PAR-4 overcomes chemo-resistance in breast cancer cells by antagonizing cIAP1

Most deaths from breast cancer result from tumour recurrence, which is typically an incurable disease. Down-regulation of the pro-apoptotic tumour suppressor protein prostate apoptosis response-4 (PAR-4) is required for breast cancer recurrence and resistance to chemotherapy. Recent advances in the analysis of apoptotic signalling networks have uncovered an important role for activation of caspase-8 following DNA damage by genotoxic drugs. DNA damage induces depletion of IAP proteins and causes caspase-8 activation by promoting the formation of a cytosolic cell death complex. We demonstrate that loss of PAR-4 in triple negative breast cancer cell lines (TNBC) mediates resistance to DNA damage-induced apoptosis and prevents activation of caspase-8. Moreover, loss of PAR-4 prevents DNA damage-induced cIAP1 depletion. PAR-4 functions downstream of caspase-8 by cleavage-induced nuclear translocation of the C-terminal part and we demonstrate that nuclear translocation of the C-terminal PAR-4 fragment leads to depletion of cIAP1 and subsequent caspase-8 activation. Specifically targeting cIAP1 with RNAi or Smac mimetics (LCL161) overcomes chemo-resistance induced by loss of PAR-4 and restores caspase-8 activation. Our data identify cIAP1 as important downstream mediator of PAR-4 and we provide evidence that combining Smac mimetics and genotoxic drugs creates vulnerability for synthetic lethality in TNBC cells lacking PAR-4.

with breast cancer, which confirms the results from previous studies 17,18 . Importantly, PAR-4 expression was also found to be low in highly aggressive, estrogen-receptor negative (ER−), basal-like, and high-grade (grade 3) breast cancers, which are all associated with poor clinical outcome 21 . These observations suggest a potential role for PAR-4 as a prognostic marker and as a drug target for breast cancer therapy.
PAR-4 is a ubiquitously expressed protein that was initially discovered as a pro-apoptotic protein in androgen-independent prostate cancer cells undergoing apoptosis in response to Ca 2+ elevation 22 . Consistent with its pro-apoptotic activity, ectopic expression of PAR-4 sensitizes a large subset of cancer cells to apoptosis-inducing stimuli or chemotherapeutic agents 23 . Mice lacking Par-4 show reduced life span and are prone to develop spontaneous tumours in the endometrium and prostate 10 . Importantly, in vivo delivery of PAR-4 by adenovirus injection or nanoliposome application into tumours growing in nude mice induces tumour regression and/or tumour sensitization to therapeutic agents 24,25 .
PAR-4 consists of a unique and central SAC (Selective for Apoptosis of Cancer Cells) domain, encompassing a nuclear localisation sequence (NLS), and a C-terminal leucine zipper domain (LZ), which are both 100% conserved in human and rodent orthologous 23 . The central SAC domain has been identified by serial deletions of PAR-4 and has been described to be indispensable for the pro-apoptotic activities of PAR-4 26 . Overexpression of the SAC domain alone is sufficient to induce cell death in a variety of cancer cells but not in normal or immortalized cells 26 . Moreover, transgenic mice that ubiquitously express the SAC domain of Par-4 are resistant to the development of spontaneous as well as oncogene-induced tumours 27 .
We have previously demonstrated that UV-and TNFα-induced apoptosis results in a rapid caspase-8-dependent cleavage of PAR-4 at EEPD131/G. This process leads to nuclear accumulation of the C-terminal PAR-4 fragment that includes the SAC and LZ domains, which then induces apoptosis 28 . In the current study we investigate the influence of PAR-4 on survival of TNBC cells following genotoxic stress. We show that PAR-4 overexpression sensitizes TNBCs to genotoxic drug treatment, whereas loss of PAR-4 is accompanied with drug resistance. Furthermore, we demonstrate that in response to DNA damage PAR-4 regulates the stability of cIAP1, a member of the mammalian inhibitor of apoptosis (IAP) family, and cIAP1 antagonists can overcome chemo-resistance induced by the loss of PAR-4.

PAR-4 expression alters drug sensitivity of TNBC cells to genotoxic stress.
