|Home | About | Journals | Submit | Contact Us | Français|
Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive non-Hodgkin lymphomas. It is curable but one-third of cases are refractory to therapy or relapse after initial response highlighting the urgent need for developing novel therapeutic approaches. Targeting sirtuins, particularly SIRT1 by genetic approaches or using pharmaceutical inhibitor tenovin-6, has shown promising therapeutic potential in various hematopoietic malignancies. However, it remains unknown whether these approaches are effective for DLBCL. In this study, we have found that tenovin-6 potently inhibits the proliferation and survival of DLBCL cells. Surprisingly, specific knockdown of SIRT1/2/3 has no effect on DLBCL. Mechanistically, tenovin-6 increases the level of microtubule-associated protein 1 light chain 3B (LC3B)-II in a SIRT1/2/3- and p53-independent manner in DLBCL cell lines. Tenovin-6-mediated increase of LC3B-II is through inhibition of classical autophagy pathway. Furthermore, inhibition of the autophagy pathway by using other inhibitors or by knocking down key genes in the pathway impairs cell proliferation and survival of DLBCL cells. These results indicate that targeting the autophagic pathway could be a novel therapeutic strategy for DLBCL and that precaution should be taken to interpret data where tenovin-6 was used as an inhibitor of sirtuins.
Diffuse large B cell lymphoma (DLBCL), the most common B cell tumor in adults, displays marked clinical heterogeneity. Indeed, first-line immunochemotherapy (R-CHOP-Rituximab, Cyclophosphamide, Doxorubicin Hydrochloride, Oncovin and Prednisone) cures only ~60% of the patients . An emerging consensus in the field is that a better understanding of DLBCL biology may be the most effective way to improve the survival rate of patients who are refractory or have a relapse . To that end, there has been steady progress in elucidating the pathogenesis of DLBCL. In earlier studies, gene expression profiling analyses have defined DLBCL signatures that are associated with a putative cell-of-origin (germinal center and activated B cell, GCB- and ABC-DLBCL) or mechanisms of transformation (consensus cluster classification, CCC) [3, 4], observations that have already guided rational clinical initiatives. More recently, massively parallel sequencing uncovered previous unsuspecting genetic lesions in DLBCL , thus suggesting that important elements of DLBCL biology remain to be elucidated creating novel opportunities for tailored therapeutic interventions.
Sirtuins are highly conserved NAD+-dependent protein lysine modifying enzymes with deacetylase, ADP-ribosyltransferase and/or deacylase activities. Among this family of proteins, SIRT1 is the most-studied member, and is a key regulator for a wide variety of physiological and pathological processes such as genome stability, metabolism, energy homeostasis, organ development, aging and cancer [6–8]. It has been shown that SIRT1 promotes the survival and/or chemoresistance of various hematopoietic malignancies including chronic myelogenous leukemia [9–11], acute myeloid leukemia , acute lymphoblastic leukemia , and Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells . Targeting SIRT2 can also induce granulocytic differentiation in acute promyelocytic leukemia . Despite these works, it remains unknown whether SIRT1, SIRT2 and other sirtuins have any roles in DLBCL and whether they can be exploited as therapeutic targets.
Tenovin-6, a small molecular compound, has drawn much attention because it can potently activate p53, and directly inhibit the protein deacetylase activity of purified human SIRT1, SIRT2 and SIRT3 [16, 17]. Tenovin-6 has since been used as an inhibitor of sirtuins, especially SIRT1, and has been demonstrated to have a promising anti-neoplastic effect in vitro and in vivo on various hematopoietic malignancies of both lymphoid and myeloid lineages [9–13, 15, 18, 19]. However, whether tenovin-6 is effective against DLBCL has not been investigated so far.
In this study, we aim to determine whether targeting sirtuins by using genetic approaches or pharmaceutical inhibitor tenovin-6 has any effects on DLBCL. We demonstrated that tenovin-6 could significantly inhibit the proliferation and survival of DLBCL cell lines through SIRT1/2/3-independent inhibition of autophagy.
