Ibl 437c

Recipient mice were irradiated with two split doses of 1,000 cGy from a ¹³⁷Cs source (IBL 437 C; CIS Bio International, Bangnols sur Ceze, France) with a 5-h interval. BM cells isolated from the femur and tibia of gender-matched donors were depleted of T cells by MACS and 5 × 10⁶ T cell-depleted BM (TCD-BM) cells were transferred i.v. into recipients 5 h after the second irradiation as previously described^{57 (link),58 (link)}. EL4 cells were purchased from ATCC (TIB-39; Rockville, MD, USA), and transduced to express H60, H60 and Luciferase, or H60 and Thy1.1 (H60⁺EL4) or transduced with empty vector^{32 (link)}. Original and transduced EL4 cells were periodically checked for expression of CD3 and the relevant antigens by flow cytometry. H60-positive or negative EL4 tumor cells were injected s.c. (5 × 10⁵) or i.v. (1 × 10⁶) into BMT recipients at 6 weeks after BMT. C1498 (B6 acute myeloid leukemia cells) were purchased from ATCC (TIB-49) and transduced to express Thy1.1 alone or together with H60 as described above. Original and transduced C1498 cells were periodically checked for expression of the relevant antigens by flow cytometry. H60-positive or -negative C1498 leukemic cells were injected i.v. (5 × 10⁵) into BMT recipients at 6 weeks after BMT. Both original and transduced EL4 and C1498 cell lines have been periodically tested to exclude mycoplasma contamination by PCR.

C57BL/6 wild-type mice were sublethally irradiated (7.5 Gray) using a Cesium-137 Irradiator (IBL437C, CIS Bio, Inc., Codolet, France). Donor mice (LPAR4_CreERT2_IRES_EGFP_TG mice) were euthanized, and GFP⁺ BM cells were collected from the tibia and femur. Next, 5 × 10⁷ GFP⁺ BM cells were systemically transplanted via the jugular vein into sublethally irradiated recipient mice (C57BL/6 wild-type mice). We used a mouse model and performed ligation of the left anterior descending artery after BMT. After 4 weeks of BMT, FACS analysis was performed against GFP⁺CD45⁺ cells to measure the donor engraftment rate.

L4 stage animals were irradiated with doses of 20 Gy, 40 Gy, 60 Gy and 80 Gy using a Cs-137 source (IBL 437C, CIS Bio International), and doses of 50 J, 100 J, 200 J and 500 J using a Waldman UV 236 B device with a spectrum of 280 nm to 360 nm (UV-B / UV-A light). cisplatin exposure was performed as described [27 (link)]. In short, animals were incubated under gentle shaking for 16h at 20°C in M9 liquid culture with cisplatin (Sigma-Aldrich, P4394) concentrations from 0 to 400 μM (S1 Table). Treated animals were allowed to recover and 3 times 3 adults per genotype and dose were transferred onto fresh plates 24h post treatment. Adults were removed after 4h and eggs laid within this time were allowed to hatch and scored for viability [26 (link), 27 (link)]. 2 F1 L4s per plate were then singled. For higher doses of radiation, not all F1 animals produced progeny. Progeny of only one of each set of 2 F1 lines was frozen, providing 3 independent lines per genotype and treatment. Genomic DNA was isolated using Invitrogen ChargeSwitch® gDNA Mini Tissue Kit (Thermo Fisher Scientific, CS11204) and sent for sequencing.

To test the sensitivity to γ-rays, sterilized seeds were kept in sterile water at 4°C for approximately 24 h and then were exposed to 100, 200, 300, 400, and 500 Gy doses (2.94 Gy/min) from a ¹³⁷Cs source (IBL 437C; CISBIO Bioassays). After irradiation seeds were germinated on MS agar medium. For mitomycin C (MMC, Duchefa) assay, 4-day-old seedlings were transferred to liquid GM with different MMC concentrations (3, 6, 9, and 12 μg/ml). For cisplatin [cis-diamminedichloroplatinum (II), CDDP, Sigma] assay, seeds were germinated on MS agar medium with different CDDP concentrations (15, 30, 50, and 75 μ M). The effects of the individual DNA damage agents on plant growth were evaluated 12 days (MMC) or 14 days (γ-rays, CDDP) after sowing.

Cells and mice were irradiated with total doses ranging between 0.01 and 1 Gy using a ¹³⁷Cs γ-irradiator (IBL 437C, CIS Bio International, Bangnols sur Ceze, France) with a dose rate of 0.8 Gy/min.

Nucleated bone marrow cells were collected from 8-weeks-old wild-type (WT) and Dok-3 knockout mice and intravenously injected into lethally irradiated ApcMin/+ mice (abbreviated as Apc mice). Irradiation was performed using an IBL-437C instrument (¹³⁷Cs, CIS Bio-International) at 9.5 Gy.

Irradiation treatments were performed at the Medical Physics Department of St. Anna Hospital (Ferrara, Italy) using a gamma irradiator (IBL 437C, CIS Bio International, Bagnols sur Ceze, France; 65.564 TBq 1772 Ci ± 10% Cs-137 linear source) at a dose rate of 2.2 Gy/min (± 3.5%). At the irradiation facility male pupae were transferred to Petri dishes (12 cm diameter) with a minimum amount of water and placed in the center of the irradiation chamber to minimize dose variation. Calibration of the radiation source met the requirements of French and international norms: NF M 61002, ISO 1677 ISO 2919, NF ISO 9978, ANS N542. Time of irradiation for the different doses was calculated based on the decay table of the isotopic source routinely corrected.