ILK</i>^−/− vs. QLT0267-treated <i>ILK</i>^fl/fl cells (A) or QLT0267/irradiated vs. DMSO/irradiated cells (B). *<i>P</i><0.05; **<i>P</i><0.01. (C) Total protein lysates from QLT0267- or DMSO-treated cells were analyzed by SDS-PAGE and Western blotting using indicated antibodies. (D) Cells were transfected with 20 nM ILK-specific siRNA or control siRNA. At 48 h post transfection, efficient downregulation of ILK expression was confirmed by Western blotting. Densitometric values were normalized to β-Actin. (E) For clonogenic assays, siRNA knockdown cell cultures were irradiated with 2 Gy X-rays 48 h after transfection. Results represent means±s.d. (n = 3).</p

The specific BEMER EMF pattern impacts on cancer cell metabolism.

<p>(A) Pie chart showing the number of detected metabolites categorized by pathways (Σ 225). (B) Heatmap comparing levels of metabolites in BEMER signal treated (~13 μT, 8 min) and BEMER sham-treated (sham) A549 cells. Red and blue indicate up- and downregulation, respectively. Cells were cultured in 3D lrECM for 24 h prior to BEMER treatment. (C) Amount of indicated metabolites in A549 cells without (sham) and with BEMER EMF exposure. (D) Scheme of glycolysis and TCA cycle. Metabolites in blue were downregulated, in red upregulated and in black unaffected upon BEMER therapy compared with sham-treated controls. Metabolites depicted in green were not measured in the metabolome analysis. All results represent mean ± SD. Student's t-test. n = 5. * P < 0.05; ** P < 0.01.</p

BEMER signal intensity determines radiosensitization and DSB numbers.

<p>(A) Flow chart of colony formation assay and foci assay. (B) Clonogenic survival after 6-Gy irradiation combined with BEMER therapy (2.7–35 μT; 8 min) of A549 and UTSCC15 cells. (C) Immunofluorescence images show nuclei with γH2AX/53BP1-positive foci after 6-Gy irradiation with (~13 or ~35 μT; 8 min) and without BEMER therapy in A549 cells. (D) Number of γH2AX/53BP1-positive DSBs 24 h after irradiation in A549 and UTSCC15 cells. BEMER sham-treated (sham), irradiated cells served as control. All results represent mean ± SD. Student's t-test. n = 3. * P < 0.05; ** P < 0.01.</p