Comprehensive analysis of FKBP4/NR3C1/TMEM173 signaling pathway in triple-negative breast cancer cell and dendritic cell among tumor microenvironment

TMEM173 is a pattern recognition receptor detecting cytoplasmic nucleic acids and transmits cGAS related signals that activate host innate immune responses. It has also been found to be involved in tumor immunity and tumorigenesis. In this study, we first identified that the FKBP4/NR3C1 axis was a novel negative regulator of TMEM173 in human breast cancer (BC) cells. The effect of FKBP4 appeared to be at the transcriptional level of TMEM173, because it could suppress the promoter activity of TMEM173, thereby affecting TMEM173 at mRNA and protein levels. Past studies, our bioinformatics analysis, and in vitro experiments further implied that FKBP4 regulated TMEM173 via regulating nuclear translocation of NR3C1. We then demonstrated that the FKBP4/NR3C1/TMEM173 signaling pathway could regulate autophagy and proliferation of BC cells as well as dendritic cell (DC) abundance through exosome release. Our study found an unprecedented strategy used by BC to escape from TMEM173 mediated tumor suppression. Identification of the FKBP4/NR3C1 axis as a novel TMEM173 regulator would provide insights for novel anti-tumor strategy against BC among tumor microenvironment.


INTRODUCTION
Breast cancer (BC) is a leading cause of cancer related deaths in women aged 40 years and younger. 1 Early detection and comprehensive treatments, which consist of surgery, radiation, chemotherapy, endocrine therapy, and targeted therapy, have dramatically improved the prognosis of BC patients. In recent years, immunotherapy in BC has been shown to have a promising future. Cancer vaccines, bispecific antibodies, and immune checkpoint inhibitors are verified to have potential applied value in BC immunotherapy. 2 For instance, adaptive immune checkpoint therapies, by targeting cytotoxic Tlymphocyte antigen-4 (CTLA-4), programmed cell death protein 1 (PD-1), and ligand partner for PD-1 (PD-L1) for BC, have been used in clinical trials. 3,4 Nevertheless, a portion of BC patients still cannot benefit from above-mentioned immunotherapy strategies. 5 Therefore, unraveling the potential molecular mechanisms of both the innate and adaptive immune systems in BC cells is essential to further understanding and improving immune related anti-tumor effects.
Transmembrane protein 173 (TMEM173), also named stimulator of interferon genes (STING), residing in the endoplasmic reticulum (ER), has been identified early as a critical adaptor for cyclic dinucleotides (CDNs) produced from a cellular nucleotidyltransferase, referred to as cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS), and regulates the induction of numerous host defense genes. [6][7][8] Therefore, TMEM173 has been found involved in anti-microbial innate immunity as well as the pathogenesis of some autoimmune disorders. 9,10 Recently, several studies revealed the suppressive function of TMEM173 in tumorigenesis, including BC, gastric cancer, leukemia, prostate cancer, colorectal cancer, melanomas, and so forth. [11][12][13][14][15][16] A recent study has discovered that HIV-2/simian immunodeficiency virus (SIV) Virus Protein X (Vpx) acts as a novel inhibitor of innate immune activation associated with TMEM173 signalosomes 17 ; we wondered whether and how TMEM173 performs its anti-tumor effects by connection with novel molecular chaperones in BC.
As one of the most extensively studied proteins among the 18 identified human FK506-binding proteins (FKBPs), FK506-binding protein 4 (FKBP4), also known as FKBP52, has been reported to exhibit multiple functions, which involve binding to different cellular receptors or targets in various kinds of cancers. [18][19][20][21][22] For example, FKBP4 has been demonstrated to interact with heat shock protein 90 (HSP90) to affect steroid hormone receptor function in BC. 23 In terms of immune regulation, phytanoyl-CoA alpha-hydroxylase (PAHX) has been regarded as a specific target of FKBP4 for studying the cellular signaling pathway in the presence of immunosuppressant drugs. 24 Our previous work found that FKBP4 interacted with noncoding RNA and mRNA during the occurrence and development of BC, thus playing a role in promoting cancer. 22,25 Nevertheless, current immunologic mechanism of FKBP4 is still in its infancy for BC; it is necessary to explore more newly detailed contents of its regulation of innate and adaptive immunity functions during the occurrence and development of BC.
The NR3C1 (nuclear receptor subfamily 3, group C, member 1/glucocorticoid receptor) normally resides in the cell cytoplasm; the NR3C1 protein translocates to the nucleus when bound to glucocorticoids, thus is involved in growth, reproduction, metabolism, immune, and inflammatory reactions as well as central nervous system and cardiovascular functions and tumor cellular proliferation and differentiation. 26 Research on NR3C1 and BC has also been conducted in recent years; e.g., high levels of NR3C1 expression and high concentrations of cortisol have been shown to have an anti-proliferative effect in cancerous breast tissue. 27 Some studies have preliminarily found that NR3C1 is associated with FKBPs, but the specific mechanisms remain unclear in BC. 28 Exosomes, also termed small extracellular vesicles, have a diameter ranging from 40 to 150 nm, which are secreted by different types of cells and contain various cargoes, including DNA, mRNA, noncoding RNA, proteins, and so forth. 29 Currently, research on the role of exosomes involved in cancer progression has grown exponentially, including that of immune regulation, which suggests that cancer cells could secrete large numbers of exosomes to regulate innate and adaptive immune cells among tumor microenvironment for immune escape. 30 For instance, a study by Wen et al. showed that highly metastatic murine BC cells derived exosomes directly suppressed T cell proliferation and inhibited natural killer (NK) cell cytotoxicity. 31 Although Diamond et al. identified exosome as a mechanism whereby DNA was transferred from irradiated BC cells to tumor-infiltrating dendritic cells (TIDCs), 32 whether and how FKBP4 or TMEM173 participated in exosomes secreted by BC cells might regulate dendritic cell (DC) maturation and function remain to be explored.
In this study, we showed that the newfound FKBP4/NR3C1/TMEM173 signaling pathway suppressed autophagy and promoted proliferation in luminal A and basal-like subtypes of BC cells. Meanwhile, the above-mentioned axis was also found involved in triggering BC cells to excrete exosomes to TIDCs among tumor microenvironment, thus leading to abundance and maturation of TIDCs. Mechanically, these effects relied on downregulation of FKBP4, which transcriptionally upregulated TMEM173 through intensive nuclear translocation of NR3C1. Identification of the FKBP4/NR3C1 axis as the novel TMEM173 transcriptional regulator would provide in-depth insights for immunological anti-tumor strategy to overcome BC.

