Downregulation of ubiquitin inhibits the proliferation and radioresistance of non-small cell lung cancer cells in vitro and in vivo

Radioresistance has been an important factor in restricting efficacy of radiotherapy for non-small cell lung cancer (NSCLC) patients and new approaches to inhibit cancer growth and sensitize irradiation were warranted. Despite the important role of ubiquitin/proteasome system (UPS) during cancer progression and treatment, the expression and biological role of ubiquitin (Ub) in human NSCLC has not been characterized. In this study, we found that ubiquitin was significantly overexpressed in 75 NSCLC tissues, compared to their respective benign tissues by immunohistochemistry (P < 0.0001). Knock-down of ubiquitin by mixed shRNAs targeting its coding genes ubiquitin B (UBB) and ubiquitin C (UBC) suppressed the growth and increased the radiosensitivity in NSCLC H1299 cells. Apoptosis and γ H2AX foci induced by X-ray irradiation were enhanced by knock-down of ubiquitin. Western blot and immunostaining showed that knock-down of ubiquitin decreased the expression and translocation of NF-κB to the nucleus by reduced phospho-IκBα after irradiation. Suppression of ubiquitin decreased the proliferation and radioresistance of H1299 transplanted xenografts in nude mice by promoting apoptosis. Taken together, our results demonstrate the critical role of ubiquitin in NSCLC proliferation and radiosensitivity. Targeting ubiquitin may serve as a potentially important and novel approach for NSCLC prevention and therapy.

molecule (monoubiquitination, mono-Ub) 10 or as chains (polyubiquitination, poly-Ub) 11 . Since ubiquitin plays a key role in essential intracellular functions, the most important function of ubiquitin is believed to be regulation of protein turnover through the UPS.
Radiotherapy plays a critical role in curative management for the inoperable NSCLC patients 12 . Upon irradiation, numerous genes and proteins are involved in shifting the balance between cell survival and death 13 . Cancer cells may acquire resistance to radiation-induced apoptosis by dynamic interplay and regulation of multiple pro-survival factors 14 , which is considered as the major factor that impairs the outcomes of lung cancer patients 15 . Ubiquitin also plays an important role in DNA damage and repair response 16,17 . Recent studies have shown that both monoubiquitin and polyubiquitin modifications have critical roles in the DNA damage response (DDR), including the direct controlling protein expression levels 18,19 . Nuclear factor-kB (NF-kB), an import transcriptional factor, is a typical intrinsic cellular suppressor of apoptosis. After exposure to both low and high doses of ionizing radiation (IR), DNA damageinduced NF-kB promotes the expression of anti-apoptotic genes 20,21 . Therefore, the activated NF-kB has been reported to be a novel regulator of tumor radioresistance, and targeting NF-kB-mediated pathway is likely to contribute to radiosensitizer development 20,21 .
The effect of the whole cellular ubiquitin level on the proliferation and response to ionizing radiation remains to be further explored. Herein, we assess the expression level of ubiquitin in NSCLC tumor specimens and elucidate the effect of ubiquitin-knock down on the proliferation and radiosensitivity of lung cancer cells and its underlying mechanisms.

Results
Ubiquitin expression was significantly upregulated in human lung cancer tissues. To determine the role of ubiquitin in lung cancer development, we first investigated the expression of ubiquitin in 75 paired clinical NSCLC specimens. The demographic information was summarized in Supplementary Table 1. The expression of endogenous ubiquitn between human lung cancer tissues and paired adjacent nontumor tissues was analyzed by IHC staining. As shown in Fig. 1A, results showed that ubiquitin was expressed in both the nuclei and the cytoplasm of lung cancer cells. Among the 75 cases, positive ubiquitin expression was observed in 82.7% (62/75) of tumor tissues compared with 4.0% (3/75) of the paired adjacent non-tumor tissue (P , 0.0001, Fig. 1B and Table 1). The average ubiquitin staining score in tumor tissues (2.26 6 0.60) was significantly higher than that in the paired adjacent normal tissues (0.75 6 0.61). Moreover, stage II-III tumors showed significantly increased expression of ubiquitin compared with stage I tumor samples (Fig. 1C). Altogether, these data indicated that ubiquitin was overexpressed in lung cancer, and its overexpression may be associated with the progression of this malignancy.