As down-regulation of PAR-4 serves as a mechanism for tumour cell survival, we analysed PAR-4 expression in a panel of breast cancer cell lines by immunoblotting (Fig. 1a). Compared to the immortalized, non-transformed mammary epithelial cell line MCF-10A, none of the analysed cell lines exhibited a complete loss of PAR-4 expression. Nevertheless, PAR-4 protein levels were found to be lower in ZR-75-1 cells and in the TNBC cell lines MDA-MB-468, Hs-578T www.nature.com/scientificreports www.nature.com/scientificreports/ and BT-20. To further explore the function of PAR-4 in the DNA damage response (DDR) in breast cancer, the TNBC cell lines BT-20 and MDA-MB-468 were chosen for the following studies. To investigate whether PAR-4 can sensitize TNBC cells to DNA damage, wild-type (WT) PAR-4 was overexpressed in BT-20 and MDA-MB-468 cells and subsequently treated with the topoisomerase II inhibitor Etoposide (Fig. 1b). Apoptosis was analysed by caspase-8 and PARP-1 cleavage. Forced expression of PAR-4 WT alone resulted in moderate PAR-4, caspase-8 and PARP-1 cleavage in these TNBC cells. In addition, treatment with Etoposide resulted in enhanced PAR-4, caspase-8 and PARP-1 cleavage, demonstrating that overexpression of PAR-4 sensitized these cells towards DNA damage. To analyse if PAR-4 is also required for apoptosis induction following DNA damage, we silenced PAR-4 expression using siRNA and stimulated cells with Etoposide (Fig. 2a). Whereas PAR-4 cleavage was observed simultaneously to caspase-8 and PARP-1 cleavage in control cells upon Etoposide treatment, apoptosis was inhibited in PAR-4 depleted TNBC cells (Fig. 2a). Furthermore, we quantified apoptotic TNBC cells under the same conditions by measuring the sub-G1 fraction using flow cytometry. We confirmed that PAR-4 depletion led to chemo-resistance (Fig. 2b). Overall, these data demonstrate that PAR-4 sensitizes TNBC cells to genotoxic drugs and is required for DNA damage-induced apoptosis.
Caspase-8 activity is required for DNA damage-induced cell death in TNBC cells. We have previously shown that PAR-4 is a caspase-8 substrate and that caspase-8-induced PAR-4 cleavage is required for TNFα-induced cell death 28 . Recent advances in analysing apoptotic signalling networks have also uncovered an important role for activation of caspase-8 following DNA damage caused by genotoxic drugs 7,29 . For this reason we analysed whether caspase-8 activation is required for DNA damage-induced cell death in BT-20 and www.nature.com/scientificreports www.nature.com/scientificreports/ MDA-MB-468 cells and whether it is upstream of PAR-4 processing. Therefore, we knocked down caspase-8 expression in both cell lines using lentiviral delivery of small hairpin RNA (shRNA) constructs (Fig. 3a). Cells were treated with Etoposide or Doxorubicin (Fig. 3a,b) and analysed for caspase-8, PARP-1 and PAR-4 cleavage. The loss of caspase-8 expression led to drug resistance following DNA damage induction and also interfered www.nature.com/scientificreports www.nature.com/scientificreports/ with PAR-4 cleavage (Fig. 3a,b). Moreover, the knockdown of caspase-8 prevented Etoposide-induced apoptosis (Fig. 3c). In addition, pre-incubation with the caspase-8 inhibitor, Z-IETD-FMK, abolished the cleavage of PAR-4, caspase-8 and PARP-1 upon induction of DNA damage (Fig. 3d,e), indicating that caspase-8 activation is indispensable for induction of apoptosis and PAR-4 processing. Our combined data using both knock-down and inhibition of caspase-8 demonstrate that this enzyme is required for DNA damage-induced cell death and is upstream of PAR-4.

The C-terminal PAR-4 cleavage fragment translocates into the nucleus in response to DNA damage-induced apoptosis and its expression is sufficient to induce apoptosis.
