A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog, EB 1089

Influence of 1,25-D₃ and the vitamin D analog EB 1089 on sensitivity to radiation in non-small cell lung cancer cells

Figure S1A shows expression levels for VDR, which is generally considered to be required for vitamin D to affect cell function,Citation²⁰ in a number of NSCLC cell lines. High levels of the receptor are evident in the A549, H460, and H358 cell lines and the receptor is also clearly expressed in the H1299 and H520 cell lines but is barely detectable in the H838 or H2030 cells.

Figure 1A and B present clonogenic survival studies performed at different doses of radiation alone (varying from 2Gy to 6Gy) and in combination with either the vitamin D analog, EB 1089, or 1,25-D₃ in the H460 and A549 cells, respectively. The inclusion of either 1,25-D₃ or EB 1089 resulted in a significantly greater loss of clonogenicity at every radiation dose, with sensitization factors of between 1.4 and 2.0. Temporal response studies assessing cell viability in Figure 1C and D corroborated the results of the clonogenic survival study. While a single dose of 6Gy results in transient growth inhibition that is followed by proliferative recovery, cell growth inhibition is more pronounced and proliferative recovery is significantly impaired with the combination treatment of radiation + 1,25-D₃ or EB 1089. These results differ from our findings in breast tumor cells where the combination treatment resulted in clear evidence of cell killing (a time-dependent decline in viable cell number).Citation^18,19,21

Clonogenic survival studies shown in Figure S1B and C indicate that neither 1,25-D₃ (the active form of vitamin D) nor EB 1089 (a synthetic analog of vitamin D) alone, at concentrations of 10 nM, 50 nM and 100 nM, significantly interfered with growth of the A549 or H460 NSCLC cells. Similarly, cell viability studies shown in Figure S1D and E indicated that neither 1,25-D₃ nor EB 1089 inhibited growth or survival of H460 and A549 cells at 100 nM, the concentration used in the current work as well as our previous studies in breast cancer. The lack of effect of these agents alone establishes that the sensitization effects observed are not simply the consequence of additive antiproliferative or cytotoxic interactions. Furthermore, treatment with 1,25-D₃ and EB 1089 had an essentially identical impact in terms of tumor cell sensitization (sensitization by 1,25-D3 and EB 1089 was significant for both cell lines; this becomes apparent when the data is plotted on an expanded scale). Consequently, while the bulk of studies in A549 cells were performed using 1,25-D₃, studies in H460 cells were performed using EB 1089, which tends to be more stable for prolonged periods in solution. Additionally, in terms of potential clinical utility, EB 1089 has reduced calcemic effects.Citation²²

1,25-D₃ and the vitamin D analog EB 1089 fail to increase the extent of DNA damage induced by IR or interfere with DNA repair in non-small cell lung cancer cells

In our previous studies with breast tumor cells, we have found no evidence for increased DNA damage or decreased DNA repair associated with sensitization to radiation by 1,25-D₃/EB 1089.Citation^8,19,21 Figure S2 presents studies in which DNA damage was assessed by measuring levels of phosphorylated H2AFX/γH2AX in response to radiation and radiation + EB 1089 in the H460 cells. DNA damage was observed within 1 h after irradiation and declined thereafter, but neither the initial extent of damage nor the rate of decline, indicative of DNA repair, was affected by the presence of the EB 1089. It is also worth noting that there was no effect of EB 1089 alone, consistent with the lack of effect on cell growth.

