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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 1  |  Page : 68-74

Investigation of apoptotic effect of juglone on CCL-228-SW 480 colon cancer cell line


1 Department of Histology and Embryology Faculty of Medicine, Süleyman Demirel University, Isparta, Turkey
2 Department of Medical Biology, Faculty of Medicine, Süleyman Demirel University, Isparta, Turkey
3 Department of Nutrition and Dietetics, Faculty of Health Sciences, Süleyman Demirel University, Isparta, Turkey

Date of Web Publication13-Mar-2019

Correspondence Address:
Dr. Dilek Bayram
Department of Histology and Embryology, Faculty of Medicine, Süleyman Demirel University, Isparta-32260
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_880_17

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 > Abstract 


Background: Colon cancer is a major cause of morbidity and mortality in the world. Juglone is a natural compound which has been isolated from Juglans mandshurica Maxim, and it has various pharmacological effects such as antiviral, antibacterial, and anticancer. In our study, we aimed to investigate the effect of juglone on CCL-228-SW 480 colon carcinoma cell line in monolayer and spheroid culture medium.
Materials and Methods: The CCL-228-SW 480 cell lines were cultured in both monolayer and spheroid cultures. Cells were treated with juglone at 24, 48, and 72 h of incubation. ID50 inhibition was determined on the dose for juglone and after it was found 20 μM was applied to the cells to examine the effect of juglone on CCL-228-SW 480 colon carcinoma cell line. After Juglone was applied the BrdU marking index, Transferase dUTP Nick ends Labeling (TUNEL) assay, active caspase-3 assay, apoptosis-inducing factor (AIF) assay were determined by immunohistochemistry in both the monolayer and spheroid cultures.
Results: The control group had a healthy pattern of S-phase fraction, and many of the CCL-228-SW 480 cells nuclei were observed to be positive for BrdU. Terminal Deoxynucleotidyl TUNEL-positive cells, active Caspase-3, and AIF were detected after treatment with juglone in both the monolayer and spheroid cultures.
Conclusions: The dead cell count was higher in the CCL-228-SW 480 cell lines with juglone applied than in the controls. Juglone significantly inhibits the proliferation and induces the apoptosis of CCL-228-SW 480 cells in vitro.

Keywords: Apoptosis, CCL-228-SW 480 cell line, colon cancer, in vitro, juglone


How to cite this article:
Bayram D, Özgöçmen M, Armagan I, Sevimli M, Türel GY, Şenol N. Investigation of apoptotic effect of juglone on CCL-228-SW 480 colon cancer cell line. J Can Res Ther 2019;15:68-74

How to cite this URL:
Bayram D, Özgöçmen M, Armagan I, Sevimli M, Türel GY, Şenol N. Investigation of apoptotic effect of juglone on CCL-228-SW 480 colon cancer cell line. J Can Res Ther [serial online] 2019 [cited 2019 Oct 18];15:68-74. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/68/244483




 > Introduction Top


Colon cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer-related death globally.[1] The most efficient therapy for colon cancer is surgery combined with radiotherapy, immunotherapy, chemotherapy, or traditional Chinese herbal medicine. However, side effects can be severe for both radiotherapy and chemotherapy and the survival rate remains low because of the reoccurrence and migration of colon tumors. Thus, finding an appropriate way to improve therapeutic efficiency without side effects is crucial.[2]

Quinones are common secondary metabolic products that possess a variety of pharmacological properties, such as antibacterial, antifungal, antioxidant, anti-inflammatory, and anticancer activity.[3] Juglone (5-hydroxy-1,4-naphthoquinone) is a natural 1,4-naphthoquinone found in the Juglandaceae family, particularly in the roots, leaves, bark, and wood of Juglans regia [Figure 1]. Juglone has shown cytotoxic effects against various tumor cells and also has antifungal, antibacterial, and antiviral activities, as well as allelopathic activities.[4],[5],[6],[7],[8] Although juglone should be a promising agent for oncotherapy, its clinical applications have been limited due to its poor solubility in aqueous solutions, poor absorption, and generalized cytotoxicity to normal tissues.[9]
Figure 1: Structure of juglone (5-hydroxy-1,4-naphthoquinone)