To illustrate whether the increased ubiquitin level is attributed to UBB or/and UBC mRNA level, we performed reverse transcriptase-PCR (RT-PCR) to analyze the expression of UBB and UBC genes in 24 paired NSCLC tissues and corresponding normal tissues. As  shown in Fig. 2A, 2B and Supplementary Fig. 1, results revealed that the expression of UBC gene remarkably increased in lung tumor tissues (P 5 0.019), while UBB mRNA did not show statistical significance between the two groups (P 5 0.167). The above results suggested that the increased ubiquitin in lung cancer tissues is likely to be ascribed to UBC transcripts.
Knock-down of ubiquitin inhibited the proliferation of NSCLC in vitro. Ubiquitin mRNA and protein expression level in human lung cancer cell lines (H1299, A549, H460 and H1975) and normal bronchial epithelial cell lines (BEAS-2B and HBE) were analyzed by RT-PCR and Western blot. Results showed that both UBB and UBC transcripts and ubiquitin protein levels were generally more pronounced in human lung cancer cell lines (Supplementary Fig.  2A and 2B). Among the lung cancer cells, H1299 cells exhibited the highest ubiquitin level, possibly due to the UBB transcription. Moreover, the expression of ubiquitin in H1299 cells could be further induced by X-ray irradiation. (Supplementary Fig. 2C and 2D). We next investigate the biological consequences of specific knockdown of ubiquitin in the NSCLC H1299 cells. Four different shRNA vectors were designed to silence the coding gene of UBB and UBC. After transfection with shRNA-NC, shRNA-UBB, shRNA-UBC or shRNA-UBB/UBC for 48 h, RT-PCR was performed to determine their silencing efficacy. The results revealed that shRNA-UBB-4 (shRNA-B4) targeting UBB gene and shRNA-UBC-2 (shRNA-C2) targeting UBC gene showed respective strongest inhibitory effect in H1299 cells ( Fig. 2C and 2D). Western blot further confirmed that mixed transfection of shRNA-B4 and shRNA-C2 (shRNA-B4/C2) showed 76% inhibition of ubiquitin expression, compared with shRNA-NC transfected cells (Fig. 2E). Hence, shRNA-B4, shRNA-C2 and a combination of both was selected to perform the following experiments.
Then we evaluated the effect of ubiquitin silencing on the proliferation and colony formation of H1299 cells. Results revealed that ubiquitin downregulation generated less and smaller plate colonies ( Fig. 3A and 3B). Cell growth monitored by MTT assay showed that knock-down of ubiquitin by shRNA-B4/C2 exhibited 32.9% reduction of cell viability compared with control shRNA-transfected cells (Fig. 3C), while treatment with either shRNA-B4 or shRNA-C2 had modest effects. Importantly, flow cytometry demonstrated that the  percentage of cells in S phase was significantly decreased in ubiquitin knock-down cells, and the population of cells in G0/G1 phase was increased ( Fig. 3D and 3E). Taken together, these results indicated that knock-down of ubiquitin remarkably inhibited cell growth by modulating the cell cycle in H1299 cells.