RESULTS
Negative correlation of FKBP4 and TMEM173 in triple-negative breast cancer First, the FKBP4 protein topology revealed intracellular membrane (cytosol and nucleoplasm) localization (Figures S1A and S1B), while the TMEM173 protein topology revealed extracellular membrane (cytosol and nucleoplasm) localization (Figures S1A and S1B); we also observed that FKBP4 and TMEM173 colocalized with the nuclear marker in different cells by immunofluorescence assay of the Human Protein Atlas (HPA) database, suggesting the subcellular localization of FKBP4 and TMEM173 in nuclei ( Figure S1C). Then, we used UALCAN, 33 a portal for facilitating tumor subgroup gene expression and survival analyses, to explore the clinicopathological characteristics of FKBP4 and TMEM173. Upregulated FKBP4 was found significantly related to luminal, HER2-positive, and basal-like subtypes of BC patients than the normal group ( Figure 1A), and downregulated TMEM173 was only significantly related to the basal-like subtype of BC patients than the normal group ( Figure 1A). Furthermore, we validated a significant negative association between both FKBP4 and TMEM173 in the basal-like subtype of BC patients ( Figure 1B). Then, the prognostic merits of FKBP4 and TMEM173 in the basal-like subtype of BC patients were further analyzed by using bc-GenExMiner v4.7 34 ; the Kaplan-Meier curve showed that increased levels of FKBP4 and decreased levels of TMEM173 were strongly correlated with worse survival in the basal-like subtype of BC patients ( Figure 1C).
To confirm the specific interaction between FKBP4 and TMEM173 at the molecular level, we first used siRNA specifically targeting FKBP4 in BT549 cells (representing the basal-like subtype of BC), which led to upregulation of TMEM173 at the protein level ( Figure 1D). In addition, we transfected FKBP4-HA plasmid in BT549 cells, which resulted in downregulation of endogenous TMEM173 ( Figure 1D). These results clearly indicated that FKBP4 had a role in negatively regulating TMEM173 protein expression. We also found silencing of FKBP4 led to upregulation of TMEM173 at the mRNA level in BT549 cells ( Figure 1E), whereas overexpressed FKBP4 resulted in downregulation of TMEM173 at the mRNA level in BT549 cells ( Figure 1E). To further determine the effect of FKBP4 on TMEM173 transcriptional repression, we constructed a TMEM173 promoter luciferase reporter plasmid. As shown in Figure 1F, siRNA specifically targeting FKBP4 enhanced TMEM173 promoter activity in BT549 cells, and FKBP4-HA plasmid inhibited TMEM173 promoter activity in BT549 cells; therefore, FKBP4 was first found to have an impact on regulating TMEM173 promoter.