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Apoptosis occurs normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues. Apoptosis also occurs as a defense mechanism such as in immune reactions or when cells are damaged by disease or noxious agents.[10] The irradiation and drugs used in cancer chemotherapy result in DNA damage in some cells, which can lead to apoptotic death through a p53-dependent pathway.[11]

In the present study, juglone was evaluated for its apoptotic effects in vitro in CCL-228-SW 480 colon cancer cells through two-dimensional (2D) monolayer and 3D spheroid cultures using immunohistochemical techniques. According to our research, to date, there have been no investigations of the apoptotic effects of juglone based on a spheroid model. Cancer cells grown in vitro as spheroids more accurately mimic the drug sensitivity and/or drug resistance behaviors of solid tumors in vivo, as compared to cancer cells cultured under monolayer conditions.[12]


 > Materials and Methods Top


Cell culture

The human colon cancer cell lines, CCL-228-SW 480, were supplied by the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ; Germany). Cell lines were cultured and maintained in monolayers in RPMI 1640 (Thermo Fisher Scientific, Gibco, USA) supplemented with penicillin G (100 U/mL) (Sigma-Aldrich Co), streptomycin (100 μg/mL) (Sigma-Aldrich Co), L-glutamine (Thermo Fisher Scientific, Gibco, USA), and 5% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco, USA) maintained at 37°C in a 5% CO2 humidified incubator (Heal Force, HF90, Serial No: 1103K0092CE).

Constitution of a three-dimensional spheroid model (spheroid culture)

An in vitro multicellular CCL-228-SW 480 human colon spheroid model were designated using a liquid overlay technique.[13] When the monolayer cultures became semiconfluent, the cells were trypsinized and single cells with 100% viability were cultured over a thin layer of agar-coated (1 mL of a 1,5% (w/v) solution of agar in RPMI-FBS) 6-well culture plates containing 5 mL RPMI 1640 medium at a concentration of 5 × 105 cells/well. Six well plates were kept in an electronic incubator (Heal Force, HF90, Serial No: 1103K0092CE) under a humidified atmosphere of 5% CO2 and air mix for 10 days.

Experimental design

The investigated juglone (5-hydroxy-1,4-naphtha-quinone) (H28343 Alfa Aesar, Germany) at a volume of 20 μM were applied to monolayer cultures of colon cancer cells at the following final concentrations: 0.1–200 μM, except for the control cells, to which nutrient medium was applied. After 24, 48, and 72 h incubation the cells were trypsinized and the cytotoxicity was determined by trypan blue dye exclusion test. All experiments were repeated three times and representative data are presented.

DNA synthesis (S phase) analyses of BrdU labeling index in monolayer culture (immunohistochemistry)

CCL-228-SW 480 colon cancer cells were cultivated on glass coverslips and treated with juglone, as described in the survival studies, every 24 h for a total of 72 h. Cells were washed in phosphate buffered saline (PBS) and fixed in 70% alcohol. Then, Invitrogen BrdU Staining Kit was used to detect BrDU labeled cells in S phase. BrDU labeled cells in S phase indicated brown-stained nuclei.

DNA synthesis (S phase) analysis of BrdU labeling index in spheroid cultures (immunohistochemistry)

Spheroids were harvested from the surface of the solidified agarose, fixed 10% Neutral Buffered Formalin, washed tap water, was passed a graded ethanol series and xylene, then embedded in paraffin. Sections were cut by the microtome (Leica SM 2000R, Serial No: 1985/11.2000 Heerbrugg, Switzerland). Sections were deparaffinized with xylene and rehydrated in a series of graded alcohol. Then, the same protocol was applied to spheroid cultures.

