The DUB siRNA library (ON-TARGETplus), USP9X siRNA pool and individual siRNA targeting USP9X (USP9X_6 5′-AGAAAUCGCUGGUAUAAAU-3′ and USP9X_8 5′-GUACGACGAUGUAUUCUCA-3′) were from Horizon Discovery (Cambridge, UK). The non-targeting control siRNA (AllStars Negative Control siRNA) was from Qiagen (Manchester, UK). The following antibodies were used: PARP-1 (sc-53643) and Mcl-1 (sc-819; both Santa Cruz Biotechnology, Heidelberg, Germany), γH2AX (05-636; Merck-Millipore, Watford, UK), USP9X (A301-350A) and 53BP1 (A300-272A; both Bethyl Labs, Montgomery, AL), LC3B (2775) and β-Galactosidase (9860; both Cell Signaling Technology, London, UK), Pericentrin (ab4448; Abcam, Cambridge, UK), CEP55 (23891-1-AP) and CEP131 (25735-1-AP; both Proteintech, Manchester, UK) and tubulin (T6199; Sigma-Aldrich, Gillingham, UK). Goat anti-mouse Alexa Fluor 555 (A21422) or goat anti-rabbit Alexa Fluor 488 (A11008) secondary antibodies for immunofluorescence were from Life Technologies (Paisley, UK).

Oropharyngeal squamous cell carcinoma cells (UMSCC74A) were kindly provided by Prof T. Carey, University of Michigan, USA. HeLa and UMSCC74A cells were routinely cultured as monolayers in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 1× penicillin-streptomycin and 1× non-essential amino acids. siRNA knockdowns were performed for 48 h using Lipofectamine RNAiMAX (Life Technologies, Paisley, UK). Irradiation sources are as previously described (6). In brief, cells were exposed to either low-LET X-rays (100 kV; CellRad X-ray irradiator, Faxitron Bioptics, Tucson, USA; dose rate of ~3 Gy/min), or with a horizontal, passive-scattered proton beam line of 60 MeV maximal energy at the Clatterbridge Cancer Centre. These low-energy X-rays will generate higher LET photoelectrons and therefore a raised RBE when compared to higher energy photon sources (25, 26). For low-LET proton irradiations, cells were irradiated directly by a ~1 keV/µm pristine beam of 58 MeV effective energy (dose rate of ~5 Gy/min). For high-LET proton irradiations, a modulator was utilized to generate a 27-mm spread-out Bragg peak and a 24.4-mm absorber was used to position the cells at the distal edge, corresponding to a mean proton energy of 11 MeV at a dose averaged LET of 12 keV/µm (dose rate of ~5 Gy/min). The LET was an estimation based on previous work utilizing the Clatterbridge beam (27) and considering the proton stopping power at this range. RBE values of 1.67 ± 0.14 (Hela) and 1.85 ± 0.15 (UMSCC74A), comparing high-LET versus low-LET protons, have been previously determined (6). High-LET α-particle irradiations (3.26 MeV, 121 keV/µm) were performed on cells grown on Mylar sealed on glass cylinders using a ²³⁸Pu irradiator at the CRUK/MRC Oxford Institute for Radiation Oncology, as previously described (28, 29). Assuming a typical cross-sectional nuclear area of an attached Hela and UMSCC74A cell of ~150 µm², these irradiations correspond to a mean of ~940, 78, and 7.7 nuclear traversals per Gy for low-LET protons, high-LET protons, and α-particle irradiations, respectively (29), with the number following a Poisson distribution and dependent on the natural variation in nuclear cross-sectional area with the cell cycle.

Clonogenic assays were performed as recently described (6, 7). In brief, following siRNA treatments, cells were irradiated in 35-mm dishes, harvested and a defined number seeded in triplicate into 6-well plates. Plating efficiencies for the untreated cells were as follows: HeLa (~40%), UMSCC74A (~10%). Plating efficiencies for USP9X siRNA-treated cells were as follows: HeLa (~30%), UMSCC74A (~4%). Increasing cell numbers were plated for increasing IR doses to allow for reductions in plating efficiencies. Colonies were allowed to grow for 7 to 10 days prior to fixing and staining with 6% glutaraldehyde and 0.5% crystal violet for 30 min. Dishes were washed, left to air dry overnight and colonies counted using the GelCount colony analyzer (Oxford Optronics, Oxford, UK). Colony counting settings were optimized for both cell lines, based on inclusion of distinct colonies of specific size and intensity, although the same settings were used across the various treatments. A colony is defined as containing 50 or more cells. Relative colony formation (surviving fraction) was expressed as colonies per treatment level versus colonies that appeared in the untreated control. Results were accumulated from at least three independent biological experiments, apart from the siRNA screen which was from a single experiment (but containing triplicate samples).