1,25-D₃ or EB 1089 fail to increase the extent of senescence induction by radiation in NSCLC cells

In studies that were performed prior to recognition of the potential role of autophagy in the response to radiation, we demonstrated that the primary response to radiation is senescence,Citation^23,24 in the absence of significant apoptosis, in cells expressing wild-type TP53/TRP53/p53 (note that the mouse nomenclature is TRP53, but we will use TP53 for either the human and mouse genes/proteins for simplicity). Since the temporal cell viability studies in Figure 1 indicated that the cells were undergoing primarily growth arrest, we evaluated whether the extent of senescence might have been increased for the combination treatments. Figure S3A and B present GLB (galactosidase, β)-staining images in A549 and H460 NSCLC cell lines, which indicates that both radiation treatment alone and the combination of radiation with 1,25-D₃ or EB 1089 leads to an increase in GLB staining and alterations in morphology indicative of senescence. Figure S3C and D provide quantification of the time-dependent increase in senescence induced by radiation and the combination treatments in A549 (left panel) and H460 (right panel) cell lines. These studies fail to demonstrate any significant differences between radiation treatment alone and the combination of radiation + 1,25-D₃ or EB 1089 in terms of the extent of senescence.

Minimal induction of apoptosis or necrosis by radiation and radiation + 1,25-D₃ or EB 1089 in NSCLC cells

Although the primary responses observed either with radiation alone or radiation + EB 1089 or radiation + 1,25-D₃ appeared to be growth arrest, it was formally possible that a fraction of the cell population might be undergoing apoptosis (or necrosis). Apoptosis and necrosis were evaluated by ANXA5 and PtdIns staining and flow cytometry. Figures S4A and Figure S4B show that there was minimal induction of apoptosis or necrosis (data for necrosis not shown) by radiation and radiation + 1,25-D₃ or EB 1089 treatments in A549 and H460 cell lines. This outcome was further confirmed by the lack of PARP (poly ADP ribose polymerase) cleavage where staurosporine was used as a positive control (data not shown).

Increased sensitivity to radiation is associated with a novel form of autophagy

We have previously reported quite extensively on the capacity of 1,25-D₃ and vitamin D analogs to sensitize breast tumor cells to radiation.Citation^{8-10,18,19,21} In recent work, we have confirmed that this sensitization, a decline in viable cell number, occurs through the promotion of autophagy based on reversal of sensitization through either pharmacological or genetic interference with autophagy.Citation^18,19 The studies presented below indicate that sensitization in the NSCLC cells is related to conversion of the cytoprotective autophagy induced by radiation alone to what we have termed “cytostatic autophagy” when the combination of radiation and radiation + 1,25-D₃ or EB 1089 is used.

Studies in the A549 cells exposed to either radiation alone or radiation + 1,25-D₃ indicated that radiation alone promotes autophagy, based on acridine orange staining and formation of autophagic puncta (Fig. 2A). Figure 2B indicates that the extent of autophagic vesicle formation (as quantified by flow cytometry) was significantly increased by the combination of radiation and 1,25-D₃ in the A549 cells. GFP-LC3 puncta formation shown in Figure 2C further corroborates the flow cytometry data indicative of the promotion of autophagy.

Figure 3A and B show autophagic vesicle formation by acridine orange staining and quantification by flow cytometry in the H460 cells; GFP-LC3 puncta formation further indicative of autophagy is shown in Figure 3C and endogenous LC3-II levels assessed using confocal microscopy are shown in Figure 3E. However, as shown in Figure 3B, the extent of autophagy was not significantly different for radiation alone and EB 1089 + radiation in the H460 cells. Consequently, despite the evidence for increased autophagy for the combination treatment in the A549 cells, these findings in H460 cells argue against the simple explanation that sensitization might be caused solely by an increase in the extent of autophagy.

Experiments were also performed to determine whether the autophagy observed reflected autophagic flux, where autophagy proceeds to completion in terms of the degradation of select protein or organelle substrates.Citation²⁵ Figure 2D and Figure 3D demonstrate that autophagic flux markers SQSTM1/p62 and LC3-II were degraded both with radiation alone and the combination treatment in A549 and H460 cells. However, there was no apparent difference in the extent or time course of degradation for radiation alone versus the combination treatment with either 1,25-D₃ or EB 1089. Furthermore, as shown in Figure 4A and B, respectively, the autophagy inhibitors bafilomycin A₁ (Baf) and chloroquine interfered with SQSTM1 degradation by radiation alone as well as the combination of radiation with EB 1089, consistent with both treatments promoting autophagic flux.