Detection of apoptotic cell deathin situ using the terminal deoxynucleotidyl transferase dUTP nick end labeling method in monolayer culture

The in situ Apoptosis Detection Kit (DeadEnd Colorimetric transferase dUTP nick end labeling (TUNEL) system, Promega G7130, Ca No: S701) was used to detect apoptosis. Staining was performed with DAB and counterstaining was performed in methyl green.

Detection of apoptotic cell deathin situ using the transferase dUTP nick end labeling method in spheroid cultures

Five μm sections were cut from the paraffin blocks of the samples. The sections were deparaffinized in xylene, rehydrated. And then, the same protocol was applied to spheroid cultures.

Immunohistochemical staining with caspase-3 antibody in monolayer culture

CCL-228-SW 480 colon carcinoma cells were seeded on glass coverslips and treated with drugs, as described in the survival studies, every 24 h for a total of 72 h. Medium was removed from cells and was washed in several changes of PBS. Cells were fixed in 70% alcohol. Sections were stained with primary antibodies to caspase-3 (Thermofisher Scientific, Waltham, MA USA, Ca No: PA5-16335). Finally, sections were counterstained with Mayer's hematoxylin.

Immunohistochemical staining with caspase-3 antibody in spheroid cultures

For immunohistochemical analysis, the spheroids were fixed in 4% paraformaldehyde (Sigma) in PBS, then washed in PBS. Following fixation the spheroids were dehydrated through graded ethanol, cleared in xylene, embedded in paraffin, and 5 μm coronal sections were cut on microtome. Sections were delineated with a Dako pen (Dako, Glostrup, Denmark). Then, the same protocol was applied to spheroid cultures.

Immunohistochemical staining with apoptosis-inducing factor antibody in monolayer culture

CCL-228-SW 480 colon carcinoma cells were seeded on glass coverslips and treated with drugs, as described in the survival studies; every 24 h for a total of 72 h. Medium was removed from cells and was wash in several changes of PBS. Cells were fixed in 70% alcohol. Sections were stained with primary antibodies to apoptosis-inducing factor (AIF) (Thermofisher Scientific, Waltham, MA USA, Ca No: PA5-17638), slides were incubated with a solution containing DAB. Finally, sections were counterstained with Mayer's hematoxylin.

Immunohistochemical staining with apoptosis-inducing factor antibody in spheroid cultures

For immunohistochemical analysis, the spheroids were fixed in 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS, pH 7.4) for 24 h at 4°C, then washed in PBS. Following fixation the spheroids were dehydrated, cleared in xylene, embedded in paraffin, and 5 μm coronal sections were cut on microtome. Sections were delineated with a Dako pen (Dako, Glostrup, Denmark) and incubated in a solution of 3% H2O2. Then, the same protocol was applied to spheroid cultures.

Hematoxylin and eosin staining in spheroid cultures

To stain with hematoxylin and eosin (H and E), the spheroids were fixed in 4% paraformaldehyde (Sigma), then washed tap water. After fixation the samples were dehydrated through graded ethanol, cleared in xylene, embedded in paraffin, and 5 μm sections were cut on microtome. Then sections were stained H and E.

Statistical analysis

All data are presented as the means ± standard error. Statistical evaluation of the data was performed with independent samples t-test using SPSS v. 20 (IBM-SPSS Statistics, Armonk, NY, USA). Differences were considered statistically significant when P ≤ 0.05.


 > Results Top


Cell proliferation

Juglone effective dose on CCL-228-SW 480 cells were determined as 20 μM. All experiments were repeated three times and data are presented. The effects of juglone on cell proliferation of CCL-228-SW 480 are shown in [Figure 2]. In comparison with the control group, juglone treatment groups inhibited cell proliferation of CCL-228-SW 480 cells for all time intervals (P < 0.05).
Figure 2: Effect of juglone on proliferation of the monolayer culture CCL-228-SW 480 Cells. Cells were initially plated at a seeding density of 5 × 105 cells per well