To test whether tenovin-6 had an inhibitory effect on DLBCL, we treated 2 GCB-type DLBCL cell lines OCI-Ly1 and DHL-10, and 4 ABC-type DLBCL cell lines U2932, RIVA, HBL1 and OCI-Ly10 with 0, 1, 2.5, 5 or 10 μM tenovin-6, and counted the viable cells every day for 3 days. Tenovin-6 potently inhibited cell proliferation in a dose- and time-dependent manner in all 6 cell lines (Figure (Figure1A).1A). Examination of cell cycle progression by BrdU (5-bromo-2′-deoxyuridine) and PI (propidium iodide) staining showed that the percentages of cells in G1 phase were significantly increased while the percentages of cells in S phase were significantly decreased in a dose-dependent manner in all 6 cell lines at 24 h post-treatment (Figure (Figure1B1B and and1C).1C). Furthermore, tenovin-6 induced apoptosis in a dose- and time-dependent manner in all 6 cell lines (Figure (Figure1D1D and and1E).1E). These results indicated that tenovin-6 potently inhibited cell proliferation and survival of DLBCL cells.
To determine the roles of sirtuins in DLBCL, we examined the expression of sirtuins in 10 different cell lines (Supplementary Figure 1). All seven members of sirtuins were expressed in all the 10 cell lines examined though the expression levels varied among the cell lines. Since tenovin-6 is thought to be specific to SIRT1, 2 and 3 , we performed knockdown of these sirtuins with specific shRNAs. We achieved 90% knockdown of SIRT1 in OCI-Ly1, U2932 and RIVA cells, and SIRT2 or SIRT3 in OCI-Ly1 cells (Figure (Figure2A).2A). However, knockdown of SIRT1, 2 or 3 affected neither the cell proliferation (Figure (Figure2B)2B) nor apoptosis (Figure (Figure2C)2C) of these cells. We then assessed the effects by combining shRNAs to different sirtuins (Figure (Figure2D).2D). Again, we failed to observe any significant effect on cell proliferation (Figure (Figure2E)2E) and apoptosis (Figure (Figure2F).2F). These results indicated that the effect of tenovin-6 was not due to its inhibitory effect on SIRT1, 2 and 3.
To identify the mechanism of tenovin-6 inhibition of DLBCL cells, we examined p53 acetylation as tenovin-6 was initially identified as a p53 activator through inhibition of SIRT1-mediated deacetylation of p53 . We detected increased p53 acetylation in OCI-Ly1, HBL1 and OCI-Ly10 cells but not in DHL-10, U2932 and RIVA cells (Figure (Figure3A).3A). p53 is mutated in OCI-Ly1 , DHL-10 , U2932 [22, 23], RIVA (also known as RI-1, Broad-Novartis Cancer Cell Line Encyclopedia, https://www.broadinstitute.org/ccle), and HBL1  while it is intact in OCI-Ly10 cells . To further determine whether p53 was activated through other mechanisms following tenovin-6 treatment, we examined p53 phosphorylation and p53 downstream targets Bax, Puma and p21 in OCI-Ly1 cells. While p53 phosphorylation (Ser15) and Puma level increased at 48 h after tenovin-6 treatment, the levels of other p53 downstream targets Bax and p21 did not (Figure (Figure3B).3B). These results indicated that p53 activation was unlikely the mechanism that mediated the observed effect of tenovin-6 on DLBCL cells.
In contrast to the conflicting results of p53 activation, tenovin-6 consistently increased the level of autophagy marker LC3B-II in all the DLBCL cell lines (Figure (Figure3A).3A). In addition, tenovin-6 increased the expression of LC3A-II in U2932, RIVA and HBL1 cells but not in OCI-Ly1 and OCI-Ly10 cells (Supplementary Figure 2). Interestingly, we did not observe any obvious SQSTM1/p62 degradation (Figure (Figure3A3A and and3C),3C), suggesting that tenovin-6 might inhibit autophagy in these cells. To confirm the increase of LC3B-II, we performed immunofluorescence assay in OCI-Ly1 cells using a LC3B specific antibody. The increased signal of LC3B and the puncta structures were readily observed under confocal microscope after treatment with 2.5 μM and 5 μM tenovin-6 for 16 h (Figure (Figure3D).3D). Together, these results indicated that LC3B-II but not p53 activation was consistently increased after tenovin-6 treatment in DLBCL cell lines.