We have recently demonstrated that caspase-8-mediated PAR-4 cleavage results in the nuclear accumulation of the C-terminal fragment of PAR-4 in HEK293 cells upon TNFα/CHX or UV-induced apoptosis 28 . To study the localisation of the PAR-4 cleavage products upon DNA damage in BT-20 and MBA-MD-468 cells, wild-type PAR-4, a PAR-4 cleavage resistant mutant (PAR-4-D131G), and N-and C-terminal deletion mutants (PAR-4-132-340 and PAR-4-1-131, respectively) with either a C-terminal eCFP tag or an N-terminal eYFP tag were generated and expressed in BT-20 and MDA-MB-468 cells (Fig. 4a). Whereas PAR-4-CFP and PAR-4-D131G-CFP, the latter is resistant to caspase-8 cleavage, showed cytosolic localization in both TNBC cell lines, the PAR-4 mutant lacking the amino-terminal part (PAR-4-132-340-CFP) localized to the nuclear compartment. The PAR-4 mutant lacking the C-terminal part (YFP-PAR-4-1-131) was equally distributed throughout the cell (Fig. 4a). Stimulation with Etoposide triggered a nuclear accumulation of PAR-4-CFP but was prevented in cells expressing PAR-4-D131G-CFP (Fig. 4a). In contrast, DNA damage had no influence on the localisation of the two deletion mutants. PAR-4-132-340-CFP remained nuclear and the cellular distribution of YFP-PAR-4-1-131 was unchanged (Fig. 4a). We also analysed the potential of the two PAR-4 cleavage products to induce apoptosis in TNBC cells. We observed that overexpression of the C-terminal part resulted in the activation of apoptosis, while the N-terminal region failed to do so, as documented by PARP-1 and caspase-8 cleavage as well as the accumulation of sub-G1 cells (Fig. 4b-d and Supplementary Fig. S1). To analyse whether the localisation of ectopically expressed PAR-4 behaved consistently with endogenous PAR-4, we used antibodies recognizing the C-terminal part of PAR-4 ( Fig. 4e). In the absence of Etoposide, PAR-4 mainly localized to the cytosolic compartment, whereas the staining accumulated in the nucleus after Etoposide treatment, confirming that the overexpressed PAR-4 behaves comparable to the endogenous protein (Fig. 4e). Given that the cleaved C-terminal fragment, which contains the SAC domain, is sufficient to induce apoptosis in cancer cells (Fig. 4b,c) 24 , we investigated whether overexpression of the PAR-4 cleavage resistant mutant was capable to evoke apoptosis in BT-20 and MDA-MB-468 cells. The pro-apoptotic properties of PAR-4 involve inhibition of the NFκB pathway 30 . Indeed, PAR-4-D131G was unable to inhibit NFκB activity and induce apoptosis, in contrast to PAR-4 and PAR-4-131-340 (Fig. 4f). Together, these findings are in line with a pro-apoptotic function of the C-terminal PAR-4 fragment after localisation to the nuclear compartment.

PAR-4 regulates cIAP1 depletion upon DNA damage-induced apoptosis.
Our data so far indicates, that loss of PAR-4 interferes with the activation of caspase-8 in breast cancer cells after stimulation with Etoposide (Fig. 2a). Genotoxic stress induces the depletion of cIAP1, cIAP2 and XIAP, which triggers the assembly of a RIP-1/FADD/caspase-8 complex and can initiate autocatalysis and activation of caspase-8 31 . We noticed in our experiments a reduced expression of cIAP1 in response to transiently expressed PAR-4 and the C-terminal PAR-4 fragment (Fig. 4b,d,f). To address the involvement of IAP proteins more broadly, the expression of cIAP1, cIAP2 and XIAP, was analysed in BT-20 and MDA-MB-468 cells in response to treatment with Doxorubicin or Etoposide (Fig. 5a). We found that cIAP1 and XIAP were expressed in both BT-20 and MDA-MB-468 cells, while cIAP2 was only detectable in the latter. All three IAPs were reduced to different degrees in response to DNA damage (Fig. 5a). As we had observed before that the C-terminal PAR-4 fragment was sufficient to deplete cIAP1 in TNBCs (Fig. 4) and because cIAP1 was efficiently depleted after cytotoxic drug treatment (Fig. 5a), we focused on the correlation of PAR-4 and cIAP1. First, we analysed whether PAR-4 cleavage correlated with cIAP1 depletion in a time-dependent manner. BT-20 cells were stimulated with Etoposide for increasing length of time (Fig. 5b). PAR-4 cleavage was detectable after 12-14 hours (Fig. 5b). The appearance of cleaved PAR-4 correlated with a decrease in cIAP-1 expression while at earlier time points no effect of Etoposide on cIAP1 could be measured (Fig. 5b). To analyse whether PAR-4 was required for DNA damage-induced cIAP1 depletion, we compared PAR-4-deficient cells with control cells after Etoposide treatment. Whereas caspase-8 activation and cIAP1 depletion was efficiently induced in control TNBCs, it was significantly inhibited in PAR-4-depleted cells (Fig. 5c). To determine whether cIAP1 overexpression was capable to protect BT-20 cells from apoptosis and caspase-8 activation, we overexpressed cIAP1 WT and a catalytic inactive cIAP1-H588A mutant and stimulated the cells with Etoposide. Only the wild-type cIAP1 was able to inhibit induction of apoptosis, caspase-8 activation and PAR-4 cleavage, whereas the catalytically inactive mutant failed to do so (Fig. 5d). In summary, these data suggested that PAR-4 expression is required to induce cIAP1 depletion in response to DNA damage and subsequent caspase-8 activation. Furthermore, ectopic expression of cIAP1 interferes with these processes.