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Cell cycle kinetics

DNA synthesis (S-phase) analysis for monolayer cultures

DNA synthesis (S-phase) analysis of colon cancer cells was determined by BrdU staining. Cells with brown-stained nuclei considered BrdU positive. A large number of BRDU-marked cells were observed in the synthesis group at all hours in the control group. There was a significant decrease in the BRDU marking index at all hours compared to the control group in the juglone-treated group [Figure 3]a and [Figure 4]a,[Figure 4]b,[Figure 4]c,[Figure 4]d (P < 0.05).
Figure 3: Two-dimensional cell culture. BrdU staining (a). Transferase dUTP nick end labeling staining (b). Caspase-3 staining (c). Apoptosis-inducing factor staining (d). Three-dimensional cell culture. BrdU staining (e). Transferase dUTP nick end labelling staining (f). Caspase-3 staining (g). Apoptosis inducing factor staining (h)

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Figure 4: Two-dimensional cell culture. BrdU staining (a-d). Control group (a). Experimental groups (b-d). Transferase dUTP nick end labeling staining (e-h). Control group (e). Experimental groups (f-h) Caspase-3 staining (i-l). Control group (i). Experimental groups (j-l) apoptosis-inducing factor staining (m-p). Control group (m). Experimental groups (n-p) (×40)

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DNA synthesis (S-phase) analysis for spheroid cultures

Twenty-five spheroids from all experimental groups, including the control group, were evaluated for three times. Cells with brown-stained nuclei assumed BrdU positive. The control group had a normal pattern of S-phase fraction and many of the CCL-228-SW 480 cell nuclei were observed to be positive for BrdU with BrdU-LI values of 20.2%, 19.5%, 19.9%, respectively. In comparison the controls, juglone decreased the BrdU-LI values of CCL-228-SW 480 spheroids to 8.1%, for 24 h, 8.4%, for 48 h and 8.3% for 72 h [Figure 3]e and [Figure 5]a,[Figure 5]b,[Figure 5]c,[Figure 5]d.
Figure 5: Three-dimensional cell culture. BrdU staining (a-d). Control group (a). Experimental groups (b-d). Transferase dUTP nick end labeling staining (e-h). Control group (e). Experimental groups (f-h). Caspase-3 staining (i-l). Control group (i). Experimental groups (j-l). Apoptosis-inducing factor staining (m-p). Control group (m). Experimental groups (n-p) (×40)

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Transferase dUTP nick end labeling analysis for monolayer cultures

To confirm the cell death mechanism on CCL-228-SW 480, monolayer cultures were used to determine differences in their sensitivity in the molecular process initiated by juglone for three times. In the control group, very few apoptotic cells (positive staining cells with TUNEL) were observed at all hours. More TUNEL-positive cells were seen in the juglone groups compared with control group (P < 0.05) [Figure 3]b and [Figure 4]e,[Figure 4]f,[Figure 4]g,[Figure 4]h.

Transferase dUTP nick end labeling analysis for spheroid cultures

While TUNEL-positive cells were observed slightly staining in the control group, there were more TUNEL-positive cells in the juglone-treated spheroids. These results supported our monolayer culture results that the spheroids of juglone group showed more TUNEL-positive CCL-228-SW 480 cells [Figure 3]f and [Figure 5]e,[Figure 5]f,[Figure 5]g,[Figure 5]h.

Caspase-3 immunohistochemical staining for monolayer cultures

Caspase-3 immunohistochemical staining for all intervals were separately identified as control and experimental groups. We observed whether juglone activate caspase-dependent apoptosis by measuring the cleavage of caspase-3. Juglone induced the cleavage of caspase-3 in CCL-228-SW 480 cells for 24 h, 48 h, and 72 h [Figure 3]c and [Figure 4]i,[Figure 4]j,[Figure 4]k,[Figure 4]l.

Caspase-3 immunohistochemical staining for spheroid cultures

We investigated juglone caspase-dependent apoptosis by measuring the caspase-3 in 3D culture. This finding supported our monolayer culture results that the spheroids of juglone group showed more caspase-3-positive CCL-228-SW 480 cells [Figure 3]g and [Figure 5]i,[Figure 5]j,[Figure 5]k,[Figure 5]l.

Apoptosis-inducing factor immunohistochemical staining for monolayer cultures

In this step, we investigated whether juglone activate caspase-independent apoptosis by measuring the AIF receptor activity. We observed that juglone induced the AIF activity in CCL-228-SW 480 cells in all intervals [Figure 3]d and [Figure 4]m,[Figure 4]n,[Figure 4]o,[Figure 4]p.

Apoptosis-inducing factor immunohistochemical staining for spheroid cultures

We were observed juglone caspase-independent apoptosis by measuring the AIF in 3D culture. This finding supported our monolayer culture results that the spheroids of juglone group showed AIF-positive CCL-228-SW 480 cells [Figure 3]h and [Figure 5]m,[Figure 5]n,[Figure 5]o,[Figure 5]p.

Hematoxylin and eosin staining in spheroid cultures

Twenty-five spheroids from all juglone-treated groups, including the control group, were evaluated for three times. Spheroid structure of control group was observed normally. In experimental groups were observed both normal spheroids and separated spheroids. Spheroid structures were also degenerated [Figure 6].
Figure 6: H and E staining in spheroid culture. Spheroid structure of control group was observed normally in CCL-228-SW 480 cells (a). Group of treated with juglone was observed both normal spheroids and separated spheroids. Spheroid structures were also degenerated (b-d) (×40)

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 > Discussion Top


Currently, therapeutic chemosensitization to drugs is a challenging issue in cancer therapy. Strategies that improve the selectivity of treatments should be based on the physiological differences between normal and malignant cells.[14] In recent studies, it has been shown that low-toxicity natural compounds and herbal products can affect cancer cells through various mechanisms (such as the induction of apoptosis) and prevent the onset of carcinogenesis, have synergistic effects with standard chemotherapeutic agents, and reduce side effects.[15]

Walnut has been used as an herbal medicine for treating cancer for many years, and juglone is reported to be the main active ingredient in walnut.[16] Juglone is 5-hydroxy-1,4-naphthoquinone and acts as strong cytotoxic agent. However, little has been reported regarding the mechanism underlying the cytotoxic potential of juglone.[17] Juglone is toxic to the growth of nonwalnut plants and likely exerts its effects by inhibiting the peptidyl-prolyl isomerases found in other plants. Juglone has differential effects on cell cycling and metabolism depending on species, organ, and drug concentration,[18] and it has been reported to inhibit the intestinal and colon carcinogenesis induced by azoxymethane.[19] Juglone's anticancer effects have been shown in studies with gastric cancer cells (SGC-7901), leukemia cells (human leukemia (HL-60)), colon carcinoma cells (HCT-15), and prostate cancer cells (LNCaP). In these studies, it is reported that juglone triggers apoptosis in a dose-dependent manner through increasing the ROS level through the loss of mitochondrial membrane potential, and it affects the expression of genes associated with apoptosis, such as Bax, Caspase-3, Caspase-9, and Bcl-2.[5],[20],[21],[22] Fang et al. reported that juglone inhibited the invasion of ovarian carcinoma cells (SKOV3) and decreased MMP-2 expression.[23] In another study of breast cancer cells (MCF7Adr), it was indicated that juglone inhibited the migration and angiogenic ability of cancer cells.[24] However, the number of studies have investigated the antimetastatic effect of juglone on cancer cells is quite limited. In this study, we investigated the apoptotic effects of juglone on CCL-228-SW 480 colon cancer cells in both 2D (monolayer) and 3D (spheroid) cultures.