For cell cycle analysis, cells were trypsinized, washed twice with ice-cold PBS (100 × g for 5 min at 4°C), fixed with ice cold 70% ethanol and kept at 4°C until analysis. Fixed cells were centrifuged (200 × g for 5 min at 4°C), washed with PBS containing 0.05% Tween-20, and then resuspended in PBS containing 0.05% Tween-20, 10 µg/ml propidium iodide and 0.1 mg/ml RNase A for 1 h at room temperature. Analysis was performed using the Attune NxT Flow Cytometer (Life Technologies, Paisley, UK). Whole-cell extracts were prepared, separated by SDS-PAGE electrophoresis, and analyzed by quantitative immunoblotting using the Odyssey image analysis system (Li-cor Biosciences, Cambridge, UK), as previously described (30, 31).

Detection of CDD was achieved using enzyme treatment of DNA originally described by Sutherland et al. (32, 33), but which has been employed in development of the enzyme-modified neutral comet assay, as recently described (34). In brief, cells were trypsinized, diluted to 1 × 10⁵ cells/ml and 250-µl aliquots of the cell suspension placed into the wells of a 24-well plate, which was placed on ice. Cells were irradiated (4 Gy) and embedded on a microscope slide in low melting agarose (Bio-Rad, Hemel Hempstead, UK). The slides were incubated for up to 4 h at 37°C in a humidified chamber to allow for DNA repair, prior to cell lysis in buffer containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl pH 10.5, 1% N-lauroylsarcosine, 1% DMSO, and 1% (v/v) Triton X-100. Slides were washed three times with enzyme reaction buffer (40 mM HEPES-KOH, 100 mM KCl, 0.5 mM EDTA and 0.2 mg/ml BSA, pH 8.0), and then incubated with either buffer alone (mock-treated; revealing levels of DNA DSBs) or with buffer containing 5 pmol OGG1, 6 pmol NTH1, and 0.6 pmol APE1 (enzyme-treated; revealing levels of DNA DSBs plus CDD) for 1 h at 37°C in a humidified chamber. Following treatment, slides were placed in cold electrophoresis buffer [1 × TBE buffer (pH 8.3)] in the dark for 25 min to allow the DNA to unwind, prior to electrophoresis at 25 V, ~20 mA for 25 min. Slides were washed three times with 1× PBS before allowing to dry overnight. Slides were rehydrated for 30 min in water (pH 8.0), stained for 30 min with SYBR Gold (Life Technologies, Paisley, UK), diluted 1:10,000 in water (pH 8.0), and again dried overnight. Cells (50 per slide, in duplicate) were analyzed from the dried slides using the Komet 6.0 image analysis software (Andor Technology, Belfast, Northern Ireland) and % tail DNA values averaged from at least three independent biological experiments.

Measurement of DNA repair protein foci (γH2AX and 53BP1) was examined as previously described (30). In brief, cells were grown on 13-mm coverslips until ~70% to 80% confluent, irradiated at 4 Gy and incubated for up to 8 h in 5% CO₂ at 37°C to allow for DNA repair. Cells were washed with PBS at room temperature for 5 min, before being fixed using 4% paraformaldehyde for 10 min. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min, washed three times with 0.1% Tween-20 for 10 min, and blocked to avoid non-specific staining via incubation with 2% BSA for 30 min at room temperature on a rocking platform. γH2AX or 53BP1 antibodies in 2% BSA were subsequently added and coverslips incubated overnight at 4°C. Following three washes with PBS, coverslips were incubated with either goat anti-mouse Alexa Fluor 555 or goat anti-rabbit Alexa Fluor 488 secondary antibodies in 2% BSA for 1 h at room temperature in the dark. Finally, samples were washed with PBS for 10 min on a rocking platform and mounted on a microscope slide using Fluoroshield containing DAPI (Sigma-Aldrich, Gillingham, UK). Cells were examined using an Olympus BX61 fluorescent microscope with a Photometrics CoolSNAP HQ2 CCD camera. MicroManager software was used to capture images (~20 images/cell line/antibody). Similarly, cells were fixed on coverslips post-irradiation (4 Gy) and incubation (48 h), but then stained using Pericentrin antibodies prior to incubation with AlexaFluor488 secondary antibodies, and pericentrin-rich foci scored using fluorescence microscopy. Analysis of senescence was carried out using the β-Galactosidase Staining kit (Cell Signaling Technology, London, UK) and where hydrogen peroxide (100 µM for 2 h) was used as a positive control.

All experiments (unless defined above) were performed in at least triplicate as separate independent, biological experiments. Statistical analysis of clonogenic survival data was performed using a one-way ANOVA and comparing surviving fractions at 2 Gy (SF2) or 4 Gy (SF4). Statistical analysis of DNA damage quantified through neutral comet assays and pericentrin-rich foci through immunofluorescence staining was performed using either a one-sample or two-sample t test.

