Irilone, a Red Clover Isoflavone, Combined with Progesterone Enhances PR Signaling through the Estrogen and Glucocorticoid Receptors

To study the potentiation mechanism of action of irilone, we decided to first determine the expression of the steroid receptors in the cell lines used in this paper. The level of receptors that might bind ligands for PR, ER, and GR is important, as many ligands are capable of interacting with these receptors at different concentrations. Four cell lines were evaluated for their steroid receptor expression: Ishikawa Null, Ishikawa PR-B, OVCAR5, and T47D cells. Ishikawa PR-B is the parental cell line that is engineered to stably express PR-B through a CMV promoter and this cell line was made by the Blok lab. 29 To determine the expression of steroid receptors in the cell lines, a western blot was performed for ER, PR, and GR. T47D cells had a high expression of both PR-B and PR-A ( Figure S1A , Supporting Information ). Ishikawa PR-B express both PR-B and PR-A ( Figure S1A ). Ishikawa Null, Ishikawa PR-B, and OVCAR5 cells did not express ERα ( Figure S1B ). Ishikawa null cells do not express PR, GR, or ER ( Figure S1C ), while the Ishikawa PR-B, OVCAR5, and T47D cells express GR ( Figure S1C ). Irilone has been reported to be present at between 3-18% weight of red clover extracts. 23 , 28 Irilone (10 μM) was combined with three concentrations of progesterone (1, 5, and 100 nM) to determine whether it can potentiate lower progesterone concentrations in the hormone response element (HRE)/luciferase (Luc) assay, which was tested previously at 100 nM. 23 Ishikawa PR-B uterine epithelial cells and T47D breast cancer cells were tested in the HRE/Luc assay. The cells were treated with either progesterone alone (black bars) or irilone plus progesterone (gray bars). Irilone (10 μM) significantly potentiated progesterone luciferase activity at 5 nM in the Ishikawa PR-B and T47D cells (P <0.01 and P<0.05 respectively, Figure 1A - B ). In the Ishikawa PR-B cells, irilone (10 μM) also potentiated progesterone luciferase activity at 100 nM (P<0.05, Figure 1A ). In the T47D cells, irilone (10 μM) potentiated progesterone luciferase activity at 5 nM (P<0.05, Figure 1B ). To confirm that the activity irilone was demonstrating in the HRE/Luc assay was potentiation, irilone was treated alone in both the Ishikawa PR-B and T47D cells ( Figure 1C - D ). Progesterone (100 nM) activated HRE/Luc activity 27-fold (P<0.001) and the HRE/Luc induction of irilone (10 μM) was 1.2-fold in the Ishikawa PR-B cells ( Figure 1C ). In the T47D cells, progesterone (100 nM) induced HRE/Luc activity 26-fold (P<0.0001) and the HRE/Luc induction of irilone (10 μM) was 1.07-fold ( Figure 1D ). Irilone (10 μM) does not induce HRE/Luc activity alone. These experiments confirmed that even at lower concentrations than previously tested (50 μM), irilone can potentiate progesterone action. Lowering the concentration of progesterone to study potentiation is important to avoid saturating the assay and increases our understanding of irilone action at lower concentrations that might be more physiologically relevant. One mechanism of how irilone could be potentiating progesterone signaling is by altering post-translational modifications of the PR. One post-translational modification that has been commonly studied and has an effect on PR transcriptional activity is phosphorylation at serine 294 (Ser294) on PR. 4 T47D cells were treated with control, R5020 (10 nM), irilone (10 μM), and R5020 (10 nM) plus irilone (10 μM) for 60 min. R5020 is a synthetic progestin agonist that binds to PR with high binding affinity, behaves like progesterone in the genomic signaling pathway, and is a ligand that is typically used in the literature to understand PR biology. 