Complementation experiments revealed that while approximately half of the a/α switchers were due to EFG1 mutations, the remaining half were due to mutations in other genes. In addition, the results of competition experiments in a mouse GI tract colonization model support previous observations that efg1/efg1 cells rapidly outcompete EFG1/EFG1 strains, but direct microscopic analysis reveals that the major colonizing cells were opaque, not gray.Close to half of a collection of 27 clinical  EFG1 functions as a suppressor of white-to-opaque and white-to-gray switching in a/α strains of Candida albicans. In a collection of 27 clinical isolates, 4 of the 17 EFG1/EFG1 strains, 1 of the 2 EFG1/efg1 strains, and all 8 of the efg1/efg1 strains underwent white-to-opaque switching. The four EFG1/EFG1 strains, the one EFG1/efg1 strain, and one of the eight efg1/efg1 strains that underwent switching to opaque did not switch to gray and could not be complemented with a copy of EFG1. Competition experiments in a mouse model for gastrointestinal (GI) colonization confirmed that efg1/efg1 cells rapidly outcompete EFG1/EFG1 cells, and in plating experiments, formed colonies containing both gray and opaque cells. Direct microscopic analysis of live cells in the feces, however, revealed that the great majority of cells were opaque, suggesting opaque, not gray, may be the dominant phenotype at the site of colonization.The transcription factor IMPORTANCE Close to half of a collection of 27 clinical a/α isolates of Candida albicans underwent white-to-opaque switching. Complementation experiments revealed that while approximately half of the a/α switchers were due to EFG1 mutations, the remaining half were due to mutations in other genes. In addition, the results of competition experiments in a mouse GI tract colonization model support previous observations that efg1/efg1 cells rapidly outcompete EFG1/EFG1 strains, but direct microscopic analysis reveals that the major colonizing cells were opaque, not gray. Candida albicans remains a pervasive opportunistic fungal pathogen, colonizing a majority of humans as a commensal  regulated switching and switching in turn regulated mating. They discovered that hemizygous MTL derivatives of a heterozygous MTL (a/α) strain, the latter representing the predominant MTL genotype among clinical isolates  at 25°C in 5% CO2. Furthermore, Xie et al.  colonization model. Using a CHROMagar plating assay, Liang et al. .ommensal . It was ommensal , 2. Howe patient , 4. Soonansition . Switchiansition , 6, 7. Iansition –14, and nsition – and systnsition –. Howevernsition – discoverisolates , 19, couould not  reportede et al.  demonstrk et al.  subsequeo et al.  subsequek et al. , suggestk et al.  further Kumamoto , a/α EFGg et al.  further k et al.  presenteEFG1 in white-to-opaque or white-to-gray switching, and GI colonization, in a collection of 27 clinical a/α isolates. We found that 13 of the 27 isolates could be induced to switch to opaque. Four of the 17 EFG1/EFG1 strains, 1 of the 2 EFG1/efg1 strains, and all 8 of the efg1/efg1 strains underwent switching from white to opaque. However, only 7 of the 13 a/α isolates that switched were complemented by site-specific integration of a copy of EFG1, and all 7 were efg1/efg1 mutants. In all cases in which complementation with EFG1 reestablished repression of white-opaque switching, it also reestablished repression of white-gray switching. These results demonstrate that mutants of genes that repress white-to-opaque switching other than EFG1 may be just as prevalent as EFG1 mutants in clinical a/α strains that switch. Finally, results from competition experiments in a mouse gastrointestinal colonization model confirmed previous results , temperature (25°C versus 37°C) and atmosphere (air [0.04% CO2] versus 5% CO2)   in suppl 5% CO2) , the fou 5% CO2) , the gra 5% CO2)  and the  5% CO2) , and the 5% CO2) . On bothhenotype . Howeverue cells  and 2A. ay cells  and 2A. ay cells  and 2A. ue cells  and 2A.10.1128/mSphere.00795-19.2TABLE S1Table S1, DOCX file, 0.02 MB.Strains