Knock-down of ubiquitin increased the radiosensitivity in H1299 cells. We next performed clonogenic survival assays to investigate the impact of ubiquitin on radiosensitivity in lung cancer H1299 cells. Cells were transfected with shRNA-NC, shRNA-B4, shRNA-C2 or shRNA-B4/C2 24 h prior to irradiation at 0, 2, 4, 6 and 8 Gy. The main parameters of H1299 cells in dose-survival curves were obtained according to the multi-target single hit model. A dose-dependent radiosensitization on ubquitin-silencing was also observed with a sensitizing enhancement ratio (SER) of 1.29 and 1.45 by shRNA-B4 and shRNA-C2, respectively (Fig. 4A). This moderate radiosensitizing effect might partially due to a decrease of cellular ubiquitin level. Transient ubiquitin knock-down (shRNA-B4/C2) gave rise to a greater radiosensitization. The mean lethal dose (D 0 ) of the radiation alone group and irradiation plus ubiquitin knock-down (shRNA-B4/C2) group were 1.40 and 0.78 Gy, while the quasithreshold dose (Dq) were 1.99 Gy and 1.42 Gy, with SER value increasing to 1.80 (Fig. 4B). Thus, downregulation of ubiquitin significantly sensitized H1299 cells to irradiation.
Knock-down of ubiquitin increased radiation-induced apoptosis and DNA damage in H1299 cells. We then assessed the apoptosis of H1299 cells after knock-down of ubiquitin and/or radiation. The proportion of apoptotic cells was determined by flow cytometry after irradiation. Compared with cells treated with radiation alone, the group in which ubiquitin was knocked down combined with X ray irradiation had a clear increase of apoptosis percentage ( Fig. 5A and 5B). As shown in Fig. 5C, knock-down of ubiquitin downregulated the expression of anti-apoptotic protein Bcl-2, but did not upregulate the expression of pro-apoptotic protein Bax in our study.
Immunofluorescence was subsequently used to examine the formation of c-H2AX foci in the nuclei. The results revealed that the formation of c-H2AX foci after 6 Gy X-ray irradiation was significantly more pronounced by knock-down of ubiquitin ( Fig. 6A and 6B). Western blot analysis confirmed that knock-down of ubiquitin inhibited the clearance of nuclear c-H2AX foci induced by irradiation (Fig. 6C), which is consistent with the results from immunofluorescence staining. Meanwhile, radiation plus ubiquitin knockdown showed no change in cell cycle distribution, indicative the irrelevance of cell cycle distribution ( Supplementary Fig. 3). Taken together, these results revealed that radiation obviously increased apoptosis fraction and DNA damage.
Knock-down of ubiquitin decreased p-AKT and inhibited NF-kB translocation into the nucleus after exposed to X-ray irraddiation. NF-kB has been extensively reported to be implicated in cell radiosensitivity due to their crucial role in radiation response. To further confirm the effect of ubiquitin knock-down on NF-kB activity induced by radiation, we analyzed the expression level and distribution of NF-kB (p65 subunit) in H1299 cells. As shown in Fig. 7A, Western blot analysis showed that the expression levels of p65 in the nuclear extracts of the H1299 cell lines were increased after both ubiquitin knock-down and irradiation, while knock-down of ubiquitin decreased radiation-induced NF-kB (p65) expression in both nucleus and cytoplasm. Result from immunofluorescence also revealed that irradiation exposure induced the translocation of the p65 protein from the cytoplasm into the nucleus in H1299 cells, which was attenuated by ubiquitin knock-down (Fig. 7B). Meanwhile, phosphorylation of IkBa, which mediates the degradation of IkBa and activates NF-kB, was decreased, suggesting that decreased NF-kB expression was attributed to decreased degradation of IkBa (Fig. 7C). Moreover, Western blot also revealed that irradiation plus ubiquitin inhibition decreased Cyclin D1 and phospho-AKT expression (Fig. 7C), suggesting that AKT and Cyclin D1 pathways are also involved in ubiquitin-modulated radiosensitivity. Our results also indicated that knock-down of UBB and UBC gene progressive increased the expression of phospho-AKT, NF-kB and Cyclin D1, and a combination of the both showed the most enhancement.