30 R5020 differs from progesterone in that R5020 does not bind to membrane progesterone receptors (mPR), and therefore does not elicit the rapid non-genomic effects of progesterone. 31 When studying PR post-translational modifications like phosphorylation, R5020 is typically used, as it does not activate MAPK through mPR. R5020 (10 nM) induced phosphorylation of Ser294 of PR-B and PR-A in T47D cells after 60 min of treatment (P<0.05 and P<0.01 for PR-B and PR-A, respectively, Figure 2A - C ). Irilone (10 μM) alone did not induce phosphorylation of Ser294 of PR-B and PR-A in T47D cells after 60 minutes of treatment ( Figure 2A - C ). Irilone (10 μM) combined with R5020 (10 nM) phosphorylated Ser294 on both PR-B and PR-A (P<0.05 and P<0.01 for PR-B and PR-A, respectively, Figure 2A - C ), but did not enhance the phosphorylation of Ser294 compared to R5020 alone. Increasing phosphorylation of Ser294 of PR-B and PR-A was not a potentiation mechanism of action for irilone. Agonists and antagonists of PR reduce expression of the receptor due to ubiquitin triggered proteasomal degradation and an increase of PR protein levels could enhance PR signaling. To determine whether irilone alters PR protein levels, western blots were performed in the Ishikawa PR-B and T47D cells for PR. Ishikawa PR-B cells were treated with progesterone (100 nM), irilone (10 μM), and progesterone (100 nM) plus irilone (10 μM) for 24 h. Progesterone (100 nM) reduced PR protein levels, irilone did not affect PR protein levels, and irilone did not increase nor reduce PR protein levels when combined with 100 nM progesterone in the Ishikawa PR-B cells (P<0.05, Figure 3A - C ). To determine whether irilone was affecting PR protein levels in T47D cells that endogenously express PR, the T47D breast cancer cell line was used. T47D cells were treated with progesterone (10 nM), irilone (10 μM), and progesterone (10 nM) plus irilone (10 μM) for twenty-four hours. Progesterone (10 nM) reduced PR protein, irilone (10 μM) increased protein of PR, and irilone (10 μM) did not change PR expression when combined with 10 nM progesterone (P<0.05, Figure 3D - F ). PR after ligand binding is degraded by the 26S proteasome, and when the proteasome is inhibited, PR does not get degraded. 4 MG132 is a proteasome inhibitor that prevents PR from being degraded and was used as a positive control. 32 Ishikawa PR-B and T47D cells were treated with progesterone (100 nM or 10 nM respectively), MG132 (2.5 μM), and progesterone plus MG132 for 24 h. MG132 (2.5 μM) alone increased PR protein levels in both cell lines. When MG132 (2.5 μM) was combined with 100 nM progesterone, MG132 prevented the reduction of PR via western blot in both cell lines ( Figure 3G - H ). Irilone increased PR protein levels on a western blot after 24 h, which is suggestive of estrogenic activity, since PR is a well-known target gene of ER transcriptional activity. 8 Since T47D cells express ERα, an estrogen response element luciferase (ERE/Luc) assay was performed to confirm this action. The positive control 17β-estradiol (E2, 1 nM) induced ERE/Luc 3-fold in T47D cells (P<0.05, Figure 4A ). Irilone (10 μM) induced ERE/Luc 4-fold in T47D cells (P<0.05, Figure 4A ). These data indicate that irilone activates ER signaling in T47D cells. Western blots were performed in T47D cells to determine whether irilone continued to alter PR expression when ER was blocked with the selective estrogen receptor modulator (SERM), 4-hydroxytamoxifen (4-OH-TAM). When irilone (10 μM) was combined with 4-OH-TAM (10 μM), an increase in PR expression of both PR-B and PR-A was not observed in the T47D cells ( Figure 4B - D ). When 4-OH-TAM (10 μM) was combined with progesterone (10 nM) and irilone (10 μM), there was no change in PR levels compared to control ( Figure 4B - D ). These findings indicate that the increase of PR expression by irilone occurs through ER signaling. Even though irilone was not affecting PR protein levels when ER was blocked, some evidence suggested that ER mediated an increase of PR expression and increased overall PR transcriptional activation of the HRE/luciferase reporter. Diep et al. demonstrated that when ER is phosphorylated and PR is bound with ligand (either R5020 or progesterone), ER and PR co-localize on a ERE promoter to induce PR expression. 