used in this study. Download Copyright © 2020 Park et al.2020Park et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the a/α isolates in the collection  or from different groups . MLST typing of isolates P75063wh, P75063dk, P80021wh, P80021dk, 1298wh, 1298dk, P37037wh, and P37037dk was performed in our lab according to the consensus MLST scheme described by Bougnoux et al.  which is commonly used worldwide. The MLST clade assignment for these isolates was performed by generating a dendrogram, including MLST data retrieved from the Candida albicans MLST website  for 24 control isolates and strain 529L, which does not appear to have been previously assigned to a clade. The control isolates were selected, three per clade, from the seven most frequent clades  found among a collection of 1,391 isolates . Three isolates from clade 18, a major clade in Asia , were also used as controls. 529L did not cluster with any other strain. The Ca3 fingerprinting clades I, II, III, SA, and E have been repeatedly shown to correspond nearly perfectly to MLST clades 1, 2, 3, 4, and 11, respectively . It should be noted that clade assignments are no longer displayed on the C. albicans MLST website. Download Table S2, DOCX file, 0.01 MB.Ca3/MLST clade assignments for the 27 isolates used in this study. Ca3 fingerprinting was performed in the Soll lab. The MLST analysis and clade assignment of 10 strains were from collaboration  harbored nonsense mutations caused by point mutations or indel frameshifts, in both EFG1 alleles , clade E (MLST 11), and I (MLST 1)  . It shour clades . It shouanalyses .10.1128/mSphere.00795-19.1FIG S1EFG1 relatedness based on EFG1 polymorphisms. Clade assignments are presented immediately to the right, based on RFLPs probed with the mid-repeat sequence CA3 and, in parentheses, MLST when available. See EFG1 genotypes of the clinical isolates are presented to the far right. The efg1− alleles in the isolates are shown in red. Download FIG S1, PDF file, 0.2 MB.A dendrogram of Copyright © 2020 Park et al.2020Park et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2, 37°C and air, and 37°C and 5% CO2). However, on GlcNAc-agar, 13 of the 27 strains in the collection (48%) formed opaque colonies under one or more sets of conditions  under any of the four sets of environmental conditions  underwent switching from white to opaque on GlcNAc-agar under one or more sets of environmental conditions ; they do not include switching from white to gray. Download Table S4, DOCX file, 0.02 MB.Switching frequency of white to opaque for the Copyright © 2020 Park et al.2020Park et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the efg1/efg1 underwent switching from white to opaque on GlcNAc-agar under more than one of the four sets of conditions on GlcNAc-agar  by reintroducing a functional copy of EFG1, we performed site-specific integration at the native EFG1 locus of one copy of EFG1 from strain SC5314 (“scEFG1”), under the control of the native promoter  that switched to opaque but not gray. The addition of scEFG1 reestablished the repression of switching  in the seven strains that switched to opaque and gray   . Serum iisolates . Most nowitching , supportansition , suggestd P37039 . Seven od P37039 . Represeormation . The onef scEFG1 . These refg1/efg1 strains outcompete EFG1/EFG1 strains in mouse gastrointestinal (GI) colonization models  and between P57003 efg1/scEFG1 versus P57003 efg1/efg1 cells (50:50) in which we plated fecal samples over time on GlcNAc-agar and incubated the plates for 5 days at 25°C in air, conditions we previously found stabilized both the opaque and gray phenotype but did not induce the opaque phenotype  were efg1/efg1 deletion mutants and that 7 of the 8 could switch to gray and all 8 could switch to opaque (EFG1/EFG1 isolates and one EFG1/efg1 isolate (19%) could switch, but as a result of a mutation in a gene other than EFG1.Xie et al.  first rea/α , 19, cane et al.  further k et al.  subsequeg et al.  further g system . We repoo opaque . HoweverMTL homozygous strains of C. albicans. Subsequent