It has been shown that tenovin-6 can directly inhibit the activities of SIRT1, 2 and 3, and activate p53 . Because SIRT1, 2 and 3 as well as p53 also regulate the expression of LC3B-II [25–30], we investigated whether they might mediate LC3B-II increase in DLBCL cells following tenovin-6 treatment. The results showed that there was no correlation between the increase of LC3B-II level and p53 activation (Figure (Figure3A).3A). Furthermore, examination of time kinetic in OCI-Ly1 cells showed that tenovin-6 increased LC3B at as early as 0.5 h following treatment while it did not affect the level of p53 phosphorylation (Ser15) after up to 8 h of treatment (Figure (Figure3C).3C). Hence, p53 was not responsible for the increased LC3B-II level.
We further examined LC3B expression and p53 acetylation in OCI-Ly1 cells following knockdown of SIRT1/2/3 or treatment with tenovin-6. LC3B-II was decreased by 40% following knockdown of SIRT1 and increased by 1.6- and 1.7-fold following knockdown of SIRT2 and SIRT3, respectively (Figure (Figure4A)4A) while it was increased by 4.2- and 7.3-fold following treatment with 2.5 μM and 5 μM tenovin-6, respectively (Figure (Figure4B).4B). p53 acetylation was slightly increased following knockdown of SIRT1 but remained unchanged following knockdown of SIRT2 or SIRT3 (Figure (Figure4C).4C). p53 acetylation did not increase until 24 h following treatment with 2.5 μM or 5 μM tenovin-6. However, LC3B-II started to increase at as early as 0.5 h following treatment with tenovin-6 (Figures (Figures3C3C and and4D).4D). These results indicated that the increase of LC3B-II after tenovin-6 treatment was independent of SIRT1/2/3 and p53. Our results confirmed those of previous studies in chronic lymphocytic leukemia  and soft tissue sarcoma cells , showing that the effect of tenovin-6 was p53-independent.
It has been shown that p27 is highly correlated with LC3B-II induction . To test whether the effect of tenovin-6 on LC3B was through regulating p27 expression, we examined p27 expression at different time points after tenovin-6 treatment. The results showed that tenovin-6 treatment did not affect p27 expression (Figure (Figure3C3C).
We observed significant apoptosis at 48 h after tenovin-6 treatment (Figure (Figure1D1D and and1E).1E). Apoptosis can be initiated by extrinsic or intrinsic pathway. Induction of apoptosis leads to activation of an initiator caspase: caspase-8 for the extrinsic pathway and caspase-9 for the intrinsic pathway . To determine the apoptotic pathway triggered by tenovin-6, we examined apoptosis-related markers including cleaved PARP-1, cleaved caspase 3, cleaved caspase 8 and cleaved caspase 9 in OCI-Ly1 cells. Tenovin-6 treatment induced both cleaved PARP-1 and cleaved caspase 3 (Figure (Figure5),5), which was consistent with the detection of increased apoptotic cells (Figure (Figure1D1D and and1E).1E). Furthermore, we observed cleavage of caspase 8 but not caspase 9 after tenovin-6 treatment (Figure (Figure5),5), indicating that tenovin-6 likely activated the extrinsic but not the intrinsic cell-death pathway.