33 Since we demonstrated that irilone was increasing PR expression, irilone may be eliciting ER and PR crosstalk on the ERE promoter. Further studies are needed to confirm this hypothesis. Ishikawa Null cells also do not express GR or ER, while the Ishikawa PR-B cells express GR but not ER. Therefore, GR expression might be necessary for the potentiation mechanism of action of irilone observed in Ishikawa PR-B cells. Since ligands that activate PR can also act as ligands for GR, the HRE/Luc assay was performed with irilone in OVCAR5 cells, a cell line that only contains GR and lacks ER and PR ( Figure S1C ). The GR agonist, dexamethasone (100 nM), was used as a positive control and induced HRE/Luc activity 9.6-fold in the HRE/Luc assay (P<0.0001, Figure 5A ). Three concentrations of irilone (1, 5, and 10 μM) were tested for GR activity. In the absence of dexamethasone, irilone (1, 5, and 10 μM) did not induce HRE/Luc activity ( Figure 5A - B ). All three concentrations of irilone were combined with dexamethasone (100 nM) to determine whether irilone could repress dexamethasone’s HRE/Luc activity. Dexamethasone alone induced HRE/Luc fold-induction 15.6-fold (P<0.05, Figure 5B ). Compared to dexamethasone alone, irilone (10 μM) reduced dexamethasone’s HRE/Luc activity by 22% (P<0.05, Figure 5B ). Irilone (10 μM) represses dexamethasone’s HRE/Luc activity in the OVCAR5 cells. To test whether this was dose responsive, 1 and 5 μM irilone were combined with dexamethasone, but those concentrations did not block dexamethsone’s action ( Figure S2 ). Next, the expression levels of GR were evaluated upon irilone treatment in OVCAR5 and Ishikawa PR-B cells. OVCAR5 cells do not express PR but do express the GR, while PR-B express both. Irilone (10 μM) increased GR protein in the Ishikawa PR-B cells (P<0.05, Figure 5C - D ). Dexamethasone (100 nM) did not alter GR protein in the OVCAR5 cells ( Figure 5E - F ). Irilone did not increase GR protein levels in the OVCAR5 cells but did increase GR protein levels in the Ishikawa PR-B cells ( Figure 5C - F ). Very few compounds have been reported to increase GR expression. One compound that increases the expression of GR is a synthetic compound, GSK4716, which binds to the estrogen related receptor (ERR) and is an agonist for the ERRβ/γ isoforms. 34 PR becomes rapidly SUMOylated upon ligand binding, and becomes deSUMOylated via phosphorylation of PR. 5 When PR is not able to be deSUMOylated, there is a repression of PR transcriptional activity. 5 The site of SUMOylation on PR is at lysine 388 and when this site was mutated to arginine, PR is not SUMOylated. 5 The removal of SUMO on PR increases PR transcriptional activity. 5 Since irilone is increasing PR activity in the HRE/Luc assay, we attempted to test whether this was due to SUMOylation removal. Ishikawa Null cells were transfected with Empty Vector (EV), WT PR and PR-B K388R (PR-B KR) plasmids, and the HRE/Luc assay was performed. A western blot confirmed that the plasmids equally expressed WT PR and PR-B KR ( Figure S3 ). Progesterone, irilone, and progesterone combined with irilone did not induce HRE/Luc in the Ishikawa Null cells transfected with empty vector due to lack of PR expression ( Figure 6A ). In the cells transfected with WT PR, progesterone (5 nM) induced HRE/Luc 39-fold (P<0.05, Figure 6A ). Irilone (10 μM) induced HRE/Luc 5.8-fold in the Ishikawa Null cells transfected with WT