Negative correlation of FKBP4 and NR3C1 in triple-negative breast cancer
We continued to determine potential factors involved in the FKBP4/TMEM173 axis. First, we used PROMO, 35,36 a virtual laboratory for the identification of putative transcription factors (TFs) binding sites in DNA sequences, to find predicted TFs binding to TMEM173 promoter. After inputting TMEM173 promoter sequence, including 1,000 bases upstream and 150 bases downstream in PROMO ( Figure S2), 15 TFs were shown in order of frequency: CEBPB, YY1, ERAL1, NR3C1, TBP, IRF2, FOXA1, FOXP3, STAT4, XBP1, RXRA, PAX5, TP53, GTF2I, and GCFC2 ( Figure 2A). In addition, FKBP4 was shown to connect to NR3C1 with a higher score than FOXA1 in protein-to-protein interacting network on the STRING database 37 ( Figure 2B); we wondered whether NR3C1 was involved in FKBP4 associated TMEM173 dysregulation.
The NR3C1 protein topology also revealed intracellular membrane (cytosol and nucleoplasm) localization (Figures S3A and S3B); immunofluorescence assay of the HPA database suggested the subcellular localization of NR3C1 in nuclei ( Figure S3C). First, downregulated NR3C1 was significantly related to all molecular subtype patients than the normal group ( Figure 2C), also strongly correlated with worse survival in the basal-like subtype of BC patients ( Figure 2D). Bioinformatics results then suggested a significant negative association between FKBP4 and NR3C1 in the basal-like subtype of BC patients ( Figure 2E). Furthermore, we used siRNA specifically targeting FKBP4 in BT549 cells and found it led to upregulation of NR3C1 at the protein level ( Figure 2F). In addition, we transfected FKBP4-HA plasmid in BT549 cells, which resulted in downregulation of endogenous NR3C1 ( Figure 2F). These results clearly indicated that FKBP4 had a role in negatively regulating NR3C1 protein expression.