In our recent work, we performed an siRNA screen of 84 DUBs analyzing the survival of HeLa cells after a single dose of IR, with a particular focus on high-LET IR, which generates increased levels and complexity of CDD consisting of two or more DNA lesions within one or two helical turns of the DNA (7). We have now further interrogated these data, which demonstrates the wide range of changes in HeLa cell survival in response to a single dose of high-LET α-particles (121 keV/µm) versus low-LET X-rays following individual DUB knockdowns ( Figure 1A ). Similarly, a variability in cell survival in the absence of the DUBs is observed in response to relatively high-LET protons (12 keV/µm) generated at the Bragg peak distal edge, versus low-LET protons (1 keV/µm) at the entrance dose of a pristine beam ( Figure 1B ). Survival was normalized to the irradiated mock-treated control sample (generating ~40% cell survival), which was set to 1.0, and the data subsequently plotted (on a log₂ scale). The top 10 DUB candidates reducing cell survival in response to high-LET α-particles ( Table 1 ) and relatively high-LET protons ( Table 2 ), in addition to their impact on survival following low-LET protons and X-rays, are shown. We focused our attention on candidate enzymes whose depletion led to a specific reduction in cell survival following high-LET radiation. This led us to identify that depletion of USP9X caused no reduction in cell survival after low-LET X-ray or proton irradiation, whereas survival after high-LET α-particles and protons was reduced by ~20% and ~40%, respectively ( Tables 1 , 2 ).

Following siRNA screening, we aimed to validate that depletion of USP9X caused a decrease in cell survival following high-LET radiation, but had no impact in response to low-LET protons, in both HeLa cells and also cells derived from head and neck squamous cell carcinoma. In response to USP9X siRNA (pool of 4 siRNAs utilized in the screen), there was no impact on cell survival following a dose titration of low-LET X-rays ( Figures 2A, B ) or low-LET protons ( Figures 2E, F ) in comparison to non-targeting (NT) control siRNA-treated cells. In fact, depletion of USP9X appeared to cause a mild increase in radioresistance following low-LET protons. However, in contrast to low-LET radiation, USP9X siRNA led to reduced survival in response to high-LET α-particles ( Figures 2C, D ), and more dramatically in response to relatively high-LET protons ( Figures 2G, H ). To further validate that our observations were specific, we utilized two single siRNA sequences (USP9X_6 and USP9X_8) that were demonstrated to be effective in suppressing USP9X protein levels in both HeLa ( Figure 3A ) and UMSCC74A oropharyngeal squamous cell carcinoma cells ( Figure 3B ). We focused on the impact of protons, particularly as PBT is employed clinically for the treatment of tumors, including head and neck squamous cell carcinoma. We confirmed that targeting USP9X had no effect on the survival of HeLa cells ( Figures 3C, D ) or UMSCC74 cells ( Figures 3G, H ) following low-LET protons, in comparison to NT control siRNA-treated cells. However, significantly reduced survival of HeLa ( Figures 3E, F ) and UMSCC74A ( Figures 3I, J ) cells was observed following USP9X depletion in response to high-LET protons, compared with NT control siRNA cells. These data confirmed the importance of USP9X in maintaining cell survival following treatment with relatively high-LET protons.

Given our demonstration that USP9X is required for maintaining cell survival specifically in response to relatively high-LET protons, we sought to define its mechanism of action. In the first instance, and given the propensity for high-LET radiation to cause increases in the levels of CDD, we utilized an enzyme-modified neutral comet assay (34) to monitor the levels and kinetics of repair of CDD. This assay also has the added advantage of revealing the levels and repair of DNA DSBs (in the absence of modifying enzymes). We demonstrate that DNA DSB levels are not significantly different in USP9X siRNA versus NT control siRNA-treated cells in response to relatively high-LET protons at any time points (0-4 h) post-irradiation ( Figure 4A ; compare black and red, and Figure 4B ). We were able to confirm that relatively high-LET protons caused a significant increase in the levels of CDD in NT control siRNA-treated cells immediately post-irradiation, followed by significantly slower kinetics of repair from 1 to 4 h post-irradiation ( Figure 4A ; compare black and grey bars, and Figure 4B ), consistent with our previously published data (6, 7). Following depletion of USP9X, we observed that the efficiency of CDD repair in comparison to NT control siRNA-treated cells was not significantly different, at least at 1 to 4 h post-irradiation ( Figure 4A ; compare grey and orange bars, and Figure 4B ). Data, demonstrating a lack of effect of USP9X depletion on the repair of DNA DSBs induced by relatively high-LET protons, were supported by an absence of any differences in the induction and resolution of γH2AX and 53BP1 foci, as surrogate markers, up to 8 h post-irradiation relative to NT control siRNA-treated cells ( Figures 4C, D ). We also analyzed the progression of cells through the cell cycle post-irradiation. Similarly, this revealed no significant differences in G2/M accumulation in USP9X siRNA-treated cells either before or after high-LET proton irradiation compared to NT control siRNA-treated cells ( Figure 4E ), or in the overall cell cycle profiles ( Figure 4G ). Similar numbers of cells in G2/M phase ( Figure 4F ) and in cell cycle profiles ( Figure 4H ) were also observed in USP9X-depleted and NT control siRNA-treated cells in response to low-LET protons. These data suggest that there is no significantly different checkpoint activation or cell cycle arrest in USP9X-depleted cells following either relatively high- or low-LET protons.

To examine the potential impact of USP9X on triggering apoptosis post-irradiation with relatively high-LET protons, we analyzed the cellular protein levels of PARP-1 (including cleaved PARP-1) and Mcl-1. However, we did not observe any differences in these proteins 1 to 24 h post-irradiation, nor in the accumulation of cleaved PARP-1, in USP9X siRNA-treated cells compared to NT control siRNA-treated cells ( Figure 5A ), suggesting that apoptosis does not play a role in the differential cellular response. We also compared the expression of LC3B ( Figure 5B ; red text refers to time following relatively high-LET protons) and β-Galactosidase by both microscopy ( Figure 5C ; utilizing H2O2 as a positive control) and immunoblotting ( Figure 5D ), as markers of autophagy and senescence, respectively. Similarly, we observed no obvious differences in LC3B or β-galactosidase levels between USP9X-depleted and NT control siRNA-treated cells post-irradiation with either relatively high- or low-LET protons, suggesting that neither autophagy nor senescence are responsible for decreased cell survival specifically following relatively high-LET protons.

Since recent data have shown that USP9X is an integral component of centrosomal protein maintenance, we analyzed the impact of USP9X depletion on stabilization of centrosomal proteins. We observed that an absence of USP9X alone in HeLa cells caused reduced levels of the centrosome proteins CEP55 (~60%) and CEP131 (~40%) in comparison to NT control siRNA-treated cells ( Figure 6A ; compare lanes 1 and 4). Reduced levels of CEP55 and CEP131 (by ~50–60%) were maintained in USP9X-depleted cells 24 h post-irradiation with both low- and high-LET protons ( Figure 6A ; compare lanes 2–3 and 5–6). Similar observations were seen in UMSCC74A cells, whereby CEP55 and CEP131 protein levels were reduced by 50% and 30%, respectively, in USP9X-depleted cells ( Figure 6B ; compare lanes 1 and 4). CEP55 and CEP131 protein levels were further reduced (by ~60–90%) in USP9X-depleted cells compared to NT control siRNA-treated cells 24 h post-irradiation with both low- and high-LET protons ( Figure 6B ; compare lanes 2–3 and 5–6). In addition to reduced centrosome protein stability, we discovered a ~2.6- to 2.9-fold increase in pericentrin-rich foci in NT control siRNA-treated HeLa cells 48 h post-irradiation with both low and high LET protons ( Figures 6C, D ). However, we observed that there was an additional ~1.9-fold statistically significant increase in foci in USP9X-depleted cells specifically in response to high-LET protons, but not following low-LET protons ( Figures 6C, D ). Similarly, in UMSCC74A cells, there was a ~1.8-fold increase in pericentrin-rich foci in NT control siRNA-treated HeLa cells 48 h post-irradiation with both low- and high-LET protons, but this further increased by ~2.2-fold in USP9X-depleted cells ( Figures 6E, F ). Amplification of pericentrin-foci was consequently ~6.7-fold (HeLa) and ~4.9-fold (UMSCC74A) higher in USP9X-depleted cells irradiated with high-LET protons compared with unirradiated cells.

To recapitulate the impact of USP9X in controlling sensitivity of cells to high-LET protons mediated via centrosomal protein maintenance, we specifically targeted CEP55 and CEP131 using siRNA. We identified conditions that led to ~50% reduced CEP55 and CEP131 protein levels in HeLa cells ( Figure 7A ; lanes 4 and 8), which mimic those of USP9X depletion ( Figure 6A ). We observed under these conditions that cells do not display any increase in radiosensitivity to low-LET protons ( Figure 7B ), but that there is reduced survival to relatively high-LET protons ( Figure 7C ). Additionally, we discovered that there was an a ~1.4-fold statistically significant increase in pericentrin-rich foci in USP9X-depleted cells versus NT control siRNA-treated cells specifically in response to high-LET protons, but not following low-LET protons ( Figures 7D, E ). This suggests that USP9X is required for maintenance of centrosomal proteins and for controlling pericentrin-rich foci, which is important for cell survival particularly in response to high-LET radiation.