PR ( Figure 6A ). Irilone (10 μM) combined with progesterone (5 nM) induced HRE/Luc 38-fold in the Ishikawa Null cells transfected with WT PR (P<0.05, Figure 6A ). Irilone combined with progesterone was not statistically significant compared to progesterone alone, indicating that irilone was not potentiating progesterone in the Ishikawa Null cells transfected with WT PR, which was a surprsing result and indicated that this cell model could not be used to test SUMO on irilone enhanced PR activity. We predicted that something was missing in the Ishikawa null compared to the Ishikawa PR-B cells that prevented potentiation. Ogara et al. had determined that GR agonists can block induction of genes regulated by PR. 35 Conversely, it is possible that GR antagonists could cause an enhancement of progesterone signaling, which is consistent with the result that irilone repressed luciferase activity when combined with dexamethasone in a cell line that lacked ER and PR but expressed GR. To determine whether GR was involved in irilone’s potentiation mechanism of action, GR was knocked down in the Ishikawa PR-B cells with a GR siRNA. A western blot, performed in the Ishikawa PR-B cells that were transfected with the GR siRNA, confirmed that GR was successfully knocked down in those cells ( Figure S4A ). Expression of PR also was not affected by the GR siRNA ( Figure S4B ). The HRE/Luc assay was then performed in the Ishikawa PR-B cells that were transfected with the GR siRNA or scrambled control to determine if irilone could potentiate progesterone signaling when GR was knocked down. In the Ishikawa PR-B cells that were transfected with the control siRNA, progesterone (5 nM) induced luciferase 30-fold and this effect was increased by irilone combined with progesterone to 44-fold (P<0.05, Figure 6B ). Irilone potentiated progesterone luciferase activity in Ishikawa PR-B cells that were transfected with non-target siRNA. In the Ishikawa PR-B cells with GR siRNA, progesterone (5 nM) induced luciferase activity 27-fold and irilone (10 μM) combined with progesterone (5 nM) induced luciferase activity 30-fold (P<0.05, Figure 6B ). To further confirm that GR activity was affected by the siRNA, Ishikawa PR-B cells were transfected with either a control siRNA or the GR siRNA and then treated with dexamethasone (100 nM). In the cells that were transfected with the control siRNA, dexamethasone (100 nM) induced luciferase activity 10-fold ( Figure 6C ), while the GR siRNA, had a reduced luciferase activity of 2.8-fold ( Figure 6C ). The luciferase activity of dexamethasone in the cells transfected with GR siRNA was reduced, confirming that GR was knocked down in the Ishikawa PR-B cells that were transfected with the GR siRNA. Irilone combined with progesterone was not statistically different from progesterone alone in the Ishikawa PR-B cells that were transfected with the GR siRNA. This indicates that knockdown of GR blocks the ability of irilone to potentiate progesterone transcriptional activity. One of the most interesting aspects of irilone’s mechanism of action is the role of GR and how the presence of GR is important for irilone to potentiate progesterone signaling in the Ishikawa PR-B cells. PR/GR crosstalk has not been well-documented, as there are only two published studies that address this subject. 35 , 36 Leo et al. transfected PR into MDA-MB-231 breast cancer cells and demonstrated that GR agonists are able to mimic progesterone activity by inhibiting proliferation, inducing cell spreading and focal adhesion. 36 The authors concluded that these GR agonists are involved with GR and PR crosstalk as PR antagonists, and were able to inhibit the effects of the GR agonists. 36 Ogara et al. found that GR agonists antagonize PR transcriptional activity and bind together at various genomic sites. 