POU domain motif3 (Pdm3) induces wingless (wg) transcription and is essential for development of larval neuromuscular junctions in Drosophila

Wnt is a conserved family of secreted proteins that play diverse roles in tissue growth and differentiation. Identification of transcription factors that regulate wnt expression is pivotal for understanding tissue-specific signaling pathways regulated by Wnt. We identified pdm3m7, a new allele of the pdm3 gene encoding a POU family transcription factor, in a lethality-based genetic screen for modifiers of Wingless (Wg) signaling in Drosophila. Interestingly, pdm3m7 larvae showed slow locomotion, implying neuromuscular defects. Analysis of larval neuromuscular junctions (NMJs) revealed decreased bouton number with enlarged bouton in pdm3 mutants. pdm3 NMJs also had fewer branches at axon terminals than wild-type NMJs. Consistent with pdm3m7 being a candidate wg modifier, NMJ phenotypes in pdm3 mutants were similar to those of wg mutants, implying a functional link between these two genes. Indeed, lethality caused by Pdm3 overexpression in motor neurons was completely rescued by knockdown of wg, indicating that Pdm3 acts upstream to Wg. Furthermore, transient expression of Pdm3 induced ectopic expression of wg-LacZ reporter and Wg effector proteins in wing discs. We propose that Pdm3 expressed in presynaptic NMJ neurons regulates wg transcription for growth and development of both presynaptic neurons and postsynaptic muscles.


Results
pdm3 and sona have a positive genetic interaction. As a first step toward understanding the function of sona, we carried out a lethality-based genetic screen using ethyl methanesulfonate (EMS) as a mutagen based on the late-pupal lethality induced by Sona driven by 30A-Gal4 (Fig. 1A). 89 rare survivors were obtained among 18,000 progenies from the cross between EMS-treated 30A-Gal4 males and untreated UAS-sona females. These survivors were balanced with Sco/CyO and D/TM6 for the establishment of suppressor lines whose mutations are in the second and third chromosomes, respectively. Established lines were retested for the suppression of Sonainduced lethality, and 28 suppressors were maintained for further analysis (Fig. 1A). All suppressors showed lethality, and a few suppressors produced rare homozygous adults.
To map the position of the lethal site in suppressor m7, meiotic and deficiency mappings as well as complementation analysis were performed. Meiotic mapping was carried out by crossing the m7 suppressor with a mapping line (BDRC #4347) that contains multiple morphological markers. The meiotic mapping revealed that the lethal region in m7 is located in between the cinnabar (cn) and curved (c) (Fig. 1B). Subsequent deficiency mapping identified two different regions that are responsible for lethality, one near cn and the other near c. Complementation analysis then showed that m7 has two independent mutations in pdm3 and arrow (arr) genes on the right arm of the second chromosome (Fig. 1B). Pdm3 is a class VI POU domain transcription factor 5 , and Arr is a co-receptor of Wg ligand and essential for transduction of canonical Wg signaling 29 . The m7 suppressor was crossed with Canton-S (CS) and their progeny was checked by complementation test with pdm3 f00828 and arr 2 mutants in order to find flies with a single mutation, pdm3 m7 and arr m7 .
Genomic sequencing revealed that pdm3 m7 has a defective hobo element in the first exon of the pdm3 gene that is upstream of the initiation codon (Fig. 1C) while arr m7 has a point mutation in the arr gene (in preparation). None of the other suppressors had the hobo element in the pdm3 gene, indicating that insertion of the hobo element is unique to the m7 suppressor, and occurred subsequent to the point mutation in the arr gene. We found that the level of Pdm3 is extremely low in pdm3 m7 wing discs, establishing that insertion of the hobo element negatively affects the expression of Pdm3 (Fig. S1). Transheterozygotes of the two available deficiencies, Df(2 R) BSC267 and Df(2 R)Exel6058, were missing only the pdm3 gene in the entire genome, so Df(2 R)BSC267/Df (2 R) Exel6058 flies were used as a deletion mutant of pdm3 in this study (Fig. 1C).
We found that not only pdm3 m7 but also pdm3 f00828 , pdm3 1 and pdm3 RNAi driven by 30A-Gal4 completely suppressed the Sona-induced pupal lethality (n > 200 each). Thus, pdm3 m7 is an authentic sona suppressor, and pdm3 shows a positive genetic interaction with sona.