Loss of cIAP1 sensitizes TNBCs to DNA damage-induced apoptosis when PAR-4 expression is depleted. The expression of IAP proteins, including cIAP1, is frequently altered in various human cancers and its expression is often associated with disease progression and poor prognosis 32,33 . Consequently, IAP proteins are considered as candidate drug targets for therapeutic intervention. Small-molecule inhibitors, referred to as Smac (second mitochondrial-derived activator of caspases) mimetics (SM), have been developed to target IAPs and are currently undergoing clinical trials for cancer treatment 34,35 . As loss of PAR-4 prevented cIAP1 depletion and induction of apoptosis after genotoxic drug treatment (Fig. 5c), we analysed whether depletion of cIAP1 could revert this effect. In agreement with previous reports, treatment of TNBCs with the Smac mimetic LCL161, which www.nature.com/scientificreports www.nature.com/scientificreports/ specifically promotes degradation of cIAP1 and cIAP2 36,37 , resulted in cIAP1 depletion (Fig. 6a). We observed that pre-treatment of PAR-4 depleted TNBCs with SM led to an efficient activation of caspase-8 and induction of apoptosis after Etoposide treatment, strongly suggesting that the chemo-resistance induced by PAR-4 deficiency can be rescued by cIAP1 antagonists (Fig. 6a). This was further confirmed by measuring apoptotic cell death in BT-20 and MDA-MB-468 cells using flow cytometry ( Fig. 6b and Supplementary Fig. S2). To verify these data we depleted cIAP1 expression using cIAP1-specific siRNA and confirmed that the loss of cIAP1 in PAR-4 deficient and drug resistant TNBCs sensitized both cell lines to DNA damage-induced apoptosis (Fig. 6c). Together, these data indicate that PAR-4 sensitizes TNBC cells to DNA damage via regulation of cIAP1 degradation.

Discussion
TNBCs are a heterogeneous mix of breast cancers defined by lack of ER and PR expression and absence of amplification of the HER2/neu oncogene. Therefore, patients with triple-negative breast cancer do not benefit from hormonal or trastuzumab-based therapies and standard treatment includes mainly DNA damaging chemotherapy 38 . Although TNBC patients are reported to initially respond to genotoxic chemotherapy, the overall survival of these patients is still poor 39 . Thus, identifying new strategies to overcome drug resistance of TNBC cells may have a therapeutic benefit.
In a recent study the pro-apoptotic tumour suppressor protein PAR-4 was identified as a critical negative regulator of residual cell survival and recurrence in mice and humans 21 . Furthermore, low PAR-4 expression was found to be associated with a poor response to genotoxic chemotherapy and an increased risk for disease relapse in breast cancer patients 21 . Therefore, therapies that either target PAR-4 effector pathways or restore PAR-4 expression may be promising for therapeutic intervention. Interestingly, PAR-4 is transcriptionally repressed by activation of the PI3K-AKT-mTOR signalling pathway and forkhead transcription factors like Foxo3a were identified to mediate this transcriptional regulation of PAR-4 40 . Importantly, Foxo-dependent upregulation of PAR-4 was found to prevent cell survival following treatment with drugs targeting the PI3K-AKT pathway 40 .