35 If GR agonists antagonize PR activity, it is possible that GR antagonists would be able to increase PR transcriptional activity. An animal study was performed to determine whether irilone could potentiate progesterone signaling in vivo . CD-1 mice were ovariectomized and injected intraperitoneally with either vehicle control, progesterone (1 mg/kg), irilone (4 mg/kg), or progesterone/irilone once a day for seven days. On the seventh day, the mice were sacrificed 6 h after treatment and the uteri, mammary gland, and liver were collected. Immunohistochemistry (IHC) was performed on one uterine horn, as the other horn was frozen for qPCR analysis. The PR target gene, FKBP5 was analyzed to determine whether irilone could potentiate progesterone signaling in vivo via IHC. Compared to progesterone alone, irilone combined with progesterone did not potentiate progesterone signaling at the PR target gene, FKBP5 ( Figure S5 ). Proliferation was evaluated by IHC for PCNA staining to determine whether irilone induces proliferation in vivo . In the control group, 33.3% of the epithelial cells were positively stained for PCNA ( Figure 7A - B ). There was a reduction of PCNA staining in the epithelial cells of the progesterone, irilone, and progesterone/irilone group compared to the control group. In the progesterone group, 7% of the epithelial cells were positively stained for PCNA (P<0.0001, Figure 7A - B ). In the irilone group, 18.4% of the epithelial cells were positively stained for PCNA (P<0.01, Figure 7A - B ). In the progesterone/irilone group, 0.93% of the epithelial cells were positively stained for PCNA staining (P<0.0001, Figure 7A - B ). Many women consume botanical supplements for various gynecological ailments and an increase in uterine proliferation could increase the risk of hyperplasia. It is important to determine the safety of these botanical supplements and compounds in regard to hyperplasia of reproductive tract tissues, and it supports data that red clover extracts do not cause uterine hyperplasia. 37 - 39 Irilone potentiates progesterone signaling through the ER in the T47D cells and through the GR in the Ishikawa PR-B cells. Irilone does not induce proliferation of the uterus in vivo . Irilone’s mechanism of action gives insight into PR crosstalk with other steroid hormone receptors, which can be important for understanding gynecological diseases.

Ishikawa cells that had stable expression of PR-B under the control of a CMV promoter 29 were graciously donated by Dr. Leen J. Blok, Department of Obstetrics and Gynecology, Erasmus, Medical Center, Rotterdam, The Netherlands. Ishikawa PR-B cells were maintained in DMEM/F12 phenol red free media (11039-021, Invitrogen) supplemented with 5% charcoal dextran stripped FBS and selection antibiotics (500 μg/mL G418 and 500 μg/mL hygromycin). Ishikawa Null cells were maintained in DMEM/F12 phenol red free media supplemented with 5% charcoal dextran stripped FBS and penicillin/streptomycin (final concentration: 100 I.U./mL of penicillin and 100 μg/mL of streptomycin). T47D cells were purchased from ATCC and maintained in RPMI 1640 (MT10040CV, Corning) supplemented with 10% FBS, 25 mM glucose (G8270 Sigma Aldrich), 2 mM L-glutamine (25030081, Thermo Scientific), 100 mM sodium pyruvate (11360070, Thermo Scientific), 18 mM sodium bicarbonate (S5761, Sigma Aldrich, St. Louis, MO), 10 mM HEPES (BP310, Thermo Scientific, 0.2 units/mL of insulin (700112P, Gemini Bio-products), and penicillin/streptomycin (final concentration: 100 I.U./mL of penicillin and 100 μg/mL of streptomycin). OVCAR5 cells were maintained in MEM media (11095098, Thermo Scientific) supplemented with 10% FBS, 2 mM L-glutamine, 1% non-essential amino acids (100X) (11140050, Thermo Scientific), 100 mM sodium pyruvate (11360070, Thermo Scientific), and penicillin/streptomycin (final