Boutons of pdm3 NMJs are decreased in number but increased in size, similar to wg NMJs.
Further analysis of sona suppressors revealed that sona itself and most suppressors are linked to Wg signaling 12,13 , which raised an interesting possibility that Pdm3 is also involved in Wg signaling. We noticed that pdm3 mutant larvae are slow in locomotion (Movie 1), implying a potential role of pdm3 in NMJ. To address the relationship between pdm3 and wg in NMJ, we stained pdm3 1 and wg ts /wg CX4 NMJs of the late 3rd instar larvae for a presynaptic marker Horseradish peroxidase (HRP) and a postsynaptic marker Dlg to detect Type Ib boutons at muscles 6 and 7 in the 2 nd abdominal (A2) and the 3 rd abdominal (A3) segments 20 .
We found that number of boutons in pdm3 1 NMJ was reduced by 40% and 16% compared to the wild-type counterparts in A2 and A3 segments, respectively ( Fig. 2A,B,F). Bouton numbers in pdm3 f00828 NMJ were reduced by 27% in A2 segment and those in pdm3 m7 NMJ were only mildly reduced ( Fig. S2; data not shown), so we focused our analysis on pdm3 1 NMJ that shows the most pronounced phenotype. Consistent with the previous report, number of boutons in wg ts /wg CX4 NMJ was reduced by 12% and 11% in A2 and A3 segments, respectively ( Fig. 2C-E,H).
We then carried out quantitative analysis on size of boutons in pdm3 and wg NMJs. To measure the size of boutons, serial images of boutons were taken, the images were combined, and then area of the most distal bouton in the combined image was measured (Fig. 2G,I). Size of pdm3 1 distal boutons was increased by 30% at A2 but was not increased at A3 segments compared to wild-type (Fig. 2G). Size of wg ts /wg CX4 boutons was increased by 32% and 11% in A2 and A3 segments, respectively, compared to the heterozygous controls, +/wg CX4 and +/wg ts (Fig. 2I). This is in line with a previous report that wg ts boutons are noticeably larger 20 .  Figure 1. A lethality-based genetic screen for sona suppressors and characterization of the m7 suppressor. (A) Scheme of a genetic screen for identifying suppressors that survive against late pupal lethality induced by Sona overexpression. EMS was used as a mutagen, and obtained suppressors from the screen were crossed with second and third chromosome balancers before further testing. (B) The m7 suppressor was mapped by meiotic mapping, deficiency mapping, and complementation test. Multiple morphological markers are present in the second chromosome of a mapping line BDRC 4347, and the two markers, cinnabar (cn) as an eye color mutation and curved (c) as a wing shape mutation were identified as sites closely located to the two independent lethal sites of m7 suppressor. Two lethal sites were separated by recombination with CS and subsequent complementation test with pdm3 f00828 and arr 2 identified that m7 has two lethal mutations in pdm3 and arr genes. (C) Two deficiency lines used for mapping are shown with deleted regions in red. Transheterozyogotes obtained by crossing the two deficiency lines do not have the pdm3 gene. pdm3 m7 has a defective hobo element inserted in an exon that represents the 5′ untranslated region. The blue boxes indicate remaining parts of the inserted hobo element. A red arrow marks the initiation codon and a red asterisk marks the termination codon. The scale bar is for the hobo element only.
Some pdm3 1 boutons were not clearly separated from neighboring boutons (Fig. 2B"), which is also reported in wg boutons 20 . We defined the axon branch in which more than 50% of boutons are unseparated as 'fused' branch while those in which less than 50% of boutons are fused as 'normal' branch. We found that 30.5% of axon branches in A2 segments of pdm3 1 NMJs are fused (36 out of 118). Only 3% of wild-type axon branches was fused based on this definition (8 out of 282).