Knock-down of ubiquitin inhibited tumor cell growth and enhanced radiosensitivity in a mouse xenograft model. For the purpose of examining the inhibitory effect of ubiquitin knock-down on tumor cell growth in vivo, H1299 cells were inoculated subcutaneously into the right posterior flank region of BALB/c nude mice. When tumors reached 150-200 mm 3 in volume, the mice were randomly divided to 4 groups. Either shRNA control (shRNA-NC) or shRNA targeting ubiquitin coding genes was injected intratumorally to the xenografts of nude mice once every five days for four times (Fig. 8D, indicated by the arrows). Mice in each group showed no difference in body weight and no gross pathologic abnormality, indicating no observable toxicity ( Supplementary Fig. 4). As shown in Fig. 8A and 8B, transfection of shRNA-B4/C2 significantly decreased the ubiquitin level of transplantation tumors, compared with the control group (85% reduction, P , 0.05). In comparison, transfection with either shRNA-B4 or shRNA-C2 had modest inhibitory effect. Compared with shRNA-NC group, combined silencing of UBB and UBC (shRNA-B4/C2) inhibited 51.51% of tumor volume (Fig. 8C upper panel and Fig. 8D), which was more effectively than that of shRNA-B4-transfected group (27.12%) or shRNA-C2-transfected group (45.05%). Given that downregulation of the ubiquitin level of H1299 cells decreased the tumor proliferation, the 50% reduction of tumor size is likely to be attributed to the effect of ubiquitin depletion.
The effect of combined treatment with X-ray irradiation plus ubiquitin silencing on the growth of H1299 xenografts was also determined. As shown in Fig. 8E, there is no significant difference in tumor volume between groups before treatment. The tumor volume of mice treated with radiation alone was reduced by 41.22% com-  pared with the control group. Comparatively, H1299 xenografts that received combined treatment of 8 Gy X-ray irradiation and ubiquitin knock-down exhibited much smaller tumors with a volume reduction of 79.10%, compared with mice that received radiation alone (P , 0.05, Fig. 8C lower pannel and 8E, indicated by starlike symbol).
Inhibition of ubiquitin decreased the proliferation and enhanced apoptosis in response to X-ray irradiation in vivo. To characterize the molecular changes in the ubiquitin silenced xenografts, proliferation marker Ki67 and cell apoptosis were determined. As shown in Fig. 9A and 9B, Ki67 positive cells were significantly decreased by 41.60% in H1299 tumors in the combined knockdown of UBB and UBC gene group (P , 0.01). When treated with ubiquitin silencing plus irradiation, the Ki67 positive cells in the xenografts were significantly reduced by 10.03% (P , 0.01), compared with shRNA-NC plus irradiation group. We further validated TUNEL positive cells in the nude mice xenografts treated with ubiquitin silencing and/or irradiation. TUNEL positive cells were significantly increased by 44.80% (P , 0.01) after treatment with the combinational knock-down of UBB and UBC gene, while traetment with ubiquitin silencing and irradiation significantly increased the TUNEL-positive cells by 34.86% (P , 0.05; Fig. 9C and 9D). These data indicate that combined knock-down of ubiquitin coding genes UBB and UBC reduced Ki67 expression and improved its antitumor effect in vivo. Moreover, ubiquitin silencing could enhance the radiosensitivity of H1299 xenografts, which is consistent with the results from in vitro study.

Discussion
The UPS is conservative in all eukaryotes and plays key roles in the regulation of numerous cellular functions. Accumulating evidence shows that elevated level of ubiquitin has been observed in multiple types of cancers [22][23][24][25][26] . Nevertheless, few study illustrated the influence of ubiquitin on cellular function of cancer cells. This study, for the first time to our knowledge, demonstrates the upregulation of ubiquitin in NSCLC tissues. The increased ubiquitin in lung cancer tissues is likely to be ascribed to UBC transcripts. We also showed that knock-down of UBB and UBC that coding ubiquitin resulted in dramatic growth inhibition and radiosensitivity enhancement in NSCLC cells. Moreover, we provide molecular evidences that ubiquitin knock-down weakened the resistance of lung cancer cells to Xray irradiation through apoptosis, DNA damage repair and NF-kB signaling pathways.