While LC3B-II is an autophagy marker, its level may be regulated at either transcriptional or post-translational level. Treatment of OCI-Ly1 cells with 2.5 μM or 5 μM tenovin-6 for 2 h or 4 h did not significantly alter the mRNA level of LC3B (Figure (Figure6A)6A) indicating that tenovin-6 did not transcriptionally regulate LC3B. ATG5 is essential for LC3-II processing in the autophagy pathway [34, 35]. To determine if the autophagy pathway was involved in regulating LC3B-II level, we first knocked down ATG5 in OCI-Ly1 cells and then treated the cells with tenovin-6. Knockdown of ATG5 significantly compromised the increase of LC3B-II caused by tenovin-6 treatment (Figure (Figure6B),6B), indicating that the classical autophagy pathway was required for tenovin-6-mediated increase of LC3B-II. Increased LC3B-II expression can be explained by either induction of autophagy or inhibition of autophagy flux. If autophagy is induced, SQSTM1/p62 should be degraded. On the other hand, if the autophagy flux is inhibited, SQSTM1/p62 should accumulate or remain unchanged. Since no SQSTM1/p62 degradation was observed following tenovin-6 treatment (Figures 3A, 3C, 4B and 4D), it suggested that tenovin-6 might inhibit the autophagic flux.
To confirm the inhibitory effect of tenovin-6 on autophagic flux, we treated OCI-Ly1 cells with tenovin-6 in the presence of a known autophagic flux inhibitor bafilomycin A1, and examined the effect on LC3B and SQSTM1/p62. If tenovin-6 is an inducer of the autophagic flux, the combined treatment of tenovin-6 with a saturated inhibitor like bafilomycin A1 should lead to an additive effect on LC3B expression. However, if tenovin-6 is an inhibitor of the autophagic flux, there should not be any additive effects because bafilomycin A1 should have completely blocked the autophagic flux. We did not observe any additive effect following combined treatment of 2.5 μM or 5 μM tenovin-6 with saturated bafilomycin A1, indicating that tenovin-6 was indeed an inhibitor of the autophagic flux (Figure (Figure6C,6C, Supplementary Figure 3). In contrast, combined treatment of rapamycin, an autophagy inducer, with bafilomycin A1 indeed showed an additive effect on the increased level of LC3B (Figure (Figure6C,6C, Supplementary Figure 3). Rapamycin alone induced SQSTM1/p62 degradation as expected ; however, it was inhibited by tenovin-6 or bafilomycin A1, further confirming tenovin-6 as an inhibitor of the autophagic flux.
To further confirm the inhibitory effect of tenovin-6 on autophagic flux, we examined the ultrastructure change of cultured OCI-Ly1 cells following tenovin-6 treatment by transmission electron microscopy analysis. We found that tenovin-6-treated cells had an increased number of double-membrane bound vacuoles within the cytoplasm. These vacuoles contained cellular debris and recognizable cytoplasmic organelles, confirming that they were accumulated autophagosomes (Figure (Figure6D6D).
Because both tenovin-6 and bafilomycin A1 had similar inhibitory effects on the autophagic flux, we proposed that tenovin-6 might affect the function of lysosomes/autolysosomes leading to the inhibition of autophagy. Furthermore, our recent study has revealed that tenovin-6 inhibits the classical autophagy pathway by impairing lysosomal function . LysoTracker is a cell-permeable pH-dependent dye that accumulates in acidic vesicles such as lysosomes or autolysosomes in live cells and emits a red fluorescence signal. LysoTracker staining has been commonly used to monitor the function of lysosome/autolysosome [38–41]. To determine whether tenovin-6 also inhibited the classical autophagy pathway by impairing lysosomal function in DLBCL cells, we treated OCI-Ly1 cells with tenovin-6, bafilomycin A1 or chloroquine, which is known to inhibit autophagy by elevating lysosomal pH, for 1 h, then stained the cells with LysoTracker. Tenovin-6, bafilomycin A1 or chloroquine significantly reduced the signal of LysoTracker staining (Figure (Figure6E),6E), suggesting that the function of lysosomes/autolysosomes were indeed affected by these inhibitors.