One unique phenotype of pdm3 boutons was an abnormally high level of Dlg in 42.9% of NMJs examined (33 out of 77, Fig. S3). Neither wild-type NMJs (0 out of 88) nor wg NMJs (0 out of 92) had high level of Dlg. This suggests that SSR is not properly developed in pdm3 1 NMJs. We also checked the localized pattern of a glutamate for A3 of +/wg CX4 , n = 30 for A2 and 25 for A3 of +/wg ts , n = 44 for A2 and 48 for A3 of wg ts /wg CX4 , n = 52 for A2 and 49 for A3 of +/wg CX4 , and n = 50 for A2 and A3 of +/wg ts and wg ts /wg CX4 . *Represents p < 0.05; ** represents p < 0.01; *** represents p < 0.0001. Data are presented as mean ± SEM. Scale bars: 10 µm. (2020) 10:517 | https://doi.org/10.1038/s41598-020-57425-9 www.nature.com/scientificreports www.nature.com/scientificreports/ receptor GluRIIA in pdm3 and wg boutons. It has been shown that the GluRIIA pattern in wild-type bouton is cluster-like but that in wg boutons is diffused without any clusters 20 . Consistent with this report, cluster-like pattern of GluRIIA was found in CS, +/wg CX4 , and +/wg ts control boutons but wg ts /wg CX4 boutons showed diffused pattern ( Fig. S4C-E). Unlike wg NMJs, the pattern of GluRIIA in pdm31 NMJs was not noticeably different from control NMJs (Fig. S4A,B). Taken together, the loss of pdm3 or wg phenotype decreased number but increased size of boutons, but pdm3 and wg NMJs were dissimilar in the level of Dlg and the pattern of GluRIIA.

Number of axon branches in pdm3 1 NMJ is reduced. Wg signaling is required for the formation of new
branches from an existing exon, and these new branches can be visualized by Futsch 20,22 . We found that number of axon branches in pdm3 1 NMJs was decreased by 25% at the A2 segment and was unchanged at the A3 segment ( Fig. 3A",B",F). wg ts /wg CX4 NMJs showed 40% and 33% reduction in number of axon branches at the A2 and A3 segments, respectively ( Fig. 3C"-E",H). Therefore, Pdm3 is important in A2 and Wg is important for both A2 and A3 for the formation of exon branches.
Stable microtubule-bound Futsch appears as a filamentous bundle that pass through the center of NMJ axon 20,22,26 . Interestingly, the distal bouton at the end of each axon branch visualized by Futsch shows four distinct shapes: a bundled shape and three types of unbundled shapes such as looped, splayed, and diffused/punctate 20 . Splayed or diffused/punctate axon terminals indicate that microtubules are unstable due to transition to new axonal growth, while looped axon terminals indicate paused growth cones 20,30 . Proportion of distal boutons with unbundled shape is increased by mutations that affect NMJ expansion such as wg and futsch 20,26 . Magnified images of wild-type NMJs showed that less than 10% and 20% of the axon terminals at A2 and A3 are unbundled, respectively ( Fig. 3A",A"' ,G). In contrast, number of unbundled terminals was increased 7.3 and 3.4 times in A2 and A3 segments of pdm3 1 NMJs compared to wild-type, respectively (Fig. 3B",B"' ,G; red arrow and arrowheads). All unbundled axon terminals in pdm3 NMJs were either splayed or diffused/punctate, and looped axon terminals were not detected. Number of splayed or diffused/punctate terminals in wg NMJs was also increased about two times in A2 and A3 segments compared to wild-type (Fig. 3I). Thus, proportion of splayed or diffused/punctate terminals in A2 and A3 segments was significantly increased in both pdm3 and wg NMJs.
Increase in number of splayed or diffused/punctate terminals in pdm3 NMJs suggests that microtubules in pdm3 NMJs are unstable. To directly address this point, we visualized axon terminals with α-Tubulin and Futsch. In fact, signals from α-Tubulin and Futsch staining were much weaker in axon branches of pdm3 NMJ compared to wild-type (Fig. S5A,B). In case of wg NMJs, signal from α-Tubulin staining was substantially reduced in entire axon compared to wild-type ( Fig. S5C-E). In summary, microtubules become unstable, which may lead to reduced number of axon branches in both pdm3 and wg NMJs.

Pdm3 expression in motor neuron is important for NMJ growth. Wg secreted from motor neuron
and glia is important for growth and differentiation of presynaptic terminals 20,24 . To figure out which cell type among motor neuron and glia expresses Pdm3, we expressed pdm3 RNAi in motor neurons by the OK6-Gal4 driver and in glia by the repo-Gal4 driver (Fig. 4A,B). These two Gal4 lines have been used to show cell specificity of a given protein in numerous reports 22,24,[31][32][33][34] . Knockdown of pdm3 by OK6-Gal4 caused 10% reduction in bouton number, suggesting that pdm3 is required in neurons for NMJ growth (Fig. 4A). Knockdown of pdm3 by repo-Gal4 did not change bouton number, suggesting that Pdm3 expression in glia is not required for NMJ growth (Fig. 4B).