In most cancers, tumor cells acquire their malignant capabilities primarily due to their high metabolic state and speedy material metabolism 27 . UPS is involved in busy carrying cellular garbage disposal for recycling. Moreover, cancer cells acquire their malignancy through a number of mutations and extensively reprogrammed pathways, which inevitably generate a variety of cellular stresses 28,29 . Thus, cancer cells must tolerate these increased stresses through stress-supporting systems such as the heat shock response (HSR) and the ubiquitin system to survive 30 . The elevated level of ubiquitin in cancer cells is believed to contribute to the reinforcement of the high metabolic and stress-support system through ubiquitination 30,31 .
Since the approval of clinical use of bortezomib in 2003 by FDA, there have been substantial progresses in developing potential anticancer drugs that target various key molecules in ubiquitin-proteasome pathway 32 . Deubiquitinating enzyme inhibitors and inhibitors of E1, E2 and E3 ligands involved in UPS have been now in clinical or preclinical trial [33][34][35][36] . The 76-amino acid protein tag ubiquitin affects cellular process by regulating protein degradation, activating and inactivating proteins, and modulating protein-protein interactions by the addition of a single ubiquitin molecule (monoubiquitination) or different types of ubiquitin chains (polyubiquitination). Inhibitors or activators of ubiquitin conjugation or deconjugation have been proved to be attractive cancer therapeutic targets 37 . Our study shows that ubiquitin itself can also be used as a novel lung cancer target, which supports the critical role of UPS in cancer progression and treatment.
In our study, inhibition of UBB and UBC could dramatically enhance the phosphorylation of AKT (Fig. 7C) and increase the expression of total NF-kB (p65, Fig. 7C). Moreover, ubiquitin silencing also promoted the translocation of p65 into nucleus. These results are consistent with previous report that UBB knock-down enhances p65 expression in both cytoplasm and nucleus of Hela cells 30 . Proteasome inhibitor MG132 has been reported to stabilize IkBa 38 , which is required for the activation of NF-kB and its translocation. Therefore, the increased NF-kB protein level and nuclear localization by ubiquitin knock-down may be ascribed to the stabilization of IkBa by UPS inhibition. Moreover, the degradation of NF-kB itsself may also be attenuated due to lack of ubiquitin.
Numerous studies have shown that ubiquitin is involved not only in protein degradation, but also in protein modification 39,40 . Upon irradiation, multiple molecules are activated by ubiquitination and initiate DNA repair 6,16 . For example, neither monoubiquitinylation nor polyubiquitinylation of PCNA affects its degradation, whereas blockade of K164 PCNA residue that is normally ubiquitinylated inhibits DNA repair 41 . We show that silencing ubiquitin significantly increased the number of cH2AX, indicative of inhibited DNA repair of H1299 cells exposed to irradiation. NF-kB is a well-established regulator of cell survival, transformation, differentiation, invasion and radiation response 42,43 . By inhibiting the proteasome, bortezomib redistributes the cell cycle and inhibits the transcriptional capability of NF-kB, which modulates cell proliferation and apoptosis 44,45 . Our results suggest that downregulation of ubiquitin decreased the expression of NF-kB and inhibited its translocation to the nucleus by decreased phospho-IkBa after irradiation, which may attenuated NF-kB-mediated radiation response 20,21 . Upon irradiation, ubiquitin silencing may suppress cellular response to radiation-induced DNA damage rather than protein degradation. Therefore, the effect of ubiquitin silencing on NF-kB (p65) expression with and without irradiation may be different. The underlying mechanism warrants in-depth exploration.