Positive correlation of NR3C1 and TMEM173 in triple-negative breast cancer
We further confirmed the specific interaction between NR3C1 and TMEM173 at the molecular level. First, we verified a significant positive association between NR3C1 and TMEM173 in the basal-like subtype of BC patients ( Figure 3A). We then used siRNA specifically targeting NR3C1 in BT549 cells, which led to downregulation of TMEM173 at the protein level ( Figure 3B). In addition, we transfected NR3C1-HA plasmid in BT549 cells, which resulted in upregulation of endogenous TMEM173 ( Figure 3B). Given NR3C1 was demonstrated to regulate TMEM173 at the protein level, we wondered whether it affected the mRNA level of TMEM173. We then found silencing of NR3C1 led to downregulation of TMEM173 at the mRNA level in BT549 cells ( Figure 3C), whereas overexpressed NR3C1 resulted in upregulation of TMEM173 at the mRNA level in BT549 cells ( Figure 3C).
Our previous work found that FKBP4 could bind to NR3C1 and regulate nuclear translocation of NR3C1, 38 with the above findings that FKBP4 regulated TMEM173 promoter, we wondered whether NR3C1 affected the expression of TMEM173 at the transcriptional level. Results showed siRNA specifically targeting NR3C1 decreased TMEM173 promoter activity in BT549 cells, while NR3C1-HA plasmid promoted TMEM173 promoter activity in BT549 cells (Figure 3D); therefore, NR3C1 was found to have a positive impact on regulating TMEM173 promoter.
FKBP4/NR3C1/TMEM173 signaling pathway involved in triplenegative breast cancer cell autophagy Because autophagic activity modulates many pathologies, including neurodegeneration, cancer, and infectious diseases, 39 we doubted whether the FKBP4/NR3C1/TMEM173 axis was also involved in autophagy. Using UALCAN, downregulated Beclin1 and LC3B were found significantly related to the basal-like subtype of BC patients than the normal group ( Figure 4A), but P62 was not significantly related to the basal-like subtype of BC patients than the normal group ( Figure 4A). A Kaplan-Meier curve further showed that only decreased levels of Be-clin1 were meaningfully correlated with worse survival in the basal-like subtype of BC patients ( Figure 4B). Because only Beclin1's relation to the FKBP4/NR3C1/TMEM173 axis fitted the predictions ( Figures  4C-4E), we further verified that silencing FKBP4 increased the expression of Beclin1 in BT549 cells ( Figure 4F). Similarly, NR3C1 overexpression in BT549 cells led to the upregulation of Beclin1 ( Figure 4F). Meanwhile, western blotting showed that FKBP4 overexpression decreased the expression of Beclin-1, and silencing NR3C1 led to the downregulation of Beclin1 ( Figure 4F). As shown in Figure 4F, TMEM173 had the same effects as NR3C1 on Beclin1.
Together, these results suggested that the FKBP4/NR3C1/TMEM173 signaling pathway was involved in triple-negative BC cell autophagy.
FKBP4/NR3C1/TMEM173 signaling pathway involved in triplenegative breast cancer cell proliferation Our previous work found that FKBP4 was a malignant indicator in BC 22 ; we doubted whether the FKBP4/NR3C1/TMEM173 axis was also involved in cell proliferation. Cell viability assay showed that silencing FKBP4 and overexpressing NR3C1 or TMEM173 prevented cell proliferation of BT549 cells at 72 h ( Figures 5A-5C), while overexpressing FKBP4 and silencing NR3C1 or TMEM173 promoted cell proliferation of BT549 cells at 72 h ( Figures 5A-5C).
Thus, these results indicated that the FKBP4/NR3C1/TMEM173 signaling pathway was involved in triple-negative BC cell proliferation. Exosomes secreted by BC cells had been reported as cell-to-cell mediators of oncogenic or anti-cancer information 40 and further regulated DC maturation 32 ; we wondered whether the FKBP4/NR3C1/ TMEM173 signaling pathway was also involved in exosome secretion among tumor environment. exoRBase, which is a repository of noncoding RNA and mRNA derived from RNA sequencing (RNA-seq) data analyses of human blood exosomes and published literature, featured the integration and visualization of RNA expression profiles based on normalized RNA-seq data, including patients with different tumors. 41 Using exoRBase, the expression levels of NR3C1 and TMEM173 were medium to high in BC, whereas the expression level of FKBP4 was extremely low in BC ( Figure 6A). We next tested the effect of exosomes secreted by BC cells on DC abundance. In the 20 BC patients, DCs affected by exosomes were all enriched, especially in the 11th patient; DCs were the most enriched ( Figure 6B). Thus, the FKBP4/NR3C1/TMEM173 signaling pathway involved in BC exosome release might exhibit adjuvant properties of promoting DC abundance.

FKBP4/NR3C1/TMEM173 signaling pathway involved in dendritic cell abundance
To confirm that the FKBP4/NR3C1/TMEM173 axis was also regulated in DCs, we used the tumor-immune system interactions and drug bank (TISIDB) and the Tumor Immune Estimation Resource (TIMER) databases. 42,43 Figures 7A and 7B show that FKBP4, NR3C1, and TMEM173 were all most closely associated with immune subtypes of BC. We then mined the relationship of the FKBP4/NR3C1/ TMEM173 axis and DC infiltration level and abundance, namely, FKBP4 expression, was significantly negatively correlated with DC infiltration level and abundance, whereas NR3C1 and TMEM173 expression were significantly positively correlated with DC infiltration level and abundance in two databases ( Figures 7C and 7D). These data suggested that the FKBP4/NR3C1/TMEM173 signaling pathway was involved in DC abundance.
We demonstrated in these results, together, that means of anti-FKBP4 mediated pro-autophagy, anti-proliferation of BC cells and pro-TIDC abundance and maturation in a newfound FKBP4/NR3C1/ TMEM173 dependent way (Figure 8).

