Vitamin K2 Prevents Lymphoma in Drosophila

Previous studies have established the anticancer effect of vitamin K2 (VK2). However, its effect on lymphoma induced by UBIAD1/heix mutation in Drosophila remains unknown. Therefore, we aimed to develop an in vivo model of lymphoma for the precise characterization of lymphoma phenotypes. We also aimed to improve the understanding of the mechanisms that underlie the preventative effects of VK2 on lymphoma. Our results demonstrated that VK2 prevents lymphoma by acting as an electron carrier and by correcting the function and structure of mitochondria by inhibiting mitochondrial reactive oxygen species production mtROS. Our work identifies mitochondria as a key player in cancer therapy strategies.

Previous studies have established the anticancer effect of vitamin K2 (VK2). However, its effect on lymphoma induced by UBIAD1/heix mutation in Drosophila remains unknown. Therefore, we aimed to develop an in vivo model of lymphoma for the precise characterization of lymphoma phenotypes. We also aimed to improve the understanding of the mechanisms that underlie the preventative effects of VK2 on lymphoma. Our results demonstrated that VK2 prevents lymphoma by acting as an electron carrier and by correcting the function and structure of mitochondria by inhibiting mitochondrial reactive oxygen species production mtROS. Our work identifies mitochondria as a key player in cancer therapy strategies.
Vitamin K2 (VK2) is a fat-soluble vitamin that is important for human health. It is abundantly present in a variety of foods and usually exists in three forms: phylloquinone (VK1), menaquinone (VK2), and menadione (VK3) 1 . VK2 is produced by a vast array of bacteria 2 and can be produced by animals and humans via the conversion of its other forms 3 .
The antitumor action of vitamin K has been investigated since 1947 4 . In rats, VK3 acts against adriamycin-resistant leukemia cells 5 . VK3, a radiosensitizing agent, extends the survival time of patients with bronchial carcinoma, and VK2 induces growth inhibition via cell cycle arrest 6,7 . VK2 exhibits remarkable antiproliferative effects on different cancer types 2,8 , including leukemia, lung cancer, ovarian cancer, prostate cancer, and hepatocellular cancer 9 . VK2 has anticancer effects against human bladder carcinoma 10 .
The protein product of UBIAD1/heix has multiple enzymatic activities, which include VK2 synthesis 3 , and menaquinone-4 synthesis in human 11 . Drosophila heixudian "heix" gene encodes a protein that bears high sequence identity with the human UBIAD1 protein, loss-of-function of UBIAD1 tends to progress bladder and prostate carcinoma 12 . Loss-of-function of heix leads to hemocyte overproliferation and aberrant differentiation 13 .heix mutants showed severe mitochondrial defects, and VK2 transfers electrons in Drosophila mitochondria, thus improving ATP production 14 .
During the larval stages of Drosophila, hemocytes are produced from a separate organ called the lymph gland 15 and differentiate into two classes of cells: plasmatocytes and crystal cells 16 . Crystal cells are involved in the melanization of pathogenic material in the hemolymph 15 . They are clearly visible because of the Black cell (Bc) mutation, which causes premature melanization 17 . Moreover, crystal cells express the enzyme phenoloxidase (Pro-phenoloxidase A1, PO), which is responsible for the initiation of the melanogenesis cascade 18 . Melanin biosynthesis is induced by PO, which catalyzes the oxidation of phenols to quinones; quinones are subsequently polymerized into melanin 19 . Black spots, which are regarded as melanotic tumors or pseudotumors, are usually associated with crystal cells and are found in various Drosophila mutants 20 .
Previous studies have shown that hemocyte-mediated immune response is involved in larval melanization 21 . These immune responses include cell aggregation, phagocytosis, encapsulation, and melanization cascade induction 18 . Plasmatocytes and lamellocytes, which function in encapsulation, are rare in healthy larvae 22 . The activation of the JAK/STAT and immune-related pathways Toll and IMD is associated with the loss-of-function of UBIAD1/heix 13 in lymphoma and leukemia 23 .
Interestingly, significant similarities are found between the molecular mechanisms that regulate the development of the Drosophila lymph gland and the formation of the mammalian aorta-gonad-mesonephrous region 24 . The average levels of nucleotide diversity are tenfold lower in humans than that in D. melanogaster 25 . A study that utilized next-generation sequencing to compare interspecies and intraspecies variation in humans and Drosophila revealed that 14.9% of human genes and 46.0% of fly genes have orthologs with a maximum identity of 45% 26 . A systematic analysis of human disease-associated gene sequences in Drosophila revealed that 79 out of 714 genes Results UBIAD1/heix mutation leading to development of lymphoma is prevented by VK2. This is the first study to examine the therapeutic effects of VK2 on LiD induced by the heix mutation. Multiple doses of VK2 ranging from 1 mM to 50 mM were tested, and responses were associated with the disappearance of black spots Error bars indicate SEM. Analysis of variance (ANOVA/Dunnett: *P < 0.05, **P < 0.01, ***P < 0.001).
( Fig. S2A) and improvement of the pupal pip-out rate (Fig. S2B). Dose volume was determined by selecting the volume that achieved the optimum vitamin distribution with the complete disappearance of black spots (Fig.  S2C) and improvement of the pupal pip-out rate (Fig. S2D). Moreover, treatment with 30-50 mM VK2 improved movement speed (Fig. S2E). The complete and significant disappearance of black spots was achieved with 25-100 µl of 50 mM VK2 (***P < 0.001) (Fig. 1A). heix mutant larvae often die at the pupal stage 13 . Interestingly, in our research, third-instar larvae fed with VK2 showed significantly increased pip-out rates (***P < 0.001) and a strong response to VK2 (Fig. 1B). heix mutation is associated with impaired flight 14 and reduced activity 13 . Similarly, we observed a significant decrease in movement speed (***P < 0.001) (Fig. 1C,D), which was restored by VK2 treatment (Fig. 1E) (See video in supplementary). We suggest that VK2 treatment increased energy supply, thus improving movement speed. Moreover, the rate of recently hatched larvae of parents fed and reared on VK2 significantly increased (***P < 0.001) (Fig. 1F). In addition, pupal pipped-out flies fed on VK2 showed significantly improved movement speed (Fig. 1G).
VK2 inhibit ROS release. In accordance with the hypoxia, the phenotype of lymphoma specifically the loss of survival signs, is identical to the classical phenotype of hypoxic cancer cells 11 . The production of reactive oxygen species (ROS) is an index of cellular hypoxia 32 . We assessed ROS production in the lymph gland, a main target in the hematopoietic system. We also assessed ROS production in the brain given that brain cells are highly vulnerable to the deleterious effects, such as hypoxia, of ROS production during oxidative stress 33 . The results showed that the heix mutation is closely related to the loss of survival signs as a result of increased ROS production and decreased energy supply. ROS levels in the lymph gland ( Fig. 2A) (***P < 0.001) and the brain (Fig. 2B) significantly decreased upon VK2 treatment (***P < 0.001). Mitochondrial defects associated with the heix mutation could be determined through mitochondrial ROS (mtROS) assessment. The functional status of the mitochondria could be inferred through the evaluation of ATP production.

VK2 restores mitochondrial function by lowering mtROS and increasing ATP production.
Mito-SOX was used to examine mtROS production. Our results revealed that mtROS amount increased in heix mutants, which responded positively and significantly to VK2 treatment (Fig. 3A). Furthermore, ATP production significantly (***P < 0.001) increased in the lymph glands of VK2-treated larvae (Fig. 3B). A similar response was observed in the brain ( Fig. S2A and B). heix mutations are the main stimulants of MAPK (JNK and ERK1/2) pathways 13,34 ; an evaluation confirmed that these pathways are activated in heix mutants. We then focused on the ability of VK2 to restore the normal activation of these pathways.
Moreover, we performed RT-PCR analysis to quantify the expression levels of genes associated with the Toll, JAK/STAT, IMD, and Ras/MAPK pathways. These pathways have been proposed to regulate hemocyte proliferation and immune response 13,34 . VK2 treatment significantly decreased the transcripts of genes involved in the JAK/STAT pathway (unpaired 1, 2, 3 (Upds) and hopscotch (Hop)) significantly decreased (*P < 0.05), (Fig. 4C). Furthermore, the expression of genes involved in the Toll pathway (Drosomycin, Dorsal-related immune factor, dorsal, cactus, spatzle, myd88, and Spatzle-Processing Enzyme) (*P < 0.05) (Fig. 4D). The expression of the genes of the IMD pathway (diptericin A, B (Dpt), Relish (Rel), and immune deficiency (IMD)) significantly decreased (*P < 0.01). In addition, the expression of the mitogen-activated protein kinase phosphatase 3 (Mkp3), which is a gene involved with the Ras/MAPK pathway, significantly decreased (*P < 0.01) after VK2 feeding (Fig. 4E). Dpt, Upds, and Mkp3 are often used as markers for the activation of the IMD, JAK/STAT, and Ras/MAPK pathways, respectively, and were highly responsive to VK2 treatment. As mentioned above, mitochondrial defects occur in heix mutation, and multiple transcripts are closely correlated with mitochondria. Mitochondrial function is closely associated with mitochondrial structure 35 . VK2 restores mitochondrial structure and membrane potential. Transmission electron microscopy (TEM) was performed to identify the relationship between mitochondrial function and structure and to verify the curative effects of vitamin K2 as an electron carrier. Mitochondrial swelling (MS) associated with  the disarrangement of cristae and partial or total cristolysis are the most consistent submicroscopic alterations observed in this study. The majority of mitochondria were round in shape with an electron-lucent matrix, subtotal cristolysis, and the presence of amorphous metric densities with a dilated mitochondrial matrix occupied by lipid-like material (Fig. 5A). VK2 treatment restored mitochondrial structure, thus restoring normal mitochondrial function.
We assessed ATP production in parent flies before and after VK2 treatment and found interesting differences (Fig. S5C). The results indicated that VK2 efficiently transferred electrons in the mitochondrial membrane, thus promoting efficient ATP production. The effective VK2 dose that increased ATP production was also determined and ranged from 30 mM to 50 mM (Fig. S5D). ATP production at 12, 24, 36, and 48 h after egg hatching under VK2 treatment gradually increased (Fig. 5D). This improvement was significant (*P < 0.01) after 12 and 24 h post-hatching and highly significant (***P < 0.001) at 36 and 48 h post-hatching (Fig. S4). ATP production increased at 24 and 48 h after the pupal pip out of flies both measurements were significant (***P < 0.001) (Figs 5E and S4).
VK2 prevents lymphoma phenotype caused by heix mutation. Black spots, which indicate increased hemocyte proliferation, completely disappeared after VK2 treatment. Therefore, we prepared hemocytic smears using Giemsa staining to show the rescue of hemocyte proliferation following VK2 treatment. The percentages of total hemocytes (**P < 0.01) and crystal cell counts significantly decreased (Fig. 6A). The results also revealed hyperplasic lymph glands with numerous lobes. VK2 treatment, however, restored the normal structures of lymph glands, and the percentage of lobulation significantly decreased (***P < 0.001) (Fig. 6B). Furthermore, brain hypertrophy also responded positively to VK2 treatment (Fig. 6C).
Prevention of lymphoma is VK2 dependent. We followed up second-generation larvae born from flies fed with VK2. The larvae were divided into two groups. One group was reared on VK2 medium and the other was reared on ordinary control medium (more details are shown in Fig. S6). Surprisingly, the lymphoma phenotype recurred in the second group, which exhibited the reappearance of black spots with significantly high percentage of larvae with recurrent spots (***P < 0.001) (Fig. 7A). Larvae in the first group had a higher pupal pip-out rate than those in the second group (***P < 0.001) (Fig. 7B). Furthermore, ROS levels significantly increased (***P < 0.001) in the lymph glands of larvae in second group but were lower in larvae reared on VK2 medium (Fig. 7C). Similar results were obtained for the brain (Fig. S7A). ATP production was inhibited in larvae in the second group (Fig. 7D). The same results were demonstrated in the brain (Fig. S7B). This experiment proved that VK2 can prevent the progression, but not recurrence, of lymphoma.
Prevention of lymphoma is VK2 related. We assessed HIF protein expression through western blot analysis to confirm the occurrence of hypoxia. heix mutants reared on control medium exhibited distinctive HIF protein expression, whereas control and VK2-treated groups had significantly lower (**P < 0.001) HIF expression (Fig. 8A). Hypoxia and elevated cytosolic ROS and mtROS indicate serious mitochondrial defects; thus, investigating the protein expression of mitochondrial regulator genes (Bcl2 family) 36 , provide evidence for the mitochondrial dysfunction hypothesis we suggested in our study and for the role of VK2 in the rescue of lymphoma phenotypes. Western blot analysis was performed on Bcl2, Bax, and P53. heix mutants exhibited the clear expression of Bax and P53 and fair expression of Bcl2. VK2 treatment reversed the expression of these proteins, the response of P53 was significant (**P < 0.001) and those of Bax and Bcl2 were highly significant (***P < 0.001) (Fig. 8A).
Previous studies used specific cytosolic ROS scavengers (N-acetyl-L-cysteine) (NAC) to evaluate the specificity of the effects of their tested treatment 10 ; other studies used mtROS scavengers (2(2,2,6,6-tetrameth ylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito-TEMPO) to evaluate the tumor-suppressing effects of antioxidant compounds 37 . To demonstrate that all responses, especially inhibition of cytosolic and mtROS production, are specific to VK2 treatment, we treated heix mutant larvae with NAC and Mito-TEMPO scavengers. We then divided the larvae into three treatment groups; i.e., one treated with NAC, the other with Mito-TEMPO, and the third with NAC and Mito-TEMPO. Interestingly, larvae did not show high response to scavenger treatments. Scavenger-treated larvae showed attenuated lymph gland hyperplasia; this effect, however, was not significantly different from that shown by VK2-treated larvae (Fig. 8B). In contrast to VK2, scavengers did not decrease the high lobulation of heix mutant lymph glands (Fig. 8C). Moreover, black spots in heix mutant larvae, which were unaffected by VK2 treatment, completely disappeared (Fig. 8D). Blood smears from the three treatment groups showed the persistent proliferation of hemocytes and an increased number of crystal cells, whereas those from control and VK2-treated larvae showed normal blood characteristics (Fig.  S8). Furthermore, the increased number of total hemocytes (Fig. S8B) and percentage of crystal cell (Fig. S8C) of the scavenger-treated larvae were significantly (***P < 0.001) different from that of control and VK2-treated larvae. This result indicated that VK2 treatment regulates the hematopoietic system and restores normal blood cell count, whereas the scavenger treatment failed to regulate hemocyte proliferation.
SCienTifiC RepoRts | 7: 17047 | DOI:10.1038/s41598-017-17270-9 Figure 6. Vitamin K2 rescues hematopoietic system in Drosophila third instar larvae with lymphoma induced by heix mutation. (A) Images (X40) circulating hemocytes labeled by Giemsa staining kit from larvaes in heix k11403 /heix 1 on control medium, in control (Canton S) on control medium, and in heix k11403 /heix 1 on VK2 supplemented medium. Images reveal an increase number of crystal cells, (black arrows). Total circulating hemocytes counted from at least twenty third instar larvaes of each genotype. The proportion of crystal cell observed in total circulating hemocytes. (B) Images (X20) lymph glands in third instar larvaes in heix k11403 /heix 1 on control medium, in control (Canton S) on control medium and in heix k11403 /heix 1 on VK2 supplemented medium. They show a significant increase size of mutant lymph glands indicating hyperplasia which responded to treatment with VK2. Percentage of lobulation in lymph gland reduced significantly. (C) Images (X20) brain in third instar larvae in heix k11403 /heix 1 on control medium, in control (Canton S) on control medium and in heix k11403 /heix 1 on VK2 supplemented medium. They show a significant hypertrophy in mutant brains which responded to treatment with VK2. Control media contain 50 mM Ethanol (EtOH) and treatment media contain 50 mM VK2. Means normalized to control (Canton S). Error bars indicate SEM. Analysis of variance (ANOVA/ Dunnett: *P < 0.05, **P < 0.01, ***P < 0.001).

Discussion
This study was designed to highlight the important role of VK2 in lymphoma treatment, as well as to provide insight on the mechanisms that underlie the curative effect of VK2. Interestingly, our results strongly indicated that VK2 effectively prevents lymphoma progression.
We found that heix mutant Drosophila exhibited almost all classical lymphoma phenotypes, including lymph gland hyperplasia, brain hypertrophy, hemocyte overproliferation, melanotic black spots, and poor survival signs. VK2 treatment rescued black spot formation, which is the most obvious feature of lymphoma in Drosophila.
Other studies have reported that the loss-of-function of UBIAD1/heix leads to the activation of immune-related pathways and hyperplasia of lymph glands 13 . Hemocyte proliferation is closely correlated with the formation of black spots, which can be attributed to an increase in number of crystal cells associated with melanization 17,38 . Hence, the disappearance of black spots upon VK2 treatment may be attributed to the regulation of hemocyte proliferation. The activated form of Hop interacts with the Ras/MAPK pathway and hemocyte proliferation 39 , which is positively triggered in heix mutant larvae 13,40 . This finding is consistent with the significant upregulation of Hop expression in the heix k11403 mutant. Our results demonstrated the ability of VK2 to restore lymphoma-associated alterations and indicated that similar to the heix gene, VK2 can act as a negative regulator of immune-related and cell-proliferation-associated pathways.
Furthermore, recent studies have demonstrated that the heix mutation is associated with certain acetifications, such as impaired flight 14 and limited physical and biological activity 13 . Biological activities require energy, and their alterations were rescued by VK2 treatment. This recovery resulted from the increase in energy supply, which is closely associated with the restoration of mitochondrial function. These findings indicated that VK2 is a key player in the restoration of mitochondrial function.
Our data also demonstrated that cytosolic ROS and mtROS production increased in heix mutant larvae. A recent study by Mugoni et al. showed that silencing UBIAD1 increases ROS production 41 . Under normal conditions, mitochondria receive pyruvate from the cytosol, where oxidative phosphorylation occurs, to produce ATP through the tricarboxylic acid cycle and electron transport chain (ETC) 42 . Conversely, under hypoxia, cells utilize anaerobic glycolysis, which converts pyruvate into lactate and rapidly produces ATP 43 . Although most electrons that pass through the respiratory chain are transferred to oxygen at complex IV, some electrons leak from complexes, particularly complexes I and III, and then react with molecular oxygen to produce superoxide 44 . Superoxide is converted to hydrogen peroxide by Mn-superoxide dismutase 42 and can freely diffuse across the mitochondrial membrane into the intermembrane space and cytosol 45 . Defects in the mitochondrial respiratory chain increase ROS production 34 . These modifications promote the kinase signaling cascade-like activation of MAPK (JNK and ERK) 45 . VK2 likely exerts its anticancer activity by restoring mitochondrial function through inhibiting ROS production.
Given that heix mutants suffer from serious defects in ETC 14 , the partial inhibition of ETC leads to excessive mtROS production 46 . mtROS are natural byproducts and are key steps in the generation of ATP 44 . Our results showed that VK2 decreased mtROS production and restored ATP production by acting as an electron carrier in the mitochondrial membrane.
The general role of ROS in cancer cells is a highly controversial subject. Intracellular signaling pathways, such as MAPK, are triggered by ROS 47 . Increased levels of ROS with hypoxia are direct factors that activate cell proliferation; signaling; and immune-related pathways, such as Toll, IMD, JAK/STAT, and JNK 47,48 . JNKs catalyze the phosphorylation and downregulation of Bcl-2 proteins 48 . Bcl-2 antagonizes ROS generation 49 . JNK alters the composition of the Bax/Bcl-2 complex by increasing Bax expression, which leads to the formation of Bax homodimers and in the disruption of mitochondrial membrane integrity 50 . ERK1/2 and JNK are activated through Ask-1; heix mutants showed the clear activation of these pathways. Moreover, ERK is associated with cell proliferation 51 .
We observed that all Bcl-2 family proteins, except for the Bcl-2 transcript, were significantly overexpressed in heix mutants. Bcl-2 proteins regulate ETC in the mitochondrial membrane 36 . The Ras/MAPK pathway is triggered in heix mutants, and UBIAD1/heix is a negative regulator of this pathway 40 . We also demonstrated the roles of related genes associated with MAPK pathways, such as MKP3 and Dpt, with Hop.
The activation of the JAK/STAT signaling pathway is associated with numerous malignancies, including lymphoma and leukemia 23 . Xia et al. detected the remarkably high expression of Upd3 in the JAK/STAT pathway of heix mutant larvae 13 . Our work showed high expression of Upds in the JAK/STAT pathway.
Our results indicated that heix mutations induce the aberrant activation of IMD and Toll pathways. The IMD pathway controls the expression of antimicrobial peptides 52 . We showed that activation of the IMD and Toll pathways is indicative of a hematopoietic disorder. VK2 feeding restored the normal expression of included transcripts.
The structure of the mitochondria is closely related to its function, and mitochondrial structure and functions vary from tissue to tissue 53 . Silencing UBIAD1 causes dramatic morphological changes and cholesterol storage in the mitochondria; this effect thus emphasizes the important role of UBIAD1 in mitochondrial function 54 . Interestingly, mutations in the Drosophila heix gene resulted in abnormal mitochondrial morphology and malfunction 13,14 . TEM sections revealed that MS is associated with the disarrangement of the cristae and with partial or total cristolysis. This morphological change is associated with hypoxic-ischemic conditions 55 .
Partial or total cristolysis is suggestive of the severely compromised ability of cells to generate ATP 56 . VK2 treatment, however, can restore normal mitochondrial structures by modifying the membrane potential of larvae didn't affected by scavenger's treatment as demonstrated with VK2 treatment which showed complete disappearance of them. Control media of control (Canton S) and heix mutant contain 50 mM Ethanol (EtOH), and treatment media contain 50 mM of each scavenger's. Error bars SEM. A significant difference is compared to the control (Canton S) (ANOVA/Dunnett: *P < 0.05; **P < 0.01; ***P < 0.001). mitochondria while acting as an electron carrier. The application of this vitamin recharged the mitochondrial membrane and increased its negativity. ETC, which is located in the mitochondrial inner membrane (MIM), pumps protons out of the mitochondrial matrix into the intermembrane space between the MIM and mitochondrial outer membrane. This process slightly changes pH and increases membrane potential due to the charge separation across MIM and the absence of anions that accompany positively charged protons. Thus, ΔΨm across MIM is negative on the matrix side. Therefore, proton-motive force resulting from membrane potential and pH gradient regulates ATP production from ADP and phosphate by ATP synthase 57 .
We evaluated the inhibitory effect of VK2 on the progression and recurrence of lymphoma in Drosophila. When VK2 feeding was stopped, most lymphoma phenotypes recurred. We found that VK2 prevents lymphoma by acting as a mitochondrial electron carrier, which is necessary for establishing mitochondrial membrane potential and facilitating ATP production.
Finally, to determine that the restoration of mitochondrial function and structure were specific to VK2 treatment, we introduced ROS scavengers, which are antioxidants that discard free radicals 58 . ROS scavengers suppress tumor growth 37 ; therefore, responses to these compounds indicate responses that are not specific to VK2. In this study, the nonsignificant response to ROS scavengers revealed that the restoration of mitochondrial function can be attributed toVK2 treatment.
Our work indicates that VK2 prevents the progression, but not recurrence, of lymphoma. These findings were in agreement with those of previous studies that were unable to confirm the efficacy of VK2 in suppressing HCC recurrence 59,60 . However, other studies have shown that VK2 can decrease cancer recurrence 61,62 . Therefore, further studies in this regard are still needed.
In conclusion, we confirmed that VK2 prevents the progression, but not recurrence, of lymphoma by restoring mitochondrial function. Our study suggests that the correction of mitochondrial function might be a fundamental step in tumor treatment and identifies VK2 as a key player in cancer therapy strategies.

Media preparation of Drosophila melanogaster.
Methyl-p-hydroxybenzoate (C 8 H 8 O 3 ) 10 g was dissolved in 100 ml of ethanol and configured to be a 10% methyl-p-hydroxybenzoate acid solution. Weigh the corn flour 120 g, agar 5 g, brown sugar 40 g into the pot, add water to 1 L, heat on the induction cooker, stir slowly in the heating process until the boiling. Add 10 ml of 10% methyl-p-hydroxybenzoate acid solution to 1 L of food, and add 4.3 ml of propionic acid, both to prevent fruit fly food spoilage. Pour the food into a bottle; stored in RT, it is ready for use, the flies were bred in a constant temperature and humidity incubator, temperature 25 C°, humidity 60%.
Vitamin K2 feeding. The newly generated genotype P-element allele, heix k11403 , an EMS allele, heix 1 , and Df adult virgin flies (not more than 6 h old) were placed on medium supplemented with VK2 (Sigma USA) (VK2 was dissolved in 99.9% ethanol, EtOH). The therapeutic dose determined by testing several gradual concentrations of VK2, from 1 mM to 50 mM, and identifying optimum response based on black spots disappearance (Fig. S2). Determination of dose volume ranging from 1 µl to 35 µl, were tested. The 25-µl volume showed an even distribution of vitamin on medium (2 cm in diameter &1 cm height) (Fig. S2). Adult virgin flies put in medium with VK2 for 24 hrs. Flies were placed together for mating in new tubes with VK2 for 4-5 days then removed before the eggs hatched. Only long "heix mutant" larvae were selected (100 larvae per group) for rearing and growth on VK2. Both control "Canton S, W 1118 and heix mutant" controls were reared on medium followed by the addition of 50 mM ethanol (EtOH). Molasses medium incubated for 24 h uncovered at room temperature (RT) to allow evaporation of ethanol and, thus, avoid alcohol-induced stress 63 . (The experiment was repeated several times to validate the results).
Immunostaining of Drosophila larval tissues. Drosophila third instar larvae were dissected in phosphate-buffered saline solution (1x PBS). Lymph glands and brains immediately transferred into a container of PBS and placed on ice, followed by fixation with 4% paraformaldehyde for 10-15 minutes at RT and three washes with PBST. Samples blocked in PBS +Triton-x100 0.3% +1% BSA for 1 h at RT, followed by staining with ab13847 at a 1/500 μl dilution (in PBS +Triton-x100 0.3% +1% BSA) for 16 h at 4 °C. The secondary antibody, Alexa Flour 594 WGA (reddish color), used to label plasma membrane at a 1/500 dilution for 1 hr. DAPI used to stain cell nuclei (blue) at a concentration of 1.43 µM. For ROS detection, 2′, 7′-dichlorofluorescein-diacetate (DCFH-DA) utilized. DCFH-DA, a cell membrane-permeable dye, is converted to DCFH (a non-fluorescent cell membrane-impermeable compound, Invitrogen Molecular Probes, cat no. C6827). Tissues incubated with dye for 10-15 minutes in a dark chamber on an orbital shaker at RT followed by three 5-minutes washes in 1x PBS on an orbital shaker at RT, fixation for 4-8 minutes in 7% formaldehyde in 1x PBS, and rinsing in 1x PBS immediately after fixation. Images captured using an Olympus confocal laser scanning microscope FV1000. Images were analyzed using ImageJ software and processed by Adobe Photoshop and Corel DRAW.
Mitochondrial superoxide indicator. Tissues stained with 5 μM Mito-SOX ™ prepared from 5 mM Mito-SOX ™ reagent stock solution. The contents (50 μg) of one vial of Mito-SOX ™ mitochondrial superoxide indicator (Component A) were dissolved in 13 μL of dimethyl sulfoxide (DMSO) to make the 5 mM Mito-SOX ™ reagent stock solution. The 5 mM Mito-SOX ™ reagent stock solution was dissolved in HBSS/Ca/Mg to prepare a 5 μM Mito-SOX ™ reagent working solution. Then, 0.3-0.5 µl of the 5 μM Mito-SOX ™ reagent working solution was used to cover the tissues. Tissues were incubated for 10 minutes at 37 °C in the dark. Tissues then washed gently three times with warm buffer. DAPI at a concentration of 1.43 µM used to stain cell nuclei (blue). Tissues fixed for 5 minutes in 4% formaldehyde and rinsed twice in 1x PBS immediately after fixation, then mounted for microscopy.
Mitochondrial membrane potential assay. Changes in mitochondrial membrane potential in tissues evaluated using Rhodamine 123, a mitochondrial-specific dye. Briefly, sample stained with 1.5 μM Rhodamine 123 and incubated at 37 °C for 10 minutes. Sample subsequently washed three times with warm PBS to remove unbound dye, fixed for 5 minutes in 4% formaldehyde then rinsed twice in 1x PBS after fixation. DAPI applied at 1.43 µM to stain cell nuclei (blue). Mitochondrial membrane potential assay evaluated by fluorescence of Rhodamine 123 under an Olympus confocal laser scanning microscope FV1000. Western blot analysis. Total protein from third instar larvae extracted by homogenization with (PRO PREP) protein extraction kit (INTRON Biotechnology, Korea). Samples subjected to 1x SDS-PAGE loading buffer and separated by 12% SDS-PAGE. Then transferred to a polyvinylidene fluoride (PVDF) membrane for immunoblotting. Membranes blocked in 5% non-fat dry milk in TBST (0.05% Tween, 20-50 mmol/L Tris HCL, pH 7.5 and 150 mmol/L NaCl) for 3 h, and incubated overnight with primary antibody at 4 °C and washed with TBST for 2 h. Secondary goat anti-rabbit IgG (HRP) incubated with membranes at RT for 3 h. After incubation, samples washed with TBST for 2 h. Finally, membranes visualized on X-ray films. Densitometry analysis of individual protein bands performed using Quantitative One Image software.
Real-time PCR analysis. Total RNA extracted from Drosophila third instar larvae with TRIzol reagent (Invitrogen). cDNA synthesized using cDNA synthesis kit (Promega) according to manufacturer's instruction. Real-time PCR was performed with double-stranded DNA dye SYBR Green (Roche) to quantify amount of gene expression. All samples analyzed in triplicate, and mRNA levels normalized to control Ef1α100E values (Elongation Factor1 alpha100, CG1873) as previously described. Ef1α100E primer pair used accordingly. Information for other primer pairs is shown in (Table S7).
Hemocyte collection and imaging. Circulating hemocytes obtained from Drosophila third instar larvae.
Hemolymphocytes transferred into 8 µl of 1x PBS solution on adhesion microscope slides (CITOGLAS, China). Stained using the Giemsa staining kit (Bio time Institute of Biotechnology) then imaged with an Eclipse 80i Microscope (Nikon).

Statistical analysis.
All experiments performed at least four times. Data analyzed using GraphPad Prism software. Confocal microscopy images analyzed using ImageJ and Corel draw software. Results expressed as mean ± SD, and differences determined using Analysis of variance (ANOVA). P values of *P < 0.05, **P < 0.01, and ***P < 0.001 were considered significant.