We then asked whether expression of Pdm3 by OK6-Gal4 rescues pdm3 1 NMJ phenotype. To this end, we generated UAS-pdm3 pdm3 1 /CyO-GFP and OK6-Gal4 pdm3 1 /CyO-GFP flies and checked the phenotype of their progeny, UAS-pdm3 pdm3 1 /OK6-Gal4 pdm3 1 . Unexpectedly, pdm3 1 homozygotes were cold sensitive and could not grow at the temperature lower than 22 °C, but Pdm3 overexpression by OK6-Gal4 induced lethality at the temperature higher than 24 °C. Due to this temperature restraint, UAS-pdm3 pdm3 1 /OK6-Gal4 pdm3 1 larvae were obtained only at 23 °C at a very low frequency. NMJs of these larvae showed increase in bouton number, and decrease in bouton size, and normalized level of Dlg compared to pdm3 1 NMJs (Fig. S6). Taken together, overexpressed Pdm3 in motor neurons rescued the loss of pdm3 phenotype in NMJs.
Our results so far have shown that bouton number and size of pdm3 NMJs are more severely affected in A2 than A3, so we examined the expression pattern of Pdm3 in ventral ganglion where cell bodies of motor neurons are present in order to examine the level of Pdm3 along the anterior-posterior (AP) axis. We found that Pdm3 is expressed more in the anterior part than posterior part of ventral ganglion, which is consistent with severer pdm3 phenotype in A2 than A3 (Fig. S7). Further analysis with more refined markers will help understand the effect of this AP gradient of Pdm3 on NMJ growth.
Pdm3 acts upstream to Wg in neurons. Similarity between pdm3 and wg NMJs prompted us to examine the genetic relationship between pdm3 and wg by co-expression of the two among pdm3, GFP-wg, pdm3 RNAi, and wg RNAi (Fig. 4C). As controls, bouton number of NMJs in these UAS lines was counted, which turned out to be similar to that of CS (Fig. S8). When we overexpressed pdm3 or wg by OK6-Gal4, pdm3 caused larval lethality, and wg increased bouton numbers in both A2 and A3 segments (Fig. 4C). When we knocked down pdm3 or wg by the same Gal4, the bouton number of the A2 segment was reduced by 10%, but that of the A3 segment was not changed in both cases. When GFP-wg and pdm3 RNAi were co-expressed, increase in the bouton number by GFP-Wg was not affected by pdm3i (Fig. 4C). When pdm3 and wg RNAi were co-expressed, however, lethal phenotype by overexpressed pdm3 was completely rescued by knockdown of wg. Therefore, wg is epistatically downstream to pdm3. This result raised an interesting possibility that Pdm3 may regulate wg transcription.  www.nature.com/scientificreports www.nature.com/scientificreports/ pdm3 1 adults (n = ~50 each) had other defects such as wing drooping (Fig. 5A,B), planar cell polarity (PCP) phenotype in a posterior region near the L3 vein (Fig. 5C,D), and incomplete adhesion between dorsal and ventral blades of wings (Fig. S9, Table 1 in Supplementary Information). These phenotypes of pdm3 mutants suggest that Pdm3 plays previously unidentified roles in wing development. Therefore, we decided to use wing discs to study the relationship between pdm3 and wg.
Pdm3 was highly expressed in both proximal and distal hinge regions in and near where patched (ptc) is expressed (Fig. S10A,A'). This Pdm3 pattern is genuine because Pdm3 was not detected in the ptc region of ptc > pdm3i discs (Fig. S10B,B"). Expression of Pdm3 in the hinge region may be responsible for the wing drooping phenotype of pdm3 mutants although there were no visible defects in adult wing hinges (Fig. 5C,D). No change in the level of Wg was observed in the ptc > pdm3i wing discs, suggesting that loss of pdm3 does not affect wg transcription in the DV midline of the wing pouch region (Fig. S11).