concentration: 100 I.U./mL of penicillin and 100 μg/mL of streptomycin). T47D and OVCAR5 cells were plated in phenol red free media that contained double charcoal stripped FBS for 24 h before experimental analysis. All cells were validated by STR analysis and maintained in a humidified incubator at 37 °C and 5% CO 2 . Progesterone (P0130-25G, Sigma Aldrich, St. Louis, MO), R5020 (NLP004005MG, Perkin Elmer), 17β-estradiol (10006315, Cayman Chemical), 4-OH-Tamoxifen (17308, Cayman Chemical), MG132 (M7449, Sigma Aldrich), dexamethasone (11015, Cayman Chemical), RU486 (10006317, Cayman Chemical), and irilone (BCN8188, Biocrick, 99% purity; Amb2264670, Ambinter, 90% purity) were dissolved in DMSO at 1000x final concentration to ensure 0.1% DMSO in media. Ishikawa PR-B and Ishikawa Null cells (40,000 cells/well), T47D (50,000 cells/well), or OVCAR5 cells (15,000 cells/well) were plated in 24 well plates in phenol red free media with charcoal stripped FBS. After 24 h, a luciferase construct and a β-galactosidase construct under the control of a CMV promoter were transfected into the cells. The conditions for the different types of luciferase assays are outlined below. PR and GR share the same response element on the DNA 13 - 15 called a hormone response element (HRE). For the hormone response element (HRE) luciferase (Luc) assay, transcriptional activity of PR and GR was measured using a plasmid expressing the thymidine kinase promoter with two steroid response elements (ACAAGA half-site) that binds to PR and GR 40 as previously described. 20 HRE/Luc and β-galactosidase were transfected into Ishikawa PR-B (200 ng/well of HRE/Luc and 100 ng/well of β-galactosidase), T47D cells (100 ng/well of HRE/Luc and 100 ng/well of β-galactosidase) or OVCAR5 (200 ng/well of HRE/Luc and 100 ng/well of β-galactosidase) using LT1 transfection reagent to carry out the transfections per the manufacturer’s instructions (MIR2304, Mirus Bio). For the Ishikawa Null cells, empty vector (EV), WT PR, or PR-B K388R (PR-B KR) were transfected along with the HRE/Luc and β-galactosidase (10 ng/well of receptor, 200 ng/well of HRE/Luc, and 100 ng/well of β-galactosidase) using LT1 transfection reagent to carry out the transfections per the manufacturer’s instructions (MIR2304, Mirus Bio). Transcriptional activity of ER was measured using an ERE/Luciferase plasmid. 41 ERE/Luc and β-galactosidase were transfected into T47D cells (100 ng/well of ERE/Luc and 100 ng/well of β-galactosidase) using LT1 transfection reagent to carry out the transfections per the manufacturer’s instructions (MIR2304, Mirus Bio). For the luciferase with the GR siRNA, 200 ng/μl of a NR3C1 endoribonuclease small interfering RNA (siRNA) (EHU068001, Sigma Aldrich) or 10 nM of a negative control siRNA (SIC001, Sigma Aldrich) was transfected into the Ishikawa PR-B cells along with a HRE/Luc (200 ng/well) and β-galactosidase construct (100 ng/well) using Mirus Transit-X2 (MIR6004, Mirus Bio) following manufacturer’s protocol. The next day cells were treated with pure compounds for 24 h. After treatment with pure compounds, 110 μL of lysis buffer was added to each well and cells were frozen at −80°C. Luciferase activity was measured as previously described. 24 T47D cells (500,000 cells/well), Ishikawa PR-B cells (500,000 cells per well), or OVCAR5 cells (200,000 cells/well) were plated in 6 well plates in phenol red free media for 24 h. Cells were treated with pure compounds for 24 h and then lysed with lysis buffer containing RIPA, a phosphatase inhibitor, and a protease inhibitor. To determine protein concentration, a BCA assay was performed. Protein lysate (30-35 μg) was run on SDS-PAGE gels by electrophoresis and then transferred to a nitrocellulose membrane. Membranes were blocked with 5% Milk in TBST and probed at 4 °C overnight with primary antibodies ( Table