Our results revealed that combined inhibition of UBB and UBC was similar to the effect of single UBC silencing (Fig. 7C), indicating that UBC gene represented a more effective radiosensitizing target. Cyclin D1, a NF-kB downstream effector, was inhibited after ubiquitin knock-down. The expression of Cyclin D1 has been implicated in cell survival 46 . Other important mechanisms associated with cancer cell radiosensitivity include apoptosis and cell cycle arrest. Both our in vitro and in vivo studies suggest that a reduced level of ubiquitin is essential to allow H1299 cells to promote apoptosis, which is similar to a previous report of UBB-knock-down in hepatoma and cervical cancer cells 30,47 . In contrast, targeting ubiquitin showed no significant effect on cell cycle distribution after irradiation in our study, which was in discrepancy with proteasome inhibitors that modulating cell cycle arrest 48,49 . Whether other mechanisms are involved in the enhancement of radiosensitivity of UBB and UBC gene in lung cancer cells also warrants further investigation.
Taken together, our results demonstrate that ubiquitin is overexpressed in NSCLC and that downregulation of ubiquitin inhibits the proliferation and enhances the efficacy of radiotherapy via multiple pathways in NSCLC cells both in vitro and in vivo. Targeting  ubiquitin may serve as a potentially important and novel approach for human NSCLC prevention and therapy.

Methods
Clinical specimens. Fresh lung cancer tissues were collected between 2010 and 2012 from Suzhou Municipal Hospital (Suzhou, China) and Changzhou Tumor Hospital affiliated to Medical School of Soochow University (Changzhou, China). All patients were diagnosed with histologically confirmed NSCLC, and the histological features of the specimens were evaluated by two senior pathologists according to the classification criteria from the World Health Organization (WHO) 50 . Signed informed consent for sample collection was obtained from all patients. Each lung cancer tissue sample has matched adjacent non-tumor lung tissue removed during the same surgery. For PCR analysis, specimens were snap frozen in liquid nitrogen and then stored at 280uC until use. For immunohistochemical analysis, samples were fixed with formalin and embedded with paraffin. These tissue samples were then processed into tissue arrays by Suzhou Xinxing Biotech (Suzhou, China). All experiments were performed in accordance with the relevant guidelines and regulations of Soochow University. And this study was approved by the Ethical Committee of Soochow University.
Immunohistochemical staining. Immunohistochemical (IHC) staining were performed to detect the ubiquitin expression level in tissues from lung cancer patients and samples from H1299 xenografts of nude mice were used for IHC staining of Ki67.
The paraffin-embedded sections were stained with antibodies against ubiquitin or Ki67 antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at dilution 151000 as the previously reported method 51 . Ubiquitin IHC staining was based on the proportion of cell staining and scored from 0 to 3 criteria as described previously 52 . An overall score of ,2 was defined as negative, while a score $ 2 was defined as positive. The slides were analyzed by standard light microscopy. Negative controls were stained with IgG as primary antibody.
Reverse transcriptase-PCR. Total RNA was extracted using TRIzol reagent (Tiangen, Beijing, China). RNA was reverse transcribed using a reverse transcription kit (Thermo-Fisher, Waltham, MA) under 42uC for 60 min and 70uC for 5 min. The expression level of UBB and UBC gene in each sample was measured by RT-PCR. GAPDH was used as an endogenous standard. The primer sequences were listed in Supplementary Table 3.
Cell cultures and irradiation. The human normal bronchial epithelial cell lines (BEAS-2B and HBE) and the human lung cancer cell lines (H1299, A549, H460 and H1975) were maintained in DMEM supplemented with 10% FBS and antibiotics (100 units/ml penicillin G, 100 units/ml streptomycin sulfate; Gibco, Grand Island, NY) at 37uC in 5% CO2. Cells were exposed to a single dose of X-rays using the linear accelerator (RadSource, Suwanee, GA) at a dose rate of 1.15 Gy/min. shRNA construction and transient transfection. Short hairpin RNAs (shRNAs) targeting ubiquitin coding genes UBB and UBC were designed and constructed by Cell proliferation assays. The effect of ubiquitin on cell viability was monitored by the MTT colorimetric assay. In brief, after an overnight incubation, H1299 cells (2 3 10 3 cells/well) were transfected with shRNA-UBB, shRNA-UBC or shRNA-UBB/ UBC mixture (with the same total quantity) in 96-well plates, and were incubated for another 24 or 48 h. Then the cells were incubated with 10 mL of 5 mg/mL MTT for 5 h. Then, the supernatant was removed and each well was added with 150 ml of dimethylsulfoxide (DMSO) to dissolve the formazan for 10 minutes by vibration. The optical density was measured at 492 nm with a microplate reader (Bio-Rad, Hercules, CA). The viability index was calculated as the experimental OD value/the control OD value (which were arbitrarily assigned as a viability of 100%). Three independent experiments were performed in quadruplicate.