In the present study we have elucidated the effector pathways regulated by PAR-4 in response to DNA damaging chemotherapy in two triple negative breast cancer cell lines. We found that loss of PAR-4 interferes with DNA damage-induced depletion of cIAP1 and subsequent caspase-8 activation. Moreover, targeting cIAP1 with Smac mimetics or RNA interference reverted drug resistance induced by the loss of PAR-4 in TNBC cells.
We and others have previously reported that PAR-4 is a caspase target 28,[41][42][43] . Our studies have demonstrated that caspase-8 cleaves PAR-4 in vitro and mechanistically we have reported that TNFα-induced apoptosis led to caspase-8-mediated PAR-4 cleavage followed by the nuclear accumulation of the C-terminal PAR-4 fragment, which then induces cell death 28 . Although caspase-8 activation is generally thought to be specific to receptor-mediated apoptosis, sequential application of EGFR inhibitors, combined with DNA-damaging chemotherapy has shown before to strongly enhance caspase-8 activation and subsequent cell death in TNBCs 7 . In agreement with this, treatment of TNBCs with the topoisomerase II inhibitors Doxorubicin or Etoposide resulted in an efficient activation of caspase-8 (Fig. 3a,b). Moreover, cleavage of PAR-4 coincided with PARP-1 cleavage, suggesting that DNA damage also triggers caspase-8-mediated PAR-4 cleavage (Fig. 3). Indeed, loss of caspase-8 expression or the inhibition of its activity in BT-20 and MDA-MB-468 demonstrate that DNA damage-induced cell death and PAR-4 cleavage is caspase-8 dependent (Fig. 3). Furthermore, DNA damage-induced PAR-4 cleavage in caspase-3-deficient MCF-7 cells was also sensitive to caspase-8 inhibition and hence PAR-4 functions downstream of caspase-8 (Figs 3e and 7).
To analyse the influence of PAR-4 in the DDR we depleted PAR-4 in BT-20 and MDA-MB-468 cells (Fig. 2a). Our results demonstrate that loss of PAR-4 abrogated DNA damage-induced cell death and surprisingly also prevented caspase-8 activation, indicating that PAR-4 interferes with the activation of the initiator caspase-8 via an unknown feedback mechanism (Fig. 2a). Recent evidence indicates that mammalian inhibitor of apoptosis (IAP) protein family members play a key role in regulating the ripoptosome, a novel cell death-inducing platform that includes caspase-8 31,44 . The ripoptosome assembles in response to DNA damage-induced depletion of XIAP, cIAP1 and cIAP2 and can stimulate caspase-8-mediated apoptosis 31 . A negative correlation between PAR-4 and XIAP has already been observed in Par-4 knockout mice, where Xiap levels were enhanced in primary mouse embryo fibroblasts and in the uteri of female mice 12,30 . Indeed, DNA damage resulted in depletion of XIAP, cIAP1 and cIAP2 in TNBCs with the strongest effect on cIAP1 (Fig. 5a). A time course analysis after induction of genotoxic stress confirmed simultaneous PAR-4 cleavage and cIAP1 depletion (Fig. 5b). Moreover, ectopic expression of cIAP1 prevented genotoxic stress-induced caspase-8 activation, whereas a cIAP1 inactive mutant failed to do so (Fig. 5d). Importantly, loss of PAR-4 completely blocked DNA damage-induced depletion of cIAP1, which was required for genotoxic stress-induced caspase-8 activation (Fig. 5c). DNA damage induced the cleavage of PAR-4 and only the C-terminal fragment translocated into the nucleus where it induced cell death, whereas a cleavage resistant mutant failed to do so (Fig. 4). Only the C-terminal part induced depletion of cIAP-1 and simultaneous caspase-8 activation in TNBC cells (Fig. 4f). Hence caspase-8-mediated PAR-4 cleavage resulted in nuclear translocation of the C-terminal part and induced cIAP1 depletion by an unknown mechanism, which is required for full activation of caspase-8.