The results so far suggested that the effect of tenovin-6 on DLBCL cells could be due to its inhibitory effect on autophagic flux rather than sirtuins. To test whether inhibition of the autophagic flux was sufficient to reduce cell proliferation and survival in DLBCL cells, we examined the inhibitory effects of known autophagy inhibitors bafilomycin A1 and chloroquine on cell proliferation and survival of OCI-Ly1 cells. Indeed, both bafilomycin A1 and chloroquine inhibited cell proliferation (Figure (Figure7A7A and and7B)7B) and induced apoptosis (Figure (Figure7C7C and and7D)7D) in a time- and dose-dependent manner. To further confirm these results, we performed knockdown of LC3B, ATG5 and SQSTM1/p62 genes (Figure (Figure7E),7E), which are critical for autophagosome formation at the early stage during the induction of the autophagy pathway . Knockdown of LC3B, ATG5 or SQSTM1/p62 inhibited cell proliferation (Figure (Figure7F)7F) and induced apoptosis in OCI-Ly1 cells (Figure (Figure7G).7G). Importantly, treatment with tenovin-6 together with knockdown of LC3B, ATG5 or SQSTM1/p62 had an additive effect on the survival of cells (Figure (Figure7G).7G). These results indicated that approaches by combining inhibitors targeting different stages of the autophagy pathway might lead to better therapeutic outcomes for patients with DLBCL.
The roles of sirtuins in cancer are complex and may either promote or suppress tumorigenesis depending on the cellular and molecular context . In the present study, knockdown of SIRT1, 2 and 3 in DLBCL cells has no significant effect on cell proliferation and survival. However, the inhibitor of sirtuins tenovin-6 has a dramatic inhibitory effect on all 6 DLBCL cell lines examined, of which 4 belong to the ABC-type, a subset of incurable DLBCL. Tenovin-6 induced cell proliferation arrest and apoptosis in these DLBCL cells, strongly suggesting that tenovin-6 might have a different target other than sirtuins. This assumption is further supported by other evidences: 1) While in most published studies, tenovin-6 was used in the range of 1–5 μM, which is much lower than the IC50 values of 21, 10 and 67 μM for the purified human SIRT1, SIRT2 and SIRT3, respectively, it still demonstrated a strong effect on cell proliferation and survival [9–13, 15, 18, 19, 31, 43–47]; and 2) There was little or no correlation between acetylomes of cells with specific knockout of SIRT1 or SIRT2 and tenovin-6 treatment . These results along with those of the current study suggest that precaution should be taken to interpret the data where tenovin-6 was used as an inhibitor of sirtuins. In the current study, we have revealed that tenovin-6 impairs autophagy, which is the underlying mechanism for its effect on DLBCL cells. In a recent study, we have examined the effect of tenovin-6 in several types of normal cells, mouse embryonic fibroblasts, a hepatocarcinoma cell line Huh7, a lung adenocarcinoma epithelial cell lines A549, a KSHV-transformed cell line KMM, and a DLBCL cell line OCI-Ly1 . The results also showed that tenovin-6 inhibited the autophagy pathway. However, the effectiveness of using tenovin-6 as a potential therapeutic agent for DLBCL remains unknown. In the current study, we have, for the first time, shown tenovin-6 is effective against diverse cell lines of DLBCL of different genotypes, and hence can be used as a potential therapeutic agent for DLBCL.
Autophagy is an evolutionarily conserved cellular homeostatic process in response to starvation or other stress conditions in which proteins, organelles and intracellular pathogens are engulfed into autophagosomes and eventually delivered to lysosomes for degradation. The autophagy pathways are involved with a large number of protein complexes. The ATG1 complex, the ATG2-ATG18 complex, the class III phosphatidylinositol 3-kinase (PI3K) complex, the transmembrane protein mATG9 complex (and its vesicular trafficking along with ATG16L1), and the ATG8 and ATG12 ubiquitin-like conjugation complexes are critical for autophagosome formation. Cytoskeletons, RAB GTPases, HOPS (homotypic fusion and vacuolar protein sorting) complex, and SNAREs are important for autophagosome-lysosome fusion. The blockage of autophagy can disrupt its protective effect against starvation and other stresses, and cause cell cycle arrest and cell death .