studies then revealed that EFG1 null mutants outcompeted EFG1/EFG1 cells in mouse models of gastrointestinal (GI) colonization   . There aWH11) , wall ulWH11) , the relWH11) , and the11) OP4 , 13, 38., 38.WH11EFG1 deletion mutants outcompete wild-type EFG1/EFG1 cells in competition experiments in a mouse GI model for colonization  in combination with EFG1/EFG1 white cells, the efg1Δ/efg1Δ cells rapidly dominated colonization in the mouse GI colonization model. Also, in the case of the combination of efg1/scEFG1 white cells and efg1/efg1 gray cells, the efg1/efg1 cells dominated. These results were consistent with those of Liang et al.  in a/α cells causes a competitive advantage over wild-type a/α cells in the mouse GI colonization model and that the colonizing WOR1OE cells formed opaque-like cells without wall pimples that were unstable on glucose-based agar at 25°C , used to generate and maintain plasmids, was grown in LB medium  plus 100 μg/ml ampicillin.The EFG1 obtained from strain SC5314 (scEFG1), was used to test for complementation by site-specific integration at the EFG1 locus. A pmCherry-HygB plasmid  gene was used to confer resistance to nourseothricin, and the CaHygB gene was used to confer resistance to hygromycin B. Integration into proper loci was verified by PCR. The genotypes of the constructed strains are provided in The plasmid pEFG1C , which c plasmid  was emplEFG1 in the 27 clinical isolates, we prepared genomic DNA from strains grown in 5 ml of a YPD suspension culture as described previously . The unrooted dendrogram . Cells from YPD suspension cultures were grown overnight (∼20 h) at 25°C and then plated on Gluc-agar or GlcNAc-agar plates. Colony phenotypes were analyzed after 5 days of incubation. Homogeneous opaque colonies containing exclusively opaque cells and white colonies with opaque sectors with the latter containing exclusively opaque cells were counted as opaque. Representative colonies were analyzed for cellular phenotypes as described in a previous study (Switching was tested on agar containing 1.25% glucose (Gluc-agar) or 2.00% GlcNAc (GlcNAc-agar), with the agar containing supplemented Lee’s medium (sLee’s)  and 5 μgus study . In all 2 for 24 h or by plating on nonnutrient agar containing 10% serum and incubating at 37°C in 5% CO2 for 3 days.Hypha formation was assessed in two ways, in suspension cultures containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) in 24-well plates and incubated at 37°C in 5% COa/α collection were genotyped for the MTL configuration by PCR with the two primer pairs, MTLa1F/R and MTLα2F/R  and fluorescence microscopic images of cells expressing 7 cells) of a cell combination (efg1Δ/efg1Δ and efg1/efg1 mutants and does not induce switching to opaque or gray when incubation is performed at 25°C in air (All procedures complied with regulatory guidelines defined by the Iowa University IACUC committee. C57BL/6J (Jackson Laboratory) female mice 6 to 7 weeks old were treated with 1 mg/ml of penicillin G and 2 mg/ml of streptomycin in their drinking water for 4 days. Mice were orally inoculated with 0.2 ml (3 × 10bination  in PBS. bination . All agabination . GlcNAc-C in air . The dat


The result was showed CQDs/Cu2O possessed anticancer properties against SKOV3 cells with IC50 = 0.85 μg mL−1, which was approximately threefold lower than other tested cancer cells and approximately 12-fold lower than normal cells. Compared with popular anticancer drugs, the IC50 of CQDs/Cu2O was approximately 114-fold and 75-fold lower than the IC50 of commercial artesunate (ART) and oxaliplatin (OXA). Furthermore, CQDs/Cu2O possessed the ability to decrease the expression of MMP-2/9 and induced alterations in the cytoskeleton of SKOV3 cells by disruption of F-actin. It also exhibited stronger antiangiogenic effects than commercial antiangiogenic inhibitor (SU5416) through down-regulating the expression of VEGFR2. In addition, CQDs/Cu2O has a vital function on transcriptional regulation of multiple genes in SKOV3 cells, where 495 genes were up-regulated and 756 genes