Transient expression of Pdm3 induces wg transcription. To understand the role of Pdm3 in relation
with Wg, we carried out gain of function analyses using multiple Gal4 lines. Overexpression of Pdm3 induced embryonic to pupal lethality with all Gal4 lines used in this study ( Table 2 in supplementary information). In case of en-Gal4 driver that caused embryonic lethality, some rare larval escapers were shorter than controls and had abnormal denticle patterns in the ventral epidermis (Fig. 5E,F). Pdm3 overexpression by other tissue-specific Gal4 lines also reduced size of affected tissues. For instance, Pdm3 expression by GMR-Gal4 generated small eyes (Fig. 5G,H), and that by nub-Gal4 caused mostly pupal lethality and loss of wings in rare adults ( Table 2 in supplementary information). Consistent with this phenotype of nub > pdm3 wings, size of all nub > pdm3 wing discs examined was smaller than control wing discs (n = 14 each, Fig. 6A,B).
One interesting finding was increase in the level of Wg at the DV midline of nub > pdm3 wing discs (Fig. 6A,B). To examine this phenomenon further, we transiently expressed pdm3 with Gal80 ts system using ptc-Gal4 for 6, 12, 24, 36 and 48 hours at 30 °C in order to avoid lethality by Pdm3 overexpression, and checked the level of wg-LacZ as a marker for wg transcription. We found that wg-LacZ was ectopically expressed in the ptc region after 36 or 48 hours but not before 36 hours of transient Pdm3 expression (Fig. 6C,D). The downstream effector proteins of Wg signaling, Distal-less (Dll) and Sensless (Sens), were also induced at the ptc region (Fig. 6E,F). Thus, pdm3 directly or indirectly activates transcription of wg.

Discussion
We report here that Pdm3 regulates growth and development of NMJs. pdm3 mutants showed increase in bouton size and decrease in bouton number, which are similar to the phenotype of wg mutants. Lethality induced by the overexpression of Pdm3 was rescued by knockdown of wg in NMJ, indicating that Pdm3 functions upstream to  www.nature.com/scientificreports www.nature.com/scientificreports/ Wg. Furthermore, overexpression of Pdm3 induced wg transcription in wing discs. We propose here that a major function of Pdm3 in motor neurons is to induce wg transcription, and secreted Wg from motor neurons regulates growth, development, and maturation of both pre-and post-synaptic regions of NMJ.
The mammalian homolog of Pdm3 is Brain-5 (Brn-5)/POU class 6 homeobox 1 (POU6F1) mainly expressed in brain and spinal cord. Brn-5 is heavily expressed in embryonic brain but also expressed in adult brain and multiple adult organs such as kidney, lung, testis, and anterior pituitary 35 . In developing brain, Brn-5 is expressed in postmitotic neurons after neuronal progenitor cells exit cell cycle in the early process of terminal neuronal differentiation 36 . Therefore, both Pdm3 and Brn-5 function in differentiation of neurons. Interestingly, ectopic expression of Brn-5 inhibits DNA synthesis 37 , which is similar to cell cycle arrest phenotype by Wg overexpression 38 . Given the homology between Pdm3 and Brn-5 as well as functional similarities, Brn-5 may also induce wnt transcription.
Most of Pdm3 functions identified so far are related to the maturation of neurons such as olfactory neurons, R neurons and td neurons as well as their postsynaptic partners [7][8][9] . Ectopic expression of Pdm3 induced lethality without exception, indicating that expression of Pdm3 in fly tissues is generally repressed in vivo in order to express Wg under the strict spatiotemporal control. An important question is whether Pdm3 directly transcribe wg. We found that wg transcription is induced only after 36 hours of transient overexpression of Pdm3. It is possible that the level of Pdm3 needs to be over a threshold to induce wg transcription. Alternatively, Pdm3 may need to turn on other components to indirectly induce wg transcription. DNA sequence of Brn-5 binding site has been reported [39][40][41] , so analysis on wg and wnt regulatory regions will help understand the mechanism of wnt induction by Pdm3 and Brn-5.