The role of autophagy in cancer has been extensively studied and the results have been reviewed [50–54]. It is now well established that autophagy functions as a suppressor in tumor initiation at the early stage of tumor progression while it promotes tumor progression at the advanced stages of tumor development. The established tumors appear to utilize autophagy for surviving the stresses in the tumor microenvironment including low oxygen, low nutrients, excess toxicities and attack of immune cells [50–54]. Autophagy is activated in cancer cells undergoing treatment with different chemotherapeutic drugs such as cisplatin, etoposide, 5-fluorouracil and doxorubicin, and genetic knockdown of autophagy genes or pharmacological inhibition of autophagy with the use of chloroquine, 3-methyladenine or bafilomycin A1 sensitizes cancer cells to chemotherapeutic drugs [55–60]. These studies suggest that suppression of the autophagic pathway can be a novel strategy for anticancer therapy. Currently, various clinical trials are investigating the effects of combining autophagy inhibitors with cytotoxic chemotherapies and targeted agents in solid and hematological cancers. The early results of some of these clinical studies indicate that autophagy inhibition in combination with anticancer therapies seem to be safe and can augment the efficacies of various anticancer therapies in melanoma, glioblastoma and multiple myeloma [50, 53]. In addition to the existing autophagy inhibitors such as hydroxychloroquine and chloroquine, which are approved for treatment of malaria by US FDA, identification of novel and highly effective autophagy inhibitors is obviously needed. Tenovin-6 seems to be a good candidate for use in combined chemotherapies: First, this study has demonstrated tenovin-6 as a potent autophagy inhibitor; Second, tenovin-6 seems to be safe since no obvious adverse effect has been reported so far in multiple studies in the mice [9, 11, 12, 17, 31]; and Third, tenovin-6 alone has shown a promising anti-neoplastic effect in vitro or in vivo on various hematopoietic and solid malignancies [9–13, 15, 18, 19, 31, 43–47].
The information on the role of autophagy in DLBCL cells is very limited so far. Only one specific study has been published, showing that proteasome inhibitor bortezomib-induced autophagy conferred relative drug resistance to DLBCL cells by eliminating IκBα, and the inhibition of both autophagy and proteasome had demonstrated a great potential for killing apoptosis-resistant DLBCL cells . In a relevant clinical trial in dogs with non-Hodgkin's lymphoma, the combined treatment of autophagy inhibitor hydroxychloroquine (12.5 mg/kg/d) with a lower dose of doxorubicin (25 mg/m2) has resulted in clinical benefit with superior overall response rate (93.3%) and comparable median progression-free interval (5 months) compared to standard single agent doxorubicin (30 mg/m2) treatment . In the current study, we have found that inhibition of autophagy, either by using inhibitors or by specifically knocking down key genes of the autophagy pathway, can induce cell proliferation arrest and apoptosis in DLBCL cells (Figure (Figure7H).7H). Our results have illustrated an essential protective role of autophagy in DLBCL cells and strongly suggested that targeting autophagy may represent a novel strategy for overcoming the chemoresistance, which is the major current challenge for treating DLBCL.
The following reagents were used in this study: tenovin-6 (BSCC-37, Agave Pharm, Seattle, WA), chloroquine diphosphate (C6628, Sigma, St. Louis, MO), bafilomycin A1 (B1793, Sigma), rapamycin (R8781, Sigma), and LysoTracker Red DND-99 (L7528, Thermo Fisher Scientific, Waltham, MA).
