Experimental cell-cycle analysis

In this work a primary culture of IMR90 human fibroblast cells (ATCC CCL-186, USA) originated from lung tissue is used. Cells were cultured in complete medium (EMEM supplemented with 12.5% of FBS, 1% of L-glutamine 2 mM and 1%of NEAA) at 37∘C and 5% CO2, until confluence was reached at 80-90%. Cells used for the measurements were always between the 8th and the 25th passage. IMR90 cells were plated at a density of 105 per T25 flask (Greiner Bio-One, Germany).

Independent measurements on cellular growth curves were performed in order to estimate the doubling time (TD) of an unirradiated population. 105 cells were seeded per T25 flask, they were cultivated and harvested at the following time points: 6 h, 16 h, 24 h, 48 h, 72 h (the same time points for the following study of the radiation effects). The samples were trypsinized, 10μl of the pellet was placed in a Bürker counting chamber. The number of cells were counted under an inverted microscope. Experiments were repeated in biological triplicate, each of them was performed in technical duplicate. The data were fitted to the following exponential function via non-linear least squares methods: fit(t)=N0exp(γt), where N0 is the number of cells at t=0, and γ is the growth rate.

Irradiations were performed at the Radiotherapy facility of IRCCS Istituti Clinici Maugeri (Pavia, Italy), with a 6 MV linear accelerator Clinac (Varian, USA), regularly used for radiotherapy treatments. Cell samples were exposed to X-rays at two different doses of 2 Gy and 5 Gy, with a dose rate of 3 Gy/min. Irradiations were performed as previously described in detail²¹. Control samples, so-called “sham” samples, underwent the same environmental stress as the irradiated samples, except for exposure to radiation. After irradiation samples were transferred in the incubator at 37∘C with 5% CO2 level. At each time point (6 h, 16 h, 24 h, 48 h, 72 h) cells were treated for flow-cytometry measurements, as described in the next section. Experiments were repeated in biological triplicate, each of them was performed in technical duplicate.

After irradiation, cells were incubated with 2μg/μl EdU (Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit, Invitrogen, USA) for 1 h, then fixed following the kit manufacture instructions with minor changes, to discriminate S-phase. M-phase was discriminated with the phospho-Histone H3 (Ser-10) primary antibody (1:25 in PBS with 1% of NGS, Cell Signaling Technology, USA [RRID:AB_331748]) and the secondary anti-mouse IgG antibody (1:500, Alexa Fluor 555 Conjugate, Cell Signaling Technology, USA [RRID:AB_1904022]). FxCycle Violet dye (4’,6-diamidino-2-phenylindole, dihydrochloride) was used to measure the total DNA content, following manufacture instructions. The Attune NxT Acoustic Focusing flow cytometer (Thermo Fisher Scientific, USA) employed for these experiments is equipped with two lasers emitting, respectively, in the blue (488 nm, 50 mW) and in the violet (405 nm, 50 mW).

The full gating strategy to discriminate the four cell-cycle phases is as follows: after the identification of the cell population and of singlets (Fig. ​(Fig.2a,b,2a,b, respectively), the characteristic cell-cycle spectrum, as measured with FxCycle Violet, (Fig. ​(Fig.2c)2c) is analyzed. A further gate is applied, to select the whole spectrum where the signal of FxCycle Violet is proportional to the amount of DNA: this has the typical form of two Gaussians, the second with a mean value equal to the double of the first peak, and a plateau in between. These three distributions represent respectively cells in G1-phase, G2/M-phase and S-phase. The cell-cycle spectrum in VL1-A channel alone cannot provide a quantitative discrimination of the four phases, so stainings with EdU and pH3 antibody were introduced. In the bi-parametric plane BL1-A versus VL1-A (EdU vs. FxCycle Violet, Fig. ​Fig.2d)2d) there are three distinct clouds of events: the cloud in the bottom left represents cells in G1-phase, that have a low signal of FxCycle Violet and low signal of EdU. The events in the bottom right part of the graph represent cells in G2 or M-phase, while the horseshoe-like subset with a high signal of EdU represents cells in S-phase. The events collected in the gate “G2-M” are visualized in the VL1-A versus BL2-A plane (pH3 vs. FxCycle Violet): here events with a double positive fluorescence correspond to cells in M-phase, that have a double content of DNA and a high content of phosphorylated H3 (Fig. ​(Fig.22e).

Data acquisition was performed with the Attune NxT software. Data analysis was carried out with FlowJo, a specialised software used for flow-cytometry applications. As a result of the gating procedure described above, we obtained cell numbers in each of the four cell-cycle phases. Data were then expressed in terms of percentages with respect to the whole cell population (hence normalized to the sum of cells in all gates used to identify cell-cycle phases). Data are given as average between both biological (3) and technical (2) replicates, and all experimental uncertainties reported are intended as standard error of the mean.