## 2 Materials and Methods
### Irradiation System and Dosimetry  
The proton beam is produced by sputtering graphite cathodes; the extracted \({}^{12}\)CH molecules are dissociated in passing through a gas stripper (Ar) and subsequently the hydrogen ions are selected using the appropriate values for the analysis magnets [25]. Before dosimetry and irradiation, the beam passes through a 3-mm-diameter collimator mounted on the target-holder present at the scattering chamber centre. The FC, placed at the end of the 0\({}^{\circ}\) forward channel, is connected to a current digitizer for beam focusing and control; the overall beam alignment is obtained by using both a telescopic alignment system and the proton beam current, with an external micrometric device additionally used for refining the collimator alignment. All current intensities were obtained as an average of at least ten samples. A couple of SSBDs and several SSTDs were used for dosimetry. The FAst Intercrate Readout (FAIR) data acquisition system was used to collect data from the silicon detectors as described in [26].  
Figure 1: Scheme of the: radiation biophysics beamline scattering chamber; the cell sample holder assembly; and the knife-edge collimator.  
The different detectors can be mounted on all outputs of the scattering chamber or on the output of the cell channel. In particular, beam dosimetry is performed measuring the proton energy and fluence with the two SSBDs, one placed on the channel at 60\({}^{\circ}\) (SSBD\({}_{60^{\circ}}\)) and the other at 15\({}^{\circ}\) (SSBD\({}_{15^{\circ}}\)) and in the same position of the sample to be irradiated. They are encapsulated within an insulating support or connected to the cell channel body using an insulating O-ring, respectively (Figure 2a). The relationship between the counts provided by the two SSBDs is as follows:  
\[\text{R}_{60^{\circ}/15^{\circ}}=\text{Count}\;\text{SSBD}_{60^{\circ}}/\text{ Count}\;\text{SSBD}_{15^{\circ}} \tag{1}\]  
This is determined before each experimental run. During irradiation of the samples, proton fluence and energy are measured by the SSDB\({}_{60^{\circ}}\).  
Particle fluence and beam spatial uniformity, before and after irradiation, can be also measured and monitored by means of the CR-39 detectors placed at the same position of the sample-holder (SSTD\({}_{15^{\circ}}\)) using a special centering tool (Figure 2b).  
After exposure to protons, SSTDs are scored by manually counting the number of tracks, made visible through an etching process [27; 28] of 90 min in NaOH 6N solution at 80\({}^{\circ}\)C, on at least 20 random fields observed by a light microscope using a 32X objective, corresponding to a field area of \(1.25\cdot 10^{-4}\) cm\({}^{2}\). Using the obtained value of the R\({}_{60^{\circ}/15^{\circ}}\) and the count values of the spectrum at 60\({}^{\circ}\), the expected dose on the SSBD\({}_{15^{\circ}}\) or the SSTD\({}_{15^{\circ}}\), and therefore the expected nominal dose given to the cells, can be calculated using the formulas:  
\[\text{Counts}\;\text{SSBD}_{15^{\circ}}\left[\text{s}^{-1}\right]=\text{Counts}\; \text{SSBD}_{60^{\circ}}\left[\text{s}^{-1}\right]/\text{R}_{60^{\circ}/15^{ \circ}} \tag{2}\]  
\[\text{Dose}\;\left[\text{Gy}\right]=1.6\cdot 10^{-9}\cdot\text{LET}\;\left[\text{keV}/ \text{\mu m}\right]\cdot\text{Fluence}\;\text{SSBD}_{15^{\circ}}\left[\text{cm} ^{-2}\right] \tag{3}\]  
where the fluence values are obtained normalizing the number of counts to the areas of the irradiated detector and the LET values are derived from the SRIM library [29]; the numerical factor in Formula (3) include the appropriate conversions between the units of measurement chosen [23; 30]. Fast shutters on each FC allow the irradiation of the samples to be stopped when the desired number of counts is reached on the monitoring SSBD.