The antibodies used were: LC3B (CTB-LC3-1-50, Cosmo Bio, Carlsbad, CA), LC3A (ab62720, Abcam, Cambridge, MA), SQSTM1/p62 (PM045, MBL, New York, NY), ATG5 (#2630, Cell Signaling Technology, Danvers, MA), β-actin (sc-8432, Santa Cruz, Dallas, TX), β-tubulin (T8453, Sigma), p53 (DO-1, sc-126, Santa Cruz), acetyl-p53 (K382) (#2524, Cell Signaling Technology), Phospho-p53 (S15) (#9284, Cell Signaling Technology), Bax (#2772, Cell Signaling Technology), Puma (#4976, Cell Signaling Technology), p21 (#556430, BD Biosciences, Franklin Lakes, NJ), PARP-1 (#9532, Cell Signaling Technology), c-caspase 3 (#9664, Cell Signaling Technology), caspase 8 (#9746, Cell Signaling Technology), c-caspase 9 (SC-7885, Santa Cruz), SIRT1 (#8469, Cell Signaling Technology), SIRT2 (#12672, Cell Signaling Technology), SIRT3 (#2627, Cell Signaling Technology), SIRT4 (NB100-1406, Novus, St Charles, MO), SIRT5 (#8782, Cell Signaling Technology), SIRT6 (#2590, Cell Signaling Technology), and SIRT7 (#5360, Cell Signaling Technology).
HEK 293T cells were cultured in Dulbecco's Modified Eagle's Medium. RIVA, U2932, HBL1, SU-DHL4, SU-DHL5, SU-DHL6 and SU-DHL10 cells were cultured in RPMI-1640 media. OCI-Ly1, OCI-Ly3 and OCI-Ly10 were cultured in Iscove's Modified Dulbecco's Medium. All media was supplemented with 100 μg/mL penicillin, 100 μg/mL streptomycin and 10% fetal bovine serum (#26140-079, Thermo Fisher Scientific) except that 20% fetal bovine serum was used for OCI-Ly3 and OCI-Ly10. All cells were maintained in a 5% CO2 atmosphere at 37°C.
The ATG5 shRNA (TRCN0000150940, 5′-GCAGAACCATACTATTTGCTT-3′), LC3B shRNA (#1: TRCN0000154060, 5′-CGCTTACAGCTCAATGC TAAT-3′ and #2: TRCN0000155417, 5′-CCTGACCAT GTCAACATGAGT-3′), and SQSTM1/p62 shRNA (TRCN0000007236, 5′-CCGAATCTACATTAAAGAGA A-3′) were purchased from Sigma. The SIRT1 shRNA (#1: 5′-GAAGTGCCTCAGATATTAA-3′ and #2: 5′-GTTGACCTCCTCATTGTTA-3′), SIRT2 shRNA (#1: 5′-GCTTATTGGAGACAAATTA-3′ and #2: 5′-GAAACATCCGGAACCCTTC-3′) and SIRT3 shRNA (5′-GGAGTGGCCTGTACAGCAA-3′) were constructed as previously described . A scrambled shRNA (5′-TTGTACTACACAAAAGTACTG-3′) was constructed as a control. The lentivirus was generated and concentrated as previously described . Cells were transduced with the lentivirus particles by spinning infection at 1,800 rpm for 60 min in the presence of 8 μg/ml polybrene at MOI 12. At 48 h post-transduction, cells were selected with 2.5 μg/ml puromycin for 3 days, cultured for another 3 days and used for the experiments.
BrdU-PI staining was performed as previously described . Briefly, 20 μM BrdU (B5002, Sigma) was added into the culture media for 4 h, fixed and stained with a Pacific Blue monoclonal antibody to BrdU (B35129, Thermo Fisher Scientific) and propidium iodide (P4864, Sigma) for DNA. PE-Cyanine 7-conjugated anti-annexin V antibody (25-8103-74, eBioscience, San Diego) was used to detect apoptosis following the instructions of the manufacturer. Samples were run in a FACSCanto System (BD Biosciences) and analyzed with FlowJo (Treestar, Ashland, OR).
qPCR was performed as previously described . Briefly, total RNA was isolated with TRI reagent (T9424, Sigma). Reverse transcription was performed with total RNA using Maxima H Minus First Strand cDNA Synthesis Kit (K1652, Thermo Fisher Scientific,). qPCR analysis was performed on Eppendorf Real Plex using KAPA SYBR FAST qPCR Kits (KK4602, Kapa Biosystems, Wilmington, MA). The relative expression levels of the targeted genes were normalized to the expression of internal control 18S rRNA gene. All reactions were run in triplicate. Sequences for LC3B primers were: human LC3B_F: 5′-CCGCACCTTCGAACAAAGAGT-3′ and LC3B_R: 5′-CACCCTTGTATCGTTCTATTATCACC-3′. Sequences for 18S rRNA primers were: human 18S rRNA_F: 5′-ATCAACTTTCGATGGTAGTCG-3′ and 18S rRNA_R: 5′-TCCTTGGATGTGGTAGCCG-3′.