were down-regulated. It is worth noting that CQDs/Cu2O also regulated angiogenesis-related genes in SKOV3 cells, such as Maspin and TSP1 gene, to suppress angiogenesis. Therefore, CQDs/Cu2O selectively mediated of ovarian cancer SKOV3 cells death mainly through decreasing the expression of MMP-2, MMP-9, F-actin, and VEGFR2, meanwhile CQDs/Cu2O caused apoptosis of SKOV3 via S phase cell cycle arrest. These findings reveal a new application for the use of CQDs/Cu2O composite as potential therapeutic interventions in ovarian cancer SKOV3 cells.It was shown that some nanomaterials may have anticancer properties, but lack of selectivity is one of challenges, let alone selective suppression of cancer growth by regulating the cellular microenvironment. Herein, we demonstrated for the first time that carbon quantum dots/CuThe online version contains supplementary material available at 10.1186/s12951-021-00813-8. In gynecological malignancies, ovarian cancer is the one of the leading causes of death , 2. Alth2, As2O3, graphene oxide-silver nanoparticle, and iron core-gold shell nanoparticles have been reported to cause the apoptosis of cancer cells [Compared with traditional treatments, nanomaterials offer new opportunities for the development of diagnostic and therapeutic tools for cancer and other diseases –8. Ag, Aer cells –13. It wMore and more evidences indicated that cancer progression is closely related to the tumour microenvironment, including the extracellular matrix (ECM) deregulation, blood vessels expanding and immune response suppression . Matrix In addition, the regulation of cytoskeleton plays a key role in the process of metastasis in cancer cells . Actin, Carbon quantum dots (CQDs) are currently eliciting much attention in cancer therapy due to outstanding properties including biocompatibility, low cytotoxicity, water solubility and unique photoluminescence . CQDs an2O NPs has been extensively researched to elucidate their significance in cancer therapy. Cu2O NPs significantly reduce the growth and metastasis of melanoma, improve the viability of tumor-bearing mice, and induce the mitochondrion-mediated apoptosis of cancer cells [2O NPs was more sensitive to rapidly proliferating HeLa cancer cells than normal human kidney 293 T cells, indicating Cu2O NPs exert distinct effects on different cells [Cuer cells . Cu2O NPnt cells . Since ont cells .Gene expression experiments, as a method for detection of disease markers, has been used to assessment the toxicity mechanism of nanomaterials . For exa2O composite possessed an intrinsic protease-mimicking activity for hydrolyzing proteins under physiological conditions, ultimately exhibiting a surprisingly higher catalytic activity than Cu2O and CQDs [2O composite on human ovarian cancer SKOV3 cells. It will be attractive to elucidative the potential roles of the CQDs/Cu2O composite in the regulation of cancer-related proteins. Furthermore, only a few nanoparticles have been reported to interact with the tumour microenvironment. As far as we know, this is the first attempt to reveal CQDs/Cu2O composite mediates tumor microenvironment and cytoskeleton.On the other hand, the proteases naturally expressed by living organisms participate in all stages of tumor progression. In our previous study, we found that the CQDs/Cuand CQDs . Based o4, poly-vinylpyrrolidone, and C6H12O6 were obtained from Shanghai Tian Scientific Co. Ltd. Cell culture medium  were from Hyclone Laboratories. Fetal bovine serum (FBS) were got from Gibico. Acridine orange, ethidium bromide, FITC-Annexin-V/PI apoptosis assay kit, WST-1 assay kit and Hoechst 33342 were obtained from Beyotime Company of China. Antibody VEGFR2 . MMP-2 . MMP-9 . GAPDH . F-actin . β-actin . Secondary antibodies goat anti‑mouse . Goat anti‑rabbit .CuSO2O have been discussed in our previous publications. [Synthesis and characterization of CQDs/Cucations. , 43.2 atmosphere. HUVEC cells were used within 6 passages All cells were grown at 37 °C with 5% CO2 in a humidified incubator.All cell lines including human ovarian cancer cells (SKOV3), human cervical cancer cells (HeLa), human lung adenocarcinoma cells (A549), human colorectal cancer cells , normal mouse embryonic fibroblasts cells (BABL-3T3), normal human epithelial kidney cells HEK293T, normal mouse macrophage cells J774A1) were purchased from shanghai life science of chinese academy of sciences. The BABL-3T3 cells were grown in high glucose DMEM containing 10% FBS , while other cells were grown in low glucose DMEM containing 10% FBS. HUVECs were isolated from term umbilical cord veins using collagenase and cultured in DMEM supplemented with 20% FBS. All cell lines were grown at 37 °C in a humidified 5% CO4 cells were grown in 96-well plates for 24 h. Cells were incubated with 1.56, 3.12, 6.25, 12.5 and 25 µg mL−1 of CQDs/Cu2O for 24–72 h. Cells incubated PBS instead of CQDs/Cu2O were used as control. Then cells were treated with MTT  for 4 h. 150 μL of DMSO was added after removing the medium. Microplate spectrophotometer (Spectra Max 190) was used to measure the absorbance at 490 nm. The assay was repeated 3 times and all experiments were carried out in duplicate. The inhibition rate (%) = (ODcontrol – OD sample)/ODcontrol50) was measured when the inhibition rate to half that of the control.The cytotoxicity of all cells was detected by MTT assay. 1 4 SKOV3 cells were grown in 96-well plates for 24 h. Cells were incubated with1.56, 3.12, 6.25, 12.5 and 25 µg mL−1 of CQDs/Cu2O for 24 h. 20 μL of WST-1 was added and incubated 1 h. The absorbance were measured by Microplate spectrophotometer (Spectra Max 190) at 450 nm. The assay was repeated 3 times and all experiments were carried out in duplicate. The inhibition rate (%) = (ODcontrol – OD sample)/ ODcontrol × 100%. The half-maximal inhibitory concentration (IC50) was measured when the inhibition rate to half that of the control.1 −1 CQDs/Cu2O, CQDs, or Cu2O for 24 h, SKOV3 cells were trypsinized and harvested. Cells in suspension were stained with 5 μg mL−1 AO/ EB for 10 min. Then cells were placed on a glass slide and analyzed using an Olympus IX73 fluorescent microscope at 545 nm.After treatment with 12.5 µg mL−1 CQDs/Cu2O, CQDs, or Cu2O for 24 h, SKOV3 cells were trypsinized and harvested. Cells were fixed for 10 min by 4% paraformaldehyde after washing 3 times using ice-cold PBS. SKOV3 cells were stained with Hoechst 33342 for 15 min after washing 3 times with PBS. Cells were analyzed using a Olympus IX73 fluorescent microscope at 350 nm.After treatment with 12.5 µg mL2O, CQDs, or Cu2O for 24 h, SKOV3 cells were trypsinized and harvested. Cells were fixed with 70% ethanol for 12 h at 4 °C, centrifuged  and washed using PBS, stained by PI for 20 min. Cells were subsequently analyzed by flow cytometer. The total number of cells was 10,000.After treatment with CQDs/Cu2O . Cells were trypsinized and harvested, stained with FITC-Annexin-V and PI for 15 min in dark. Then tested by a flow cytometer. The number of cells was 10,000.SKOV3 cells were treated with CQDs/Cu2O . Then cells were fixed using 4% paraformaldehyde, washed with PBS, and permeabilized by 0.1% Triton X-100 for 15 min. After washing by PBS, cells were then incubated with FITC-conjugated phalloidin. Cells were stained using PI for 10 min, then analyzed with Olympus IX73.SKOV3 were treated with CQDs/Cu2O  were added and incubated with SKOV3 cells for 0, 6, 12 and 24 h. The number of cells migrated into the scratch area was quantified under fluorescent microscope. The migration inhibition rate of a sample was calculated by the following equation: inhibition percentage = (NControl − NSample)/ NControl × 100%, where NControl is the average total number of migrated cells in three groups of control, NSample is the average total number of migrated cells in three groups of CQDs/Cu2O.SKOV3 were seeded onto six-well plates. When cells grown to 90% confluence to form monolayers, cells were scratched by a pipette tip. Subsequently, CQDs/Cu4 cells per well), VEGF (10 ng mL−1) and different concentration of CQDs/Cu2O were added into 96-well plates which coated by Matrigel for 12 h. Tubular network were observed and quantified by the measuring tool of the software in randomly chosen five fields under a fluorescence microscope . The inhibition (%) = (lengthControl − length Sample)/length ControlHUVEC cells  for 24 h. After harvested and washed by cold PBS, cells were incubated with cell lysate for 1 h at 4 °C on ice. The proteins were extracted by centrifuging for 15 min . 