It has been established by a number of laboratories, including our own, that radiation-induced autophagy frequently has a cytoprotective function.Citation^18,19 Recent studies by Kroemer's lab have shown this to be the case in the A549 and H460 cells by demonstrating that inhibition of radiation-induced autophagy increases sensitivity to radiationCitation²⁶ and this has been confirmed in our current work. Figure S5A and B show that radiation sensitivity increases (downward arrow), when autophagy is blocked by chloroquine in both A549 and H460 cell lines, respectively, consistent with the cytoprotective function of autophagy in response to radiation alone. Furthermore, Figure S5C and D demonstrate that blocking cytoprotective autophagy by either chloroquine or Baf led to a modest (but not statistically significant) increase in apoptosis. In contrast, no increase in apoptosis was observed when autophagy was inhibited for the combination treatment. We therefore hypothesized that exposure of the NSCLC cells to 1,25-D₃ or EB 1089 could be altering the function of the autophagy, converting the cytoprotective autophagy induced by radiation to what we have termed cytostatic autophagy, based on the temporal responses to treatment observed in Figure 1C and D.

To investigate whether an altered function of autophagy might be responsible for the apparent increase in sensitivity to radiation, we first determined whether interference with autophagy could reverse the impact of radiation + EB 1089, using chloroquine and Baf, both well-characterized pharmacological inhibitors of the late stages of autophagy.Citation²⁵ As indicated above, Figure 4A and B present western blots indicating that both inhibitors by themselves and in combination with radiation and EB 1089, lead to an accumulation of SQSTM1, indicative of the inhibition of autophagic flux.Citation²⁵ As shown in Figure 4C, both chloroquine and Baf, when used to block autophagy, reversed the radiosensitization effects of EB 1089 (upward arrows). This indicates that what we have termed a cytostatic form of autophagy (based on the temporal response studies presented in Fig. 1C and D) is likely to be the mode of radiosensitization with the combination treatment.

The conclusion that the cytoprotective autophagy induced by radiation has been converted to cytostatic autophagy by the inclusion of 1,25-D₃ or EB 1089 is strongly supported by complementary genetic silencing studies, where the autophagy-related genes BECN1 and ATG5 were knocked down utilizing shRNA, as shown in the immunoblots in Figure 5A and B. Figure S6 confirms successful inhibition of autophagy, based on the reduction in the extent of autophagic vesicle formation, by genetic silencing of BECN1 and ATG5. (It should also be noted that the increased cell size that is a component of autophagy induction is not observed in cells where the autophagy genes have been silenced). The temporal and cell viability studies in Figure 5D and F indicate that as was the case with pharmacological inhibition of autophagy, radiosensitization by EB 1089 is reversed when cytostatic autophagy is genetically suppressed. In dramatic contrast, Figure 5C and E demonstrate that when cytoprotective autophagy is genetically suppressed, tumor cell sensitivity to radiation is increased.

It is important to note that the sensitization to radiation that occurs when the cytoprotective autophagy induced by radiation is inhibited (Fig. 5C and 5E) is no longer evident in the cells exposed to EB 1089 + radiation; otherwise, it would be difficult to reverse the sensitization induced by EB 1089, a problem we encountered in our studies in breast tumor cells.Citation^18,19 This is the basis for our conclusion that the cytoprotective form of autophagy induced by radiation has been converted to a cytostatic form.

The effects of radiation and EB 1089 + radiation are associated with cell cycle arrest

As shown in the temporal response studies presented in Figure 1, the primary effects of radiation as well as radiation in combination with either 1,25-D₃ or EB 1089 were to arrest cell growth without evidence for cell killing in either condition. To confirm these observations, cell cycle analysis was performed in H460 cells exposed to radiation alone and EB 1089 + radiation with and without knockdown of the autophagy genes ATG5 and BECN1. As shown in Figure 6A and B, radiation alone as well as EB 1089 + radiation reduced the S phase population and increased the G₂/M phase population. Effects of EB 1089 alone on cell cycle distribution were not significant, again consistent with its lack of effect on cell growth. As would be expected, inhibition of autophagy by genetic silencing reversed the G₂/M associated growth arrest.

Requirement for functional TP53 and VDR in sensitization of NSCLC cells

In our previous work with breast tumor cells, we identified a fundamental requirement for functional TP53 in radiosensitization by 1,25-D₃ or EB 1089.Citation^8,18,19 Both A549 and H460 cells are known to express wild-type TP53.Citation^27,28 However the combination treatment of radiation +EB 1089 failed to increase sensitivity to radiation in the H358 NSCLC cell line (Fig. S7A), which has mutant TP53, despite the very high expression of the VDR (Fig. S1A). To establish the importance of the requirement for VDR in enhancement of the response to radiation, similar combination studies were performed in H838 cells, which lack VDR (Fig. S1A) but are wild type in TP53.Citation²⁹ Figure S7B indicates that these cells did not show any enhancement in the response to the combination treatment of radiation + 1,25-D₃ compared to radiation treatment alone.

To confirm this requirement for functional TP53 in radiosensitization, we demonstrate (Fig. 7A and B, where 7B is an expanded scale for IR and IR+EB 1089) that sensitization to radiation by EB 1089 also fails to occur in the H1299 NSCLC cell line, which is tp53-null,Citation³⁰ but expresses the VDR (Fig. S1A). In dramatic contrast, Figure 7C and D (7D is an expanded scale for IR and IR + EB 1089) indicate that H1299 cells could be radiosensitized by EB 1089 (downward arrow) when TP53 was expressed under the influence of doxycycline (induction of TP53 shown in figure inset).

To confirm and extend these observations in the H1299 cells, we investigated the effects of both pharmacological and genetic autophagy inhibition in both the tp53-null and inducible cell lines. As shown in Figure S8A and B, both ATG5 and BECN1 were successfully silenced for tp53-null and tp53-inducible H1299 cells. Figure S8C shows the reduction in autophagic vesicle formation when ATG5 and BECN1 were knocked down while Figure S8D shows that chloroquine effectively promoted accumulation of LC3-II, which is indicative of interference with the late stage of autophagy. Figures 8B, D and F indicate that in H1299 cells with induced TP53, sensitization by the combination treatment was reversed when autophagy was blocked. In quite dramatic contrast, blocking autophagy in the tp53-null H1299 parental cells had minimal effect (chloroquine) or no effect (genetic silencing of ATG5 or BECN1) on tumor cell sensitivity to the combination treatment of EB 1089 + radiation (Fig. 8A, C and E). It should be noted that the low cell number for radiation alone in Figures 8B, D and F reflects sensitization to radiation through interference with the cytoprotective function of radiation-induced autophagy. Conversely, the high cell numbers for the EB 1089 + radiation combination reflects the fact that sensitization to radiation by EB 1089 was eliminated through interference with cytostatic autophagy.

Studies in normal lung epithelial cells and cardiomyocytes

Drug selectivity is a critical consideration in studies related to cancer therapies; treatments should ideally be highly targeted to minimize the damage to normal cells and tissue. This is particularly true for the utilization of radiation in diseases such as non-small cell lung cancer. Consequently, studies were conducted to test for selectivity of the combination treatment of 1,25-D₃ with radiation in normal human bronchial epithelial cells and cardiomyocytes in culture. As with the experiments in H460 cells, the HBEC3-KT bronchial epithelial cells were pretreated with 100 nM EB 1089 24 h before irradiation with a single dose of 6 Gy while the H9C2 cardiomyocytes were exposed to 100 nM 1,25-D₃ for 72 h followed by a single dose of radiation (5 Gy). Figure S9A and B indicate that there were no significant differences between the impact of radiation alone and the combination of radiation + 1,25-D₃ or radiation + EB 1089 treatments in the epithelial cells or the cardiomyocytes, respectively.