S1 ). Membranes were incubated with secondary antibody ( Table S2 ) and developed with SuperSignal West Femto Substrate (34095, Thermo Scientific). FluorChem C (Protein Simple) was used to capture images. Densitometric analysis was performed by ImageJ software. Ovariectomized female CD1 mice (age 7 weeks old from Envigo) were randomly assigned into four different treatment groups with 5 mice per group. Treatments consisted of vehicle (10% DMSO), 1 mg/kg progesterone, 4 mg/kg irilone, and 1 mg/kg progesterone + 4 mg/kg irilone. Irilone was purchased from Biocrick and progesterone was purchased from Sigma Aldrich. Progesterone and irilone were dissolved in 10% DMSO and 90% sesame oil. Treatments were administered via intraperitoneal injection once daily for seven days. On the seventh day after 6 h of treatment, mice were euthanized by humane means. uteri, mammary glands, and liver were excised for immunohistochemical (IHC) and qPCR analysis. Half of the reproductive tracts, mammary glands, and a lobe of the livers were fixed in 4% paraformaldehyde (PFA) for IHC. The other half of the reproductive tracts, mammary glands, and a lobe of the livers were snap frozen in liquid nitrogen for qPCR analysis. All protocols were approved by University of Illinois at Chicago Animal Care and Use Committee and the Association for the Assessment and Accreditation of Laboratory Animal Care (Protocol #18-205). Four of the five animals of each group were stained for IHC. Uterine tissues were fixed in 10 mL of 10% buffered formalin for 24 h, transferred into 70% EtOH and processed for histology as described before. 42 The uterine tissues were processed in a Citadel 1000 processor (Thermo), embedded with paraffin into 5 mm thick blocks and then sectioned into 5 μm thick sections using a microtome. The slides were dried for at least 24 h before being processed for further analysis. IHC was performed for FKBP5 and PCNA on uterine samples as previously described. 43 Briefly, slides of uterine horns were deparaffinized using three xylene washes and rehydrated though a series of decreasing concentrations of EtOH, then subjected to heat-induced antigen retrieval with sodium citrate buffer at 100 °C for 30 min and allowed to cool to room temperature (rt). This was followed by inactivation of endogenous peroxidase activity with 0.3% H2O2/MeOH for 15 min in dark. The samples were then rinsed with phosphate buffered saline with Tween-20 (PBST) and incubated in blocking solution consisting of 5% horse serum (Vectastain ABC kit, Vector Laboratories, Inc. Burlingame, CA) diluted in 1%BSA/PBST at RT for 60 minutes. The tissue sections were incubated with primary antibodies overnight at 4 °C ( Table S3 ). Next day, slides were rinsed with PBST prior to incubation with anti-goat biotinylated secondary antibody (Vectastain ABC kit, Vector Laboratories, Inc.) at 1:200 dilution in PBST for 60 min at rt. Slides were then rinsed and incubated in ABC solution (PBS: A: B=50:1:1) (Vectastain ABC kit, Vector Laboratories, Inc.) for 30 min at rt. For visualization of the immunoreactivity, all slides were subjected to chromogen 3’3-diaminobenzidine (DAB) (Vector Laboratories, Inc.) for 30 seconds. Slides were rinsed in tap water for 10 min to stop the DAB reaction. Thereafter, the slides were counterstained with hematoxylin for one minute followed by dehydration and cover-slipping. After drying for 24 h, the slides were cleaned and imaged using Nikon E600 microscope with CMOS C-Mount microscope camera. Data is represented as mean ± standard error and replicated at least three times. The data was analyzed with Prism version 8.4.1. The data was analyzed by a one-way ANOVA followed by a Dunnett’s or Tukey’s post-hoc analysis or Student’s t-Test comparing each treatment to a control group as indicated. Significance was determined by P<0.05.