OCI-Ly1 cells were treated as indicated and cytospun onto glass slides at 800 rpm for 3 min. After fixation with 2% paraformaldehyde for 20 min at room temperature, the cells were washed and blocked with 3% BSA in PBS for 30 min at 37°C. The cells were then incubated with an anti-LC3B antibody (CTB-LC3-1-50, Cosmo Bio) at a 1:500 dilution for 1 h at 37°C. After incubation for 1 h at 37 °C with a secondary antibody (A-11001, ThermoFisher Scientific), samples were counterstained with 0.5 μg/mL 4-,6-diamidino-2-phenylindole (DAPI) in PBS for 5 min, and the slides were mounted in FluorSave Reagent (#345789, Calbiochem, Billerica, MA). Samples were observed with a laser-scanning confocal microscopy (Nikon Eclipse C1).
The cell pellets were fixed with 2% glutaraldehyte in 0.1M cacodylate buffer [Na(CH3)2AsO2·3H2O], pH7.2, at 4°C, overnight. The cell pellets were washed three times with 0.1M cacodylate buffer, pH7.2, post-fixed with 1% OsO4 in 0.1M cacodylate buffer for 30 min and washed three times with 0.1M cacodylate buffer. The samples sequentially dehydrated once with 60%, 70%, 80% and 95% ethanol, twice with 100% absolute ethanol, and twice with propylene oxide were left in propylene oxide/Eponate (1:1) overnight at room temperature. The vials were sealed. The next day the vials were left open for 2–3 h to allow the propylene oxide to evaporate. The samples were infiltrated with 100% Eponate and polymerized at ~64°C for 48 h. Ultra-thin sections (~70 nm thick) were cut using a Leica Ultra cut UCT ultramicrotome with a diamond knife and picked up on 200 mesh copper EM grids. Grids were stained with 2% uranyl acetate for 10 min followed by Reynold's lead citrate staining for 1 min. The grids were observed with an FEI Tecnai 12 transmission electron microscope equipped with a Gatan Ultrascan 2K CCD camera (FEI, Hillsboro, OR).
Cells were stained with 50 nM LysoTracker Red DND-99 (ThermoFisher Scientific) by directly adding this dye to the culture media, incubated for 30 min in a 5% CO2 atmosphere at 37°C, and observed with a Nikon Eclipse microscopy (Tokyo, Japan).
Western-blotting was performed as previously described with minor modifications . Briefly, cells were lysed in RIPA buffer supplemented with 1% SDS and proteinase inhibitor cocktail (1:100) (P8340, Sigma). Protein lysate of 10 μg for each sample was resolved by SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% skim milk, the membrane was probed with primary and then secondary antibodies. The signal was developed with the Luminiata Crescendo Western HRP substrate (WBLUR0500, EMD Millipore, Billerica, MA) and visualized with a Fujifilm LAS-3000 imaging system (Redondo Beach, CA).
Two-tailed t-test was performed, and P < 0.05 (*) and P < 0.01 (**) were considered statistically significant.
We thank members in Dr. S-J Gao's laboratory for technical assistances and helpful discussions.
CONFLICTS OF INTEREST
The authors declare that there are no conflicts of interest in this work.
This work was supported by grants from NIH (CA096512, CA124332, CA132637, CA177377, CA213275, DE025465 and CA197153) to S-J Gao; RCT Aguiar was supported by grants from the CPRIT (RP140452 and RP150277) and a VA Merit Award (I01-BX001882).