10% SDS-PAGE was used to separate the extracted proteins. After proteins transferred, membranes were treated with blocking agents. The membranes were incubated with primary antibodies  at 4˚C overnight. After washed 3 times, the membranes were incubated with secondary antibodies goat anti‑mouse or goat anti‑rabbit at room temperature for 1 h. Washing 3 times with PBST, the membranes were treated using chemiluminescence agents.SKOV3 cells treated by CQDs/Cu2O  for 24 h. Total RNA of cells treated by CQDs/Cu2O was extracted by TRIzol reagent. Sequencing libraries were tested by the Illumina HiSeq xten/NovaSeq 4000 sequencer. A fold change of 2 were used as the threshold. P < 0.05 was indicated statistically significant.SKOV3 cells were treated with CQDs/CuP < 0.05 was indicated the statistically significant.All values represent mean ± SD, n = 3. The significant levels were assessed by Student’s t-test. 2O, Cu2O on cell cytotoxicity was tested in cancer cells , and normal cells  by the MTT assay. Cells were treated with CQDs/Cu2O or Cu2O  for 24–72 h. The effect of CQDs was detected in HeLa, SKOV3 and BABL-3T3 cells by MTT assay. The results were shown CQDs were non-toxic and biocompatibility, which is agreed with previous findings [2O displayed cytotoxicity in a concentration-dependent manner in all tested cells, and SKOV3 cells displayed the highest inhibitory rate for proliferation among all cells. To further verify the results of MTT assay, a second mitochondrial activity-based assay, WST-1, was employed to measure the cytotoxicity of CQDs/ Cu2O in SKOV3 cells. WST data showed that CQDs/Cu2O inhibited the growth of SKOV3 cells in a dose-dependent manner, the value of IC50 obtained by WST assay (IC50 = 1.46 µg mL−1) was consistent with the result from MTT assay (IC50 = 1.50 µg mL−1). It was suggested the MTT assay can be used to evaluate the cytotoxicity of CQDs/Cu2O.The effect of CQDs/Cufindings . As show2O was lower than 12.5 µg mL−1, a discriminative difference was identified for the cytotoxicity between SKOV3 cancer cells and other cancer cells, even normal BABL-3T3, HEK293T and J774A1 cells. This is important because developing an anticancer drug that can effectively distinguish between tumor and normal cells is currently the greatest challenge [Interestingly, when the concentration of CQDs/Cuhallenge .2O composite displayed cytotoxic activity against cancer and normal cells in the IC50  range of 0.85 to 22.4 µg mL−1  measurement, suggesting CQDs/Cu2O displayed high stability and the CQDs/Cu2O plays a leading roles in cytotoxic activity. Moreover, the IC50 value of CQDs/Cu2O against SKOV3 cells was lower than above mentioned cancer cells and normal cells, which was approximately threefold lower than other tested cancer cells and approximately 12-fold lower than normal cells. It was further suggested CQDs/Cu2O was sensitive to SKOV3 cells.In general, the CQDs/Cu2O was better than that of ART and OXA against cancer cells (Table 50 of CQDs/Cu2O in SKOV3 cells (IC50 = 0.85 µg mL −1) was approximately 75-fold lower than that of OXA (IC50 = 64.6 µg mL −1) [50 = 96.9 µg mL −1) [2O may have a potential utility for effectively distinguishing between SKOV3 cells and other tested cells.On the other hand, compared with commercial anticancer agents such as ART and OXA at different times, the cytotoxicity of CQDs/Cug mL −1)  and 114-g mL −1)  at 72 h.2O treatment libraries  and control group libraries  were successfully constructed and subsequently sequenced  analysis indicated that a total of 1011 transcripts at a fold change > 2 and false discovery rate (FDR) < 0.05 were differentially expressed, among which, 381 genes were upregulated while 630 