We consistently found more significant NMJ phenotypes in A2 than A3 in both pdm3 and wg mutants. Therefore, pdm3 and wg may play more prominent roles in the A2 than the A3 segment. In fact, the level of Pdm3 was higher in the anterior region than the posterior region of ventral ganglion, which suggests that more Wg may be present in the NMJs of anterior abdominal segments. Consistent with this idea, the number of type Ib boutons in the A2 segment was 1.8 times more than A3 segment. One difference between pdm3 and wg mutants is the lack of certain phenotypes in the A3 segment of pdm3 NMJs: the size of boutons and the number of axon terminals in A3 were not affected in pdm3 mutant. It is possible that Pdm3 turns on both common and segment-specific www.nature.com/scientificreports www.nature.com/scientificreports/ genes besides wg, and A3 segment-specific components may alleviate the loss of wg phenotype in the A3 segment. Similarly, other proteins induced by Pdm3 may also play important roles in NMJ growth, differentiation and maintenance. In fact, multiple signaling pathways including Glass-bottom-boat (Gbb) pathway also play roles in NMJ development 42,43 . Gbb is secreted from muscles and induces development of both pre-and post-synaptic structures, similar to Wg signaling.
We identified a defective hobo element in the pdm3 m7 allele. The hobo element belongs to Ac family found in maize and has short inverted terminal repeats 44 . Laboratory and wild strains of D. melanogaster have average 28 and 22 copies of hobo elements in the genome that are either full-length or defective, respectively 45,46 . Because other suppressors identified in the genetic screen using Sona overexpression did not have hobo element in the pdm3 gene, the transposition of the hobo element to the pdm3 gene may have occurred subsequent to the generation of a point mutation in the arr gene by EMS. Since both arr and pdm3 are positively involved in Wg signaling, this hobo insertion may have helped the original arr m7 mutation to further decrease the activity of Wg signaling under the condition of Sona overexpression.
Besides the neuronal roles of Pdm3, all pdm3 mutants show minor but consistent defects in planar cell polarity in a restricted region of the wing as well as adhesion between the dorsal and ventral wing blades. Other phenotypes such as wing drooping and premature death were also observed in all pdm3 mutants, but these may be due to malformation of synaptic structures. Pdm3 also plays a role in female-limited color dimorphism in abdomen of D. montium 11 . The authors found in sexually dimorphic females that the first intron of the pdm3 gene has four tandem sets with predicted binding sites for the HOX gene Abdominal-B (Abd-B) and the sex determination gene doublesex (dsx). Interestingly, it has been shown that Wg expression is repressed by the combinatory work of Abd-B and Dsx proteins 47 . Taken together, it is possible that transcription of wg and pdm3 is co-repressed by Abd-B and Dsx. Such co-repression of wg and pdm3 transcription may be also required for synaptic growth and differentiation in neurons. Further studies on Pdm3 will help understand how this understudied transcription factor is involved in the final differentiation of various cell types.
Image capture and quantitative analysis of boutons. We stained the late 3rd instar larvae for a presynaptic marker HRP and a postsynaptic marker Dlg to detect NMJs. Type Ib boutons have more extensive SSR compared to other bouton types (Is, II, and III), so are easily detected by the high level of Dlg 19 . Therefore, type Ib boutons are defined as round-shaped structures in NMJ branches that are stained with HRP and have high level of Dlg, and only ones that were qualified to this definition were counted as boutons. To obtain images containing boutons, type Ib boutons visualized with HRP and Dlg were taken with 1 μm interval for 7-10 Z stacks at 400X magnification by a confocal laser microscope of Carl Zeiss (NFEC-2010-09-141569) with Zen 2009 program. To manually count number of boutons, all Z stack images were then merged and type Ib boutons at muscles 6 and 7 in A2 or A3 segments in a given image were counted. The number of images used for counting for each genotype was 17-52. To measure size of boutons, images of terminal boutons were captured at 2,000X magnifications and then area of boutons in merged images was measured by Zen 2009 program.
Cuticle preparation of larvae. The cuticle preparation was performed as described with slight modifications 54 . Flies were put into a chamber with a grape juice-containing agar plate that has yeast paste at the center. After 4 hours of egg-laying, plates were incubated for 20 hours at 25 °C. Larvae were transferred to distilled water on cover glass, and washed again with distilled water. Water was then removed and the 1:1 mixture of lactic acid and Hoyer's mount solution was applied. After waiting for about 1 minute, the sample on a cover glass was placed on the slide glass, and then incubated for overnight at 65 °C.
Statistical analysis. Statistical analysis was performed using ANOVA to compare different genotypes to a wild-type control within experimental groups. Data are presented as mean ± SEM. To determine statistical significance, t-test and one-way ANOVA of Microsoft Excel 2019 were used.