Finally, we analysed whether inhibition of cIAP1 can overcome the resistance to genotoxic stress induced by the loss of PAR-4 (Fig. 6). Smac mimetics are small-molecule inhibitors that mimic Smac, an endogenous antagonist of cIAP1, cIAP2 and XIAP. The interaction of Smac mimetics with cIAP proteins promotes their autoubiquitination and proteasomal degradation 34,35 . Indeed, depleting cIAP1 with the Smac mimetic compound and analysed by confocal microscopy. (f) BT-20 and MDA-MB-468 cells were transiently transfected with 1 μg of the C-terminal CFP-tagged PAR-4 WT, PAR-4 D131G and the deletion mutant PAR-4 132-340 (PAR-4 132-340-eCFP) and incubated for 24 h. Cell lysates were analysed by immunoblotting using antibodies recognizing GFP, cleaved caspase-8, PARP-1 and GAPDH. (b,d,f) Full-length blots are presented in Supplementary Fig. S5. www.nature.com/scientificreports www.nature.com/scientificreports/ LCL161 completely abrogated DNA damage-induced resistance mediated through the loss of PAR-4 (Fig. 6a,b). Furthermore, silencing cIAP1 with RNAi in TNBCs confirmed the specificity for cIAP1 as an important mediator in genotoxic stress-induced resistance as a consequence of down regulation of PAR-4 (Fig. 6c). Currently, several Smac mimetics are under evaluation in clinical trials either in monotherapy or in combination with cytotoxic therapies 34,35 .
In summary, our data suggest an important role for caspase-8 in the execution of cell death in response to DNA-damaging chemotherapy in TNBC. This is also supported by the fact that CASP8 truncation mutations were identified in a large screen aiming to identify somatic driver mutations in breast cancer 45 . Caspase-8-induced PAR-4 cleavage results in the nuclear accumulation of the C-terminal fragment, which induces cIAP1 depletion and thereby facilitates full activation of caspase-8 in a feedback loop (Fig. 7). Moreover, loss of PAR-4 in TNBCs completely prevents cIAP1 depletion induced by genotoxic stress and results in drug resistance. Importantly, targeting cIAP-1 with Smac mimetics in TNBC overcomes chemo-resistance induced by down regulation of PAR-4. Together, this suggests that tumour cells with low PAR-4 expression might be particularly vulnerable to Smac www.nature.com/scientificreports www.nature.com/scientificreports/ mimetics combined with chemotherapy. As low PAR-4 expression is associated with poor response to neoadjuvant chemotherapy and an increased risk of relapse in breast cancer patients 21 , combining Smac mimetics with chemotherapy might be a promising treatment option for these patients.  www.nature.com/scientificreports www.nature.com/scientificreports/ achieve a stable Caspase-8 knockdown the following plasmids from the Thermo Scientific GIPZ lentiviral shRNA library were used: Caspase-8 (V2LHS_112733; referred to as shCaspase-8) or non-silencing control (RHS4346; referred to as shControl). Lentiviral transduction procedures were carried out as described before 28 , and 2 μg/ ml puromycin (Sigma) to the medium of BT-20 or MDA-MB-468 cells. Transient transfections of BT-20 or MDA-MB-468 cells was carried out by using Lipofectamine ® 2000 (Invitrogen) according to the manufacturer's instructions. A transient knockdown of PAR-4 was achieved using two pre-designed siRNAs constructs directed against human PAR-4 (SI02628997 and SI03071782; referred to as siPAR-4 #1 and siPAR-4 #2) at a final concentration of 20 nM and compared to a non-silencing siRNA (SI03650325; referred to as siControl) (Qiagen). A knockdown of cIAP1 was achieved using siRNA constructs directed against human cIAP-1 (sc-29848; referred to as sicIAP1) (Santa Cruz). Transfection was achieved using Oligofectamine TM (Invitrogen) transfection reagent according to the supplier's instruction. Cloning and mutagenesis. Generation of pcDNA5/FRT/TO-PAR-4(132-340)-eCFP, pcDNA5/FRT/ TO-PAR-4wt-eCFP, pcDNA5/FRT/TO-PAR-4(D131G)-eCFP and pcDNA5/FRT/TO-eYFP-PAR-4(1-131) was described before 28 . Plasmids for pcDNA3-myc-cIAP1 and pcDNA3-myc-cIAP1 mutant (H588A) were provided by S. Schreek.
Flow cytometry analysis. TNBCs were washed once in PBS and fixed with 80% (v/v) ethanol, stored at −20 °C, for 30 min. After fixation, cells were resuspended in PBS and to avoid unspecific staining of RNA DNAse free RNAse (20 μg/ml, Roche) was added and incubated for 5 min at RT. Finally, propidium iodide (Sigma) was applied to a final concentration of 50 μg/ml and cells were incubated protected from light for 20 min at RT. Cell