Cell cycle progression analysis. The distribution of H1299 cell cycle was assessed by flow cytometry 24 h after shRNA transfection. Cells were harvested by trypsin digestion, pelleted by centrifugation, washed with ice-cold PBS, then fixed with 70% cold ethanol overnight. The staining solution containing propidium iodide (50 mg/ ml) (Sigma-Aldrich, St. Louis, MO) and DNAse-free RNAse (20 mg/ml) were added 30 min before detection. The fraction of the population in each phase of the cell cycle was determined as a function of the DNA content using a coulter flow cytometry (Beckman-Coulter, Brea, CA).
Clonogenic assay. For standard clonogenic assays, the cells were treated with shRNA transfection for 5 h. and then cells were re-suspended and seeded into six-well plates at 200-6,000 cells/well depending on the dose of radiation. The cells were irradiated with 0, 2, 4, 6 or 8 Gy X-ray irradiation by linear accelerators at a dose rate of 1.15 Gy/ min. After the irradiation, the cells were grown from 7-10 days to allow for colony formation and were subsequently fixed and stained using crystal violet. Colonies consisting of 50 or more cells were counted as a clone. The radiation sensitivity enhancement ratio (SER) was measured according to the multi-target single hit model.
Apoptosis analysis. Cells were transfected with shRNA 24 h prior to treatment with sham or 6 Gy X-ray irradiation. Apoptosis was measured using propidium iodide (PI)/Annexin-V double staining following manufacturer's instructions (BD Biosciences, San Jose, CA). Cells were harvested 48 h after irradiation and apoptotic fractions were measured using flow cytometry (Beckman, USA). The Annexin-V1/ PI-cells are early in the apoptotic process, the Annexin-V1/PI 1 cells indicating late apoptosis. The percentage of both kinds of cells was counted.
The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to detect the apoptosis of xenograft tissue samples. 5 mm sections from H1299 xenografts were immersed in xylene and hydrated in decreasing concentrations of ethanol, and the TUNEL assay was performed following manufacturer's instructions (Keygen, Nanjing, China). Ten random fields from 3 slides per group were counted. TUNEL-positive brown nuclei within tissues were counted. Data were expressed as the percentage of apoptotic cells per field.
Protein extraction and Western blot. H1299 cells (1 3 10 5 cells/dish) in 6-well plates were grown in DMEM containing10% FBS to a 70% confluent. The cells were trasfected with shRNA for 24 h and then with sham or 6 Gy X-ray irradiation. Cytosolic and nuclear protein was extracted 24 h after irradiation using the cytosolic and nuclear protein extract kit (Beyotime, Nantong, China). In brief, cells were harvested in lysis buffer with 1 mM phenylmethyl-sulfonyl fluoride (PMSF). The cells were allowed to swell on ice for 15 min. The homogenates were centrifuged for 5 min at 12,000 3 g and the supernatant was used as the cytosolic extract. The nuclear pellet was re-suspended in cold extraction buffer and the samples were centrifuged at 15,000 3 g for 30 min. The obtained supernatant was used as the nuclear extract.
Proteins were separated by sodium dodecyl sulfatepoly-acrylamide gel electrophoresis (SDS-PAGE) through 10% gel and transferred onto PVDF membranes. The gels have been run and transferred under the same experimental conditions. The membranes were blocked with 5% non-fat dried milk in TBS containing 1% Tween-20 (TBST) for 2 h at the room temperature, followed by incubation with the appropriate Bax, Bcl-2, Cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), NF-kB, AKT, phospho-AKT and phospho-IkBa primary antibodies (Epitomics, Burlingame, CA) at a 152,00-151,0000 dilution for 2 h, and either horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies for 1 h. b-actin, a-Tubulin and LaminB (all from Beyotime, Nantong, China) were served as the loading controls. Antibody-bound proteins were detected using an ECL Stable Peroxide solution (PointBio, Shanghai, China). All protein bands were visualized by a FluroChem MI imaging system (Alpha Innotech Santa Clara, CA) under the room temperature.
Immunofluorescence. Cells were were transfected with shRNA 24 h before exposed to irradiation. After irradiation, the cells were fixed with 4% paraformaldehyde for 10 min, treated with 1% Triton X-100 for 10 min, and then incubated with blocking serum for 1 h at room temperature. After washing with PBS, samples were incubated with a rabbit monoclonal against cH2AX or NF-kB antibody (both from Epitomics, Burlingame, CA) over night at 4uC, followed by FITC-conjugated goat-anti-rabbit secondary antibody (15200) for 2 h. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO). The cells were observed using an Olympus fluorescence microscope (Olympus, Tokyo, Japan). cH2AX foci were counted in each cell. At least 100 cells were counted from 10 randomly chosen fields of view.
Xenograft studies of nude mice. 4-week-old BALB/c athymic nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were maintained under standard laboratory conditions on a 12-h light-dark cycle and given access to sterilized food and water ad libitum under specific pathogenfree environment. For the subcutaneous injection, H1299 cells (4 3 10 6 ) were suspended in 100 ml PBS and then inoculated subcutaneously into the right posterior flank region of BALB/c nude mice. Each group consisted of five nude mice. Twodimensional measurements were taken with an electronic caliper, and the tumor volume in mm 3 was calculated using the formula: volume 5 a 3 b 2 3 0.52 53 , where a is the longest diameter, b is the shortest diameter. When a tumor reached 150-200 mm 3 in volume, the mice were random divided into 4 groups and injected intratumor respectively with shRNA control (shRNA-NC), shRNA-UBB, shRNA-UBC or shRNA-UBB/UBC together with transfection reagents. Tumors were irradiated by a tailor-made irradiator to a signal dose of 8 Gy X-rays from linear accelerators (Varian, Palo Alto, CA) at a dose rate of 200 cGy/min on day 7. At the terminal point when the mice were sacrificed, tumors were frozen in 280uC or fixed in 10% formalin overnight and subjected to routine histological examination.
Mice were observed for lethargy, appetite, and sacrificed timely before natural death occurred. The date on which the grossly visible tumor stabilized appeared for a volume of 150-200 mm 3 was recorded and random distributed. Both the tumor size and animal weight were measured every 3 days. All of the animals were treated according to protocols approved by the Institutional Animal Care and Use Committee of Soochow University. And this study was approved by the Institutional Animal Care and Use Committee of Soochow University.
Statistical data analyses. Data were expressed as the means 6 SEM of at least three independent experiments. The data were then analyzed using Student's t-test when only two groups were present, or assessed by one-way analysis of variance (ANOVA) when more than two groups were compared. The interaction between ubiquitin knock-down and radiation was tested using two-way ANOVA. The sensitizer enhancement ratios (SER) were measured according to the multi-target single hit model. Expression difference of ubiquitin mRNA and protein between lung tumor tissues and the adjacent normal tissues were analyzed using paired t-test and Pearson Chi-square (x 2 ) test. Differences were considered statistically significant when P , 0.05. SPSS Statistical software (version 19.0) was utilized for statistical analysis.