Additional evidence for differences in the functional forms of autophagy induced by radiation alone and by radiation + EB 1089.

AMPK, an energy-sensing protein kinase, has been shown to be involved in regulation of autophagy.Citation^31,32 Studies were performed to investigate changes in the phosphorylated (active) form of AMPK levels in response to radiation alone (cytoprotective autophagy) and radiation + EB 1089 (putatively cytostatic autophagy). Figure 9A indicates that increased phospho-AMPK levels were observed with radiation alone as well as with the combination of radiation + EB 1089 as measured by flow cytometry. After 48 h, AMPK activation by the combination treatment significantly exceeded that for radiation alone. This was confirmed by the protein gel blotting data presented in Figure 9B where p-AMPK levels are clearly higher for the combination treatment. A recent study conducted by Singh K et al. indicates that AMPK is activated upon irradiation of H1299 cells,Citation³³ consistent with our own findings.

To potentially distinguish between the involvement of AMPK in autophagy signaling by radiation alone and radiation + EB 1089, compound C was used to inhibit AMPK. Figure 9B confirms that compound C interfered with the phosphorylation of AMPK and its downstream target ACC (acetyl-CoA carboxylase) induced by radiation alone as well as EB 1089 + radiation. Figure 9D shows that inhibition of AMPK resulted in reversal of the radiosensitizing effects of EB 1089. In dramatic contrast, treatment with compound C enhanced the response to radiation alone as shown in Figure 9C. Consequently, these findings closely parallel the outcome of the experiments presented in Figures 4, 5 and 7, where autophagy inhibition enhanced sensitivity to radiation but reversed the enhancement of radiation sensitivity by EB 1089. These studies support the premise that autophagy has diametrically opposite functions in the case of radiation alone (cytoprotective) and radiation + EB 1089 (cytostatic radiosensitizing).

Cell lines

The wild-type (WT) TP53 A549 cell line, VDR null H838 cell line, TP53 mutant H358 cell line, and normal bronchial epithelial cell line HBEC3-KT originally generated by the Minna laboratoryCitation⁴⁸ were generously provided by Dr. Charles Chalfant (Virginia Commonwealth University). H460 (WT TP53) NSCLC cells were a generous gift from Dr. Richard Moran at Virginia Commonwealth University. H460 shBECN1, shATG5 cells were generated as described below. H1299 tp53-null and TP53-inducible cells were originally developed by Dr. Constantinos KoumenisCitation⁴⁹ and provided through our collaborators, Drs. Frank and Suzy Torti, (University of Connecticut).

Mission shRNA bacterial stocks for ATG5 and BECN1 were purchased from Sigma Aldrich (TRCN00151963 and TRCN0000299864, respectively). Lentiviruses were produced in HEK 293TN cells cotransfected using Lipofectamine (Invitrogen, 11668–019) with psPAX2 and pMD2.G packaging constructs (Addgene, 12260, 12259 ). Viruses shed into the media were then used to infect H460 cells. Puromycin (1 ug/ml) was used as the selection marker to enrich the infected cells.

Cell culture and treatment

H460, A549, and H1299 cells were maintained in DMEM media supplemented with 10% (v/v) fetal bovine serum (Thermo Scientific, SH30066.03), 100 U/ml penicillin G sodium (Invitrogen, 15140–122), and 100 μg/ml streptomycin sulfate (Invitrogen, 15140–122). H838 cells were maintained in RPMI 1640 media with the same supplements. HBEC3-KT cells were maintained in Keratinocyte-SFM media (Life Technologies, 12568–010) supplemented with fetal bovine serum (provided with the kit), 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate. H460 shATG5 and shBECN1 and H1299 shATG5 and shBECN1 cells were maintained with puromycin (1 μg/ml; Sigma, P8833) for selection. All cells were maintained at 37°C under a humidified, 5% CO₂ atmosphere. Except where otherwise indicated, after equilibration post-seeding, cells were treated with single dose of 6 Gy radiation alone (using a ¹³⁷Cs irradiator), 100 nM 1,25-D₃ alone, or 100 nM EB 1089 (Tocris Bioscience, 2551, 3993) alone, or the combination of EB 1089 or 1,25-D₃ with 6 Gy radiation. Cells were washed free of the 1,25-D₃ or EB 1089 after 48 h (i.e., exposure for 24 h prior to and 24 h post irradiation). To induce TP53 expression, H1299 cells were continuously incubated with doxycycline (1 μg/ml; Sigma, 09891) throughout the experiment. Media was replenished once all the treatments were completed. H1299 shATG5 and shBECN1 cell lines were maintained in doxycycline post transfection.

Cell viability and clonogenic survival

Cell viability was assessed by trypan blue exclusion at various time points after the treatment. Cells were harvested using trypsin, stained with 0.4% trypan blue (Sigma, T8154), and counted using a hemocytometer under phase contrast microscopy. For clonogenic studies, cells were plated in 6-well dishes at an appropriate density. After 2 wk, the cells were washed with phosphate-buffered saline (PBS; Life Technologies, 10010023), fixed with 100% methanol, air-dried and stained with 0.1% crystal violet (Sigma, C3886). Cells in groups of 50 or more were counted as colonies and data were normalized relative to untreated controls.

Western blotting

After the indicated treatments, cells were scraped from the culture dishes, collected, and lysed using M-PER mammalian protein extraction reagent (Thermo Scientific, 78505) containing protease and phosphatase inhibitors (Sigma Aldrich, P8340, P5726). Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories, 500–0205r). Total protein was then diluted in SDS sample buffer and dry boiled for 10 min. Protein samples were subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane, and blocked in 5% milk, 1x PBS, 0.1% Tween 20 (Fisher, BP337) for 1 h. Primary antibodies used at a 1:1000 dilution with overnight incubation were SQSTM1/p62 (BD Biosciences, 610497), ATG5 (Cell Signaling Technology, 2630), BECN1 (BD Biosciences, 612112), VDR (Santa Cruz Biotechnology, sc1008), LC3B-I/LC3B-II (Cell Signaling Technology, 3868), phospho-AMPK (Cell Signaling Technology, 2531), total-AMPK (Cell Signaling Technology, 2795s), phospho-ACAC/acetyl-CoA carboxylase (p-ACC; Cell signaling Technology, 3661s), TP53 (BD Biosciences, 554157), ACTB/β-actin (Sigma, A5316). The membrane was then incubated with secondary antibody of either horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:10000; GE Healthcare, NA434) or goat anti-mouse (1:10000; GE Healthcare, NA931) for 1 h, followed by extensive washing with Tween-PBS (PBS with 0.1% Tween 20). Blots were developed using Pierce enhanced chemiluminescence reagents and Bio-Max film (Phenix Reseach Products, F-BX57).

Detection and quantification of acidic vesicles with acridine orange staining

Cells were plated in 6-well culture dishes, stained with acridine orange (Sigma, A6014) at a final dilution of 1:10,000 in respective culture media and allowed to incubate at 37°C for 10 min. After 2 washes with PBS, cells were imaged under an inverted fluorescence microscope (Olympus, Tokyo, Japan). All images were taken at the same magnification. For quantification of autophagic vesicles (AVOs), cells were trypsinized, harvested, and washed with PBS at approximately 1 × 10⁸ cells/ml. Pellet fractions were resuspended in PBS, stained with a 1:10,000 dilution of acridine orange for 10 min and analyzed by BD FACSCanto II (Virginia Commonwealth University) using BD FACSDiva software at the Virginia Commonwealth University Flow Cytometry Core Facility. A minimum of 10,000 cells within the gated region was analyzed. Data collected was further analyzed using FCS Express 4 Flow research edition.

Detection and quantification of senescent cells

The method described by Dimri et al.Citation⁵⁰ was used for detection of GLB-positive cells. After treatment, cells were washed with PBS and fixed with 2 % formaldehyde/0.2% glutaraldehyde/PBS for 5 to 8 min. Cells were again washed with PBS and stained with 5-bromo-4-chloro-3-inolyl-β-D-galactopyranoside (Fisher, R0404) in dimethylformamide (20 mg/ml), 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl₂ and incubated at 37°C for 24 h. After incubation, the cells were washed twice with PBS and visualized using an Olympus inverted microscope. All images were taken at the same magnification. For quantification of SA-GLB/β-galactosidase staining, cells were treated as above and analyzed using a fluorescent GLB activity marker C₁₂FDG (Life Technologies, D2893). The protocol was adapted from Debacq-Chainiaux et al.Citation⁵¹

RFP-LC3 and GFP-LC3 redistribution

The RFP-LC3 construct was a generous gift from Dr. Keith Miskimins, Sanford Research, University of South Dakota. H460 cells were transfected with RFP-LC3 using lipofectamine. A549 cells were transfected with GFP-LC3 (Addgene, 22405) using lopofectamine (Invitrogen, 11668–019). Cells were treated as described above, fixed with 3% paraformaldehyde, and visualized using a Leica Confocal laser-scanning microscope (Virginia Commonwealth University). Cells were counterstained with DAPI to stain and visualize the nucleus.

FACS analysis for ANXA5 and propidium Iodide (PI)-positive cells to assess apoptosis

After treatments, cells were collected, washed with PBS, and resuspended in 500 μl of 1x Binding Buffer (ANXA5-FITC apoptosis detection kit; BD Biosciences, 556547) with 5 μl of ANXA5-FITC, and 5 μl of propidium iodide. Samples were gently mixed and incubated at room temperature in the dark for 15 min. The number of cells with increased ANXA5 and PtdIns staining was assessed by flow cytometry and analyzed by BD FACSCanto II using FACSDiva software. A minimum of 10,000 cells within the gates region was analyzed.

FACS analysis for cell cycle distribution

The protocol described by Menon VR et al.Citation⁵² was used to assess cell cycle distribution. H460 control, H460 shATG5 and shBECN1 cells were seeded in 100-mm tissue culture plates at different densities based on the time points for collection. After 24 h, cells were exposed either to radiation alone, EB 1089 alone, or the combination of EB 1089 and radiation. At indicated time points, 1 x 10Citation⁶ cells were suspended in 1 ml PI solution (Sigma, P4864) with added RNase A (Sigma, R5125) at a final concentration of 0.2 mg/ml. Cells were maintained in the dark at 4°C overnight and analyzed the next day to measure the DNA content by flow cytometry.

Assessment of AMPK phosphorylation and H2AFX/γ-H2AFX formation

After the indicated treatments, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized with 100% cold methanol for 30 min and incubated with blocking buffer (4% BSA [Fisher, NC9857891] in PBS) for 10 min. Cells were incubated with a 1:50 dilution of primary antibody for 1 h, washed with PBS and incubated with a fluorochrome-conjugated secondary antibody (1:100 dilution) for 30 min in the dark. After the second incubation, cells were washed with blocking buffer and resuspended in 400 μl PBS. Cells were analyzed using FACS with a minimum of 10,000 cells for each sample.Citation⁵³ Primary antibodies used were p-PRKAA1/PRKAA2 (AMPK α1/α2 Thr172; Cell signaling Technology, 2531), and anti-p- γ-H2AFX (γ-H2AX Ser139) (EMD Millipore, 07–627).

Statistical analysis

Statistical differences were determined by StatView statistical software (SAS Institute, Cary, NC). The data were expressed as means ± SE (as standard error of the mean). Comparisons were made using one-way analysis of variance (ANOVA) followed by Turkey-Kramer post hoc test, and P values ≤ 0.05 were taken as statistically significant.