DISCUSSION
Recently, endogenous host-derived regulators of TMEM173 have become a hotspot in related research. For instance, an inflammasome protein, called the nucleotide-binding and oligomerization domain (NOD)-like receptor family caspase activation and recruitment domain (CARD) containing 3 (NLRC3), blocked the direct trafficking of TMEM173 to the punctuated endoplasmic-associated puncta region by direct binding to TMEM173. 44 The autophagy protein P62 negatively regulated the TMEM173 signaling by initiating the ubiquitination of TMEM173 to autophagosome. 45 As an ER membrane-bound E3-ubiquitin ligase, the RING finger protein 5 (RNF5) degraded TMEM173 by mediating its K48-linked polyubiquitination. 46 Unlike the above-mentioned factors inhibiting TMEM173 at the protein level, the current study suggested that the FKBP4/NR3C1 axis might be a novel endogenous negative regulator of TMEM173 by altering its transcriptional activity in BC cells. Thus, FKBP4 and NR3C1 could serve as notable therapeutic molecules against TMEM173-dependent tumorigenesis, autoinflammation, and autoimmunity in the future.
The FK506-binding protein family in human genomes has included 18 FKBPs to date, which could target various pathways in embryonic development, stress response, cardiac function, cancer tumorigenesis, and neuronal function. 47 In colorectal cancer, silencing FKBP3 has been found to attenuate oxaliplatin resistance by regulation of the phosphatase and tensin homolog (PTEN)/AKT axis. 48 In Alzheimer's disease, FKBP12 and amyloid precursor protein (APP) interplay has suspected to affect Ab peptides expression. 49 Although FKBP4 has been demonstrated to connect mammalian target of rapamycin complex 2 (mTORC2) and phosphoinositide-3-kinase (PI3K) to enhance cell proliferation of BC, for the first time, we found that FKBP4 played a carcinogenic role by downregulating TMEM173 in BC cells.
Currently, there is no research on regulation mechanisms of the NR3C1 and cGAS-STING pathway, but only a few studies suggest that NR3C1 is involved in the innate immune response. 50,51 In our study, we demonstrated FKBP4 transcriptionally downregulated TMEM173 through binding to NR3C1, but this effect was found relatively weak because short hairpin NR3C1 (shNR3C1) could not completely inhibit FKBP4 from regulating TMEM173, according to results of Figure 3E. Therefore, we speculated that some of the predicted TFs were also involved in the FKBP4/TMEM173 pathway; subsequent mechanism studies need to be further improved to verify our conjecture.
As for the interaction of FKBP and autophagy, FKBP5 has been reported to change phosphorylation of Beclin1 by binding to Beclin1, thus triggering autophagic pathways. 52 FKBP8 was found to recruit lipidated LC3A to induce mitochondrial autophagy via the N-terminal LC3-interacting region motif of FKBP8, 53 although the regulation of autophagy by FKBP4 in tumor cells has not been reported yet. Meanwhile, TMEM173 could activate autophagy through inducing LC3B lipidation, which was independent of TANK binding kinase 1 (TBK1) activation and interferon induction. 54 In current study, we first demonstrated that inhibiting FKBP4 could inhibit autophagy of BC cells.
TMEM173 expression has been reported suppressed or lost in majority of cancers, especially in BC, [55][56][57] which suggests that during BC progression, downregulation of the proteins involved in innate immune response may be helpful for evading innate immune response pathways to facilitate tumor growth. Although hypermethylation of the promoter regions in TMEM173 has been revealed in colorectal cancer, 15 little is known about the mechanism of decreased TMEM173 expression in BC. Our results imply FKBP4 could induce TMEM173 transcriptional suppression via NR3C1; it would be interesting to further study the correlation between TMEM173 and other FKBPs involved in innate immune response and relevance during breast tumorigenesis. Diamond et al. demonstrated that interferon (IFN)-stimulatory double-stranded DNA (dsDNA) from irradiated BC cells, which are phagocytosed by DCs via stimulating the cGAS/TMEM173 pathway. 32 Thus, it is likely that different methods mediate the transfer of other cargos to DCs through TMEM173 related signaling; e.g., our study first found that exosomes secreted by BC cells induced DC abundance and maturation. Further experimentation will be required to determine the relative contribution of these different mechanisms of tumor DNA delivery to DCs.
Recently, a growing body of evidence has demonstrated that the pharmacological agents, e.g., dimethyloxoxanthenyl acetic acid, 2 0 3 0 -cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), mixed linkage (ML) RR-S2 cGAMP, and ML RR-S2 CDA, could induce activation of TMEM173. [58][59][60][61] Here we proposed anti-FKBP4 to be another kind of potential TMEM173 conditioning means. With the development of immunotherapies, such as cancer vaccine, immune checkpoint inhibitor, oncolytic virus, and chimeric antigen receptor T cell (CAR-T) therapies, 62 a combination of TMEM173targeting agonists and immunotherapies may provide multiple feasible approaches to new BC treatment strategies.

MATERIALS AND METHODS
Cell culture BT549 cells were obtained from the American Type Culture Collection (ATCC). BT549 cells were cultured in Roswell Park Memorial Institute (RPMI) medium. Growth media were supplemented with 10% fetal calf serum and penicillin/streptomycin (100 U/mL). All human cell lines were cultured at 37 C in a humidified incubator supplied with 5% CO 2 .

Gene silence
To validate hits from the genetic screens, BC cells were transduced with pLKO.1 vectors, which, in addition to the short hairpin RNA (shRNA) cassette, carried a puromycin resistance cassette (pLKO.1puro, Addgene plasmid #10878). The shRNAs against NR3C1 were cloned into pLKO.1 vectors using the Age1 and EcoR1 restriction sites. The shRNA targeting sequence of NR3C1 63 was from the

Western blotting
Knockdown efficiencies and biochemical responses were analyzed by western blotting. Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (EMD Millipore). Separated proteins were transferred to nitrocellulose filter membranes and blocked in 5% milk in Tris-buffered saline, with 0.05% Tween 20. Immunodetection was done with various primary antibodies. Appropriate horseradish peroxidase-conjugated secondary antibodies were used and signals were visualized with enhanced chemiluminescence (Bio-Rad) by the Bio-Rad Chemiluminescent Imaging System.
Target fragment amplification was carried out as follows: 95 C for 30 s, followed by 40 cycles consisting of 95 C for 5 s and 60 C for 34 s. Melting curve analysis was carried out at 90 C for 15 s and then at 60 C for 1 min and 95 C for 15 s.

Luciferase Reporter Assay
BC cells were plated into 12-well dishes and transfected the following day; 1 mg of the reporter plasmid for TMEM173 promoter, 50 ng the Renilla luciferase control plasmid, and the indicated amounts of the expression plasmids were used per well. At 24 h post-transfection, luciferase activities were then measured by using a Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity. Finally, the relative luciferase activities were expressed as fold changes over the empty-plasmid-transfected controls.

Transient transfection
BC cells cultured in 12-well tissue culture plates were transiently transfected with plasmids using Lipofectamine 2000 Reagent (Invitrogen) or small interfering RNA (siRNA), using Lipofectamine RNAiMAX Reagent (Invitrogen), as instructed by the manufacturer. The siRNA targeting FKBP4, NR3C1, and TMEM173 as well as negative control siRNA were purchased from RIBBIO (Guangzhou, China); 72 h later, the whole-cell extract was prepared for real-time qPCR or western blot analysis.

Cell proliferation assay
Cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8) (Dojindo). All cells were seeded into 96-well plates at a density of 5,000 cells/well in a 100 mL volume and incubated at 37 C under 5% CO 2 for 24, 48, and 72 h, followed by the addition of 10 mL of CCK-8 solution. The absorbance in each well was measured after 1 h incubation using a microculture plate reader at a test wavelength of 450 nm. Three replicate wells were set up in each group, and three independent experiments were performed. www.moleculartherapy.org

Statistics
A two-tailed student's t-test was used in this study. Data shown were mean ± SD from at least three independent experiments. Statistical probability was expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.