were downregulated. There was no significant difference between the high concentration group and the low concentration group. On the one hand, the enriched GO terms in the DEGs of cells treated by 3.12 μg mL−1 CQDs/Cu2O have also been provided in supporting information  were significantly upregulated after 3.12 μg mL−1 CQDs/Cu2O treatment, which was also consistent with those of higher doses treatment.In order to determine the differentially expressed genes at lower doses and elucidate the mechanism of selective inhibition of SKOV3 cells by CQDs/CuGene Ontology (GO) was used to analyze the obtained DEGs, where three main ontologies such as biological process, cellular component, and molecular function performed. As shown in Fig. 2O treatment. As shown in Table 2O plays a key role in the induction of SKOV3 cell apoptosis and the regulation of cell movement. The enriched CC terms mainly included proteinaceous extracellular matrix and integral components of the plasma membrane, suggesting that CQDs/Cu2O affected the extracellular matrix and plasma membrane. The enriched MF terms included calcium ion binding, nucleic acid binding, receptor binding, etc.GO enrichment analysis was also used to identify significantly enriched BP, CC, or MF terms in DEGs during CQDs/Cu2O on the functions of DEGs, the KEGG data base was used to analyze annotated pathways of DEGs. The result indicated that DEGs were significantly enriched in 20 pathways after treated with CQDs/Cu2O in Fig. P corrected < 0.05). According to the number of DEGs, the three pathways such as steroid biosynthesis, focal adhesion, and ECM-receptor interaction were the most enriched pathways. Based on the lowest P value, steroid biosynthesis was among the most notable enriched pathways.To determine the effect of CQDs/Cu2O samples had an orange nuclei and fragmented chromatin, indicating that they were either in necrosis or at the late stage of apoptosis. In addition, only few necrotic cells with uniformly red nuclei and an organized structure appeared after treatment with the same concentration of Cu2O. However, there was no significant change in the CQD group. These results indicate that CQDs/Cu2O induces necrosis or apoptosis of SKOV3 cells, and the effect of the CQDs/Cu2O composite is better than that of CQDs and Cu2O.Transcriptome analysis revealed that apoptotic process and programmed cell death were significantly affected. AO/EB (acridine orange/ethidium bromide), Hoechst 33342, and FITC-Annexin-V/PI were used to demonstrate apoptosis. AO is absorbed by viable and nonviable cells to emit green fluorescence, while EB is absorbed by nonviable cells to emit red fluorescence. Therefore, viable cells displayed uniform bright green fluorescence with organized structure. The apoptotic cells showed red to orange fluorescence with fragmented chromatin, and the necrotic cells showed red fluorescence with swollen . As show2O on SKOV3 cells were determined by staining with Hoechst 33342. Nuclear condensation & DNA fragmentation are considered to be typical features of apoptosis. SKOV3 cells were treated with 12.5 μg mL−1 CQDs/Cu2O for 24 h. As shown in Fig. 2O treated cells showed nuclear shrinkage, cytoplasmic blebbing, and chromatin condensation, including bright blue dots in the nuclei, thereby representing nuclear fragmentation, whereas a small number of cells were chromatin condensation in Cu2O group. These observations further indicated CQDs/Cu2O can induce SKOV3 cells apoptosis, which is better than both CQDs and Cu2O.The apoptotic effects of CQDs/Cu2O-induced apoptosis. As shown in Fig. 2O increased from 3.12 ~ 12.5 μg mL−1, the proportion of cells in the upper right quadrants increased from 3.5% to 48.4%, however, the proportions of other quadrant cells did not change significantly. This data also confirm that CQDs/Cu2O can effectively induce SKOV3 cell apoptosis.A flow cytometric analysis via FITC-Annexin-V/PI assay was used to further confirm the CQDs/Cu2 NPs, have been reported to arrest cell cycle to suppress cancer cell proliferation [2O, SKOV3 cells were treated by