Caspase-3, caspase-6, and caspase-7 function as effector or “executioner” caspases cleaving various substrates including cytokeratins, PARP, the plasma membrane cytoskeletal protein alpha fodrin, the nuclear protein NuMA, and others, which ultimately causes the morphological and biochemical changes seen in apoptotic cells.[25] Caspase-3 is considered to be the most important of the executioner caspases, and it can be activated by any of the initiator caspases (caspase-8, caspase-9, or caspase-10). Caspase-3 also induces cytoskeletal reorganization and the disintegration of the cell into apoptotic bodies.[11]

Caspase-independent mechanisms of neuronal cell death have also been identified. This specific type of programmed cell death may involve specific mitochondrial factors. In experimental models, AIF and endonuclease G promote this type of cell death.[26]

We determined that the IC50 values of juglone in CCL-228-SW 480 colon cancer cells were 20 μM at 24, 48 h, and 72 h of incubation. Therefore, 20 μM of juglone (IC50 dose) were applied to the colon cancer cells in both the monolayer and spheroid cultures. Then, TUNEL assay, caspase-3, and AIF immunohistochemical staining were used to investigate cell death in both the monolayer and spheroid cultures. For all of the incubation periods (24 h, 48 h, and 72 h), the TUNEL-positive cell indices of juglone were found to be significantly different from the TUNEL-positive cell index of the control in the monolayer cultures. Overall, juglone increased the number of TUNEL-positive cells, increased the number of caspase-3-positive cells, and increased the number of AIF-positive cells in the monolayer culture. When we compared caspase-3 and AIF staining, a larger number of cells were positively stained for caspase-3, and this result led us closer to the idea that apoptosis was carried out by the caspase pathway.

The DNA synthesis (S-phase) of colon cancer cells was also assessed through bromodeoxyuridine (BrdU) staining. Those cells with brown-stained nuclei represented BrdU uptake and were considered to be BrdU positive. Overall, juglone reduced the number of cells marked with BrdU when compared with the untreated control group. Previously, Xu et al. showed the activities of caspase-3 and-9 in LNCaP cells treated with juglone. Juglone remarkably activated caspase-3 and-9 more, with 2.5-fold increases in activity being seen after 24 h of treatment. In addition, these authors determined that, mitochondrial membrane potential decreased after treatment with juglone in a dose-dependent manner.[20] Recent studies have shown that juglone induces cell apoptosis through the mitochondria-dependent pathway in human lung cancer (A549) cells, HL - 60 cells, and human cervical carcinoma (HeLa) cells.[6],[17],[27]

Spheroids are known to be in vivo-like tissue culture representatives and the most adapted model for investigating the in vitro resistance properties of cells. Because of these characteristics, spheroids quite realistically represent the results of the drug effects by including limitations in penetration, distribution, and cell signaling feedback mechanisms. In the present study, 25 spheroids from all of the experimental groups, including the control group, were evaluated at 24, 48, and 72 h. In the spheroid culture, 20 μM of juglone were applied to the CCL-228-SW 480 colon cancer cells. Although TUNEL-positive cells were weakly detected in the untreated control group, there were more TUNEL-positive cells in the juglone-treated spheroids. We were observed juglone caspase-dependent apoptosis by measuring the caspase-3 in a 3D culture. Juglone increased the number of caspase-3-positive cells as compared with the untreated control group. In addition, AIF activity measurement was performed for the caspase-independent apoptosis effect of the juglone in 3D culture. Juglone increased the number of AIF-positive cells as compared with the untreated control group. When we compared caspase-3 and AIF immunohistochemical staining, we found that the number of caspase-3-positive staining cells was higher. This suggests that juglone is causing apoptosis through the caspase pathway. In addition, juglone reduced the number of cells marked with BrdU in the spheroid cultures. These findings supported our monolayer culture results. Overall, there have not been enough recent studies of 3D cultures, and no studies have been conducted that used methods similar to ours. In this study, the spheroids were stained with H and E, and the spheroid structure of the control group was observed to be normal. However, in the group treated with juglone, both normal and separated spheroids were seen, and the spheroid structures had degenerated. Therefore, these findings supported our other results.

In this study, the application of juglone increased TUNEL-positive cells as compared with the control in a multicellular spheroid model.


 > Conclusions Top


As a result of our study, it was observed that juglone inhibits the proliferation and proapoptotic effect on CCL-228-SW 480 cell lines by immunohistochemistry staining in both the monolayer and spheroid models.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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