2. Experimental Section
Chemicals: PpIX was purchased from Sigma (St. Louis, MO, SUA). The cell proliferation reagent 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) was purchased from Roche (Mannheim, Germany). APF was purchased from Sekisui Medical Co. Ltd. (Tokyo, Japan). DHE was purchased from Life Technologies (Tokyo, Japan). Methanol, perchloric acid, acetic acid, sodium hydrogen carbonate, DMEM medium, penicillin, streptomycin, fetal bovine serum and PBS were purchased from Wako Chemicals (Osaka, Japan).
Cell culture: The HeLa cell line was supplied by the Riken Cell Bank (Tsukuba, Japan). The cells were cultured in DMEM containing 10% FBS in a 5% CO2 humidified incubator at 37 °C. The medium was supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin. 
X-ray irradiation: HeLa cells were irradiated (0, 1, 3 or 5 Gy) using 160-kV X-rays (Faxitron®Cabinet X-ray System Model CP-160, Wheeling, IL, USA). A Unidos® E Universal Dosimeter (PTW, Freiburg, Germany) was used to measure the dose rate. The resulting dose rate was 1 Gy/min.
Determination of porphyrin concentration in cells: Intracellular porphyrin was isolated in 1 N perchloric acid and methanol (1:1 v/v) after homogenization and centrifugation at 3000 rpm for 10 min. The number of cells used to determine the porphyrin concentration was approximately 2.0 × 106 cells. The supernatant was transferred to a tube, neutralized with sodium hydrogen carbonate and acidified with acetic acid. Porphyrin concentration was determined by spectrophotometry at the Soret maximum (405 nm) and by fluorescence, using an excitation wavelength of 405 nm and an emission wavelength of 630 nm [7]. 
Cell viability assay: The WST-1 cell viability assay was performed to assess cellular responses to PpIX treatment and X-ray irradiation. WST-1 was used as described by the manufacturer. As the reagent is based on the cleavage of the tetrazolium salt, WST-1, by mitochondrial dehydrogenases in viable cells, the absorbance of the formazan dye is supposed to correlate with the number of active cells in the medium. Before X-ray exposure, HeLa cells were cultivated in 96-well plates to confluency. PpIX was added 6 h before X-ray irradiation in 100 μL of culture medium at concentrations of 0, 0.3, 1 and 3 μg/mL. The plates were irradiated at a dose of 0, 1, 3 and 5 Gy. Immediately after irradiation, the medium was removed, and 100 μL of fresh medium was added. 1, 24 and 72 h after irradiation, and 10 μL WST-1 was added to each well. The plates were incubated in a 5.0% CO2 humidified incubator at 37 °C for 1 h, following which, the absorbance at 450 nm was measured using a plate reader against a referenced absorbance at 600 nm. Relative cell viability was defined as the dye absorption ratio of treated versus untreated cells. 
Clonogenic ability: Clonogenic survival ability was detected by a colony-forming assay based on the methods of Franken et al. [8]. Briefly, cells were seeded in six-well microplates at a density of 500 cells per well. Cells were allowed to attach for 14 h and treated with PpIX and/or X-ray. PpIX was added 6 h before X-ray irradiation in 3 mL of culture medium at concentrations of 0, 0.3, 1 and 3 μg/mL. The plates were irradiated at a dose of 0, 1, 3 and 5 Gy. After X-ray irradiation, cells were washed with PBS and replaced into fresh medium. Cells were then cultured for the period required for control cells to form colonies (one colony was defined as a colony containing 50 or more cells) or for 7 days. The experiments were performed on plates kept in the dark to avoid the effects of light. After fixation with 100% methanol for 15 min, cells were stained with Giemsa solution (diluted 1:50 in water) for 15 min and rinsed with distilled water. The number of colonies was counted.
Measurement of intracellular ROS: HeLa cells were cultivated in 96-well plates up to confluency. PpIX was added 6 h before X-ray irradiation in 100 μL of culture medium at concentrations of 0, 0.3, 1 and 3 μg/mL. After washing with PBS, APF or DHE was added with fresh medium at a final concentration of 10 μM. The plates were incubated in the dark for 30 min at 37 °C and irradiated at a dose of 0, 1, 3 and 5 Gy. After irradiation, the medium was removed, and the cells were washed with PBS. Fluorescence was measured on a microplate reader (Infinite M200, TECAN) at excitation/emission and λ = 490 nm/515 nm for APF and at excitation/emission and λ = 510 nm/580 nm for DHE. 
Minimum information about a microarray experiment (MIAME) compliance and data availability: The microarray experiments described in this manuscript are MIAME compliant, and the raw data have been deposited in the Gene Expression Omnibus (GEO) database (Accession Number GSE 61805) [9]. 
Sample preparation and microarray assays: RNA was extracted from cells using the Qiagen RNeasy Mini kit (Qiagen GmbH, Germany), according to the manufacturer’s guidelines. The quality of the purified RNA was verified using an Agilent® 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA concentration was determined using a NanoDrop® ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Fluorescent cyanine 3-cytidine triphosphate (CTP)-labeled cRNA was used for hybridization to human oligo microarray slides (#G4112F, Agilent Technologies) at 65 °C for 17 h. The hybridized microarray slides were washed according to the manufacturer’s instructions and were scanned with an Agilent DNA Microarray Scanner (#G2565BA, Agilent Technologies) at a resolution of 5 μm. The scanned images were analyzed quantitatively using Agilent Feature Extraction Software version 9.5.3.1 (Agilent Technologies).
Microarray data analysis: Data were normalized by quantile normalization and were analyzed using GeneSpring GX software version 10.0.1 (Agilent Technologies). The Gene Ontology (GO) Database (http://www.geneontology.org/) was used to functionally categorize gene-expression profiles. GO terms were obtained from Agilent Technologies eArray [10]. Microarray cDNA probes were classified according to GO terms for different biological processes.
Statistical analysis: Accumulation of porphyrins in HeLa cells was analyzed by a two-tailed Student’s t-test. Data for cell viability, colony-forming ability and intracellular ROS were analyzed by one-way analysis of variance (ANOVA). The Tukey–Kramer HSD test was used for post hoc pairwise comparison. Differences were considered statistically significant at p < 0.01. 
The Student’s t-test was used to assess the significance of each gene in the microarray. We selected genes in the PpIXT, XT and PpIX-XT groups with p-values < 0.01 when compared to the NT group. After excluding overlapping probes, genes with significantly different expression in each group (PpIXT, XT and PpIX-XT) were analyzed using the functional annotation chart in the Database for Annotation, Visualization and Integrated Discovery (DAVID Bioinformatic Resources 2007, National Institute of Allergy and Infectious Disease) [11,12,13]. To characterize gene expression, the Fisher’s exact test was applied to calculate the significance for GO terms related to biological processes. Terms with adjusted p-values < 0.01 (Benjamini–Hochberg FDR correction) were defined as statistically significant.