CQDs/Cu2O and subjected to flow cytometry analysis , while there was no significant change in the G0/G1 and G2/M phase. The results showed that the SKOV3 cells are arrested at S phase after treatment by CQDs/Cu2O, indicating that mitosis and proliferation of cancer cells were inhibited.The cell cycle, which includes DNA replication, mitosis, and cytokinesis, is a major event in cell division. It has been proven that deregulation of the cell cycle is related to numerous carcinogenic processes . Nanomatferation –49. Cu-bferation . A previferation  To detersis Fig. b. As sho2O regulated cell motility. Hence, the wound-healing assay was used to detect the amount of migrated cells and the width of scratch after treatment with CQDs/Cu2O. As shown in Fig. 2O and prolonging reaction time. As well as the amount of migrated cells decreased after treated with CQDs/Cu2O 22 serve as potent migration inhibitors by downregulating MMP-2/9 [2O effectively suppressed SKOV3 cell migration by regulating the expression of metalloproteinase.We found that the expression of MMP-2/9 sharply decreased after treatment with CQDs/Cusis Fig. d. It has MMP-2/9 , 50. The2O treatment. To confirm whether CQDs/Cu2O has an anti-angiogenesis effect, a blood vessel formation in vitro model system was selected and the effect of CQDs/Cu2O on new blood vessel formation in HUVECs cells was assessed. VEGF was used to induce endothelial cell proliferation, migration, and differentiation into blood vessel structures. As shown in Fig. 2O effectively inhibited the length of blood vessel. A reduction of approximately 40 ~ 70% in total blood vessel length per field following treatment with 3.12 to 12.5 μg mL−1 CQDs/Cu2O for 6 h. When the time was extended to 12 h, the inhibition percentage increased from 60 to 90%, suggesting a dose-dependent and a time-dependent decrease. The viability of HUVEC cells was also determined by MTT assay. It was shown that the inhibition rate was lower than 20% when HUVEC cells were treated with CQDs/Cu2O for 12 h  and THBS1 (known as TSP1) genes were significantly upregulated after CQDs/Cu2O also had better antiangiogenic activity than SU5416 and Cu2O  was about 1.8-fold shorter than the length of blood vessel induced by SU5416. The length was approximately 4.2-fold shorter than that caused by Cu2O. CQDs/Cu2O inhibited tube formation by ~ 90%, which was more than that with Cu2O (53%) and SU5416 (80%) , as a selective inhibitor of VEGF, was used as the experimental control. CQDs/Cuu2O Fig. b. In Fig−1 AgNPs  suppress-MIL-101  effectiv2O. Altogether, our results indicated that angiogenesis mediated by VEGFR2 and MMP-2/9, indicating that CQDs/Cu2O may be a potential candidate in anti-angiogenic therapy.A variety of growth factors and cytokines are involved in regulating angiogenesis. VEGF, as one of the most important pro-angiogenic factors, combined to receptors VEGFR1 and VEGFR2, stimulating endothelial cell migration and blood vessel formation. , 17, 51 2O on the F-actin cytoskeleton of SKOV3 cells. First, we observed the morphological changes of the F-actin cytoskeleton by FITC-conjugated phalloidin and PI immunofluorescence staining. As shown in Fig. 2O, a large number of fluorescence spots were scattered throughout the cytoplasm, gradually disappeared as the CQDs/Cu2O concentration increased and the fluorescence intensity decreased, which significantly differed from that in normal F-actins. CQDs/Cu2O might therefore exhibit enhanced damage and disruption of F-actin. We also evaluated the content of F-actin cytoskeleton after treated by CQDs/Cu2O. The results of western blot demonstrates that the expression of F-actin in SKOV3 cells treated with CQDs/Cu2O significantly was significantly lower than that in the control group  and oxaliplatin (OXA), indicating CQDs/Cu2O possess strong antitumor activity than chemotherapeutics OXA and ART. This should be one of the most fascinating features of CQDs/Cu2O.In summary, we demonstrated for the first time that CQDs/Cu2O suppressed migration and angiogenesis processes in vitro mainly through downