Clinac 600

Any treatment started 48 h after inoculation of cells; all steps, with the exception of irradiation, were carried out at 37 °C and 5% CO₂. For gene expression analysis cells were irradiated with 4 or 10 Gy IR dose at a 1Gy/min dose rate using 6 MeV photons (generated by linear accelerator Clinac 600, Varian), the medium was replaced with a fresh one, and then cells were harvested 4 h after irradiation. For ChIP-Seq analysis, wild-type cells were harvested 2 and 4 h after irradiation at 10 Gy. Additionally, cells were incubated with TNFα cytokine (10 ng/ml) (T0157; Sigma) and harvested after 30 min. of stimulation.

ADF Cells were plated in 25 cm² tissue culture flasks, allowed to attach for 24 h and treated with three different doses of radiation (5,8 and 10 Gy) at room temperature (1.8 Gy/min, 98 cm Source Surface Distance (SSD) by using a 6 MV photon linear accelerator (CLINAC 600 Varian). After 72h, cells were processed using the Flow Cellect Oxidative Stress Characterization Kit. Flow Cellect (FCCH025111, Millipore)
Cells were analyzed on Guava EasyCyte Plus flow cytometer. The median fluorescence intensity (MFI) was calculated: an increase in oxidative stress is detected as a right shift in MFI from untreated sample to the treated sample.

Cell survival was measured by clonogenic assay, according to Franken et al. [57 (link)]. Briefly, on day 0, between 150–20,000 cells, depending on the treatment, were seeded in triplicate in six-well plates and incubated under regular culture conditions. On day 1, cells were treated with vemurafenib, PRIMA-1^Met, or both, and subsequently incubated under regular culture conditions. On day 2, cells were irradiated. Cells were irradiated in a 6-MV beam from a Clinac 600 linear accelerator (Varian Medical Systems, Palo Alto, CA, USA). The collimator opening was set to 40 × 40 cm², which gives the possibility of irradiating several plates in one batch. In order to achieve a good dose homogeneity and electronic equilibrium, a 6-mm thick polystyrene build-up was put on top of the plates. Plates were placed on a 5-cm thick polystyrene phantom for adequate backscattering conditions. The dose rate was set to 4 Gy per minute, so that the full experiment took less than 20 minutes and can be easily accommodated in a clinically used linear accelerator. On day 14, cells were fixed and stained with 6% glutaraldehyde and 0.05% crystal violet. Colonies of at least 50 cells were counted. The survival fraction was calculated either relative to untreated samples.

All chemicals were purchased from Sigma–Aldrich (Poznań, Poland). Irradiation experiments were performed using X-rays (Clinac 600, 6 MV, Varian) with dose of 2 Gy (1 Gy/min). Monomeric bovine serum albumin (BSA) was prepared from Cohn fraction V BSA suitable for radioimmunoassay (Sigma–Aldrich), as described [17 ].

Cells were plated in 100-mm Ø tissue culture dishes, allowed to attach for 24 h, and treated with different doses of radiation (5, 8 and 10 Gy) at room temperature (1.8 Gy/min, 98 cm Source Surface Distance (SSD) by using a 6 MV photon linear accelerator (CLINAC 600 Varian) [29 (link)].

Cells were irradiated in a 6MV photon beam at a dose rate of about 4Gy/minon a Clinac 600 linear accelerator (Varian Medical Systems, Palo Alto, CA, USA). The collimator opening was set to 40 × 40 cm², which allows the possibility of irradiating several plates at the same time. For adequate backscattering conditions, plates were placed on a 5 cm-thick polystyrene phantom. To achieve dose homogeneity and ensure electronic equilibrium, a 6 mm-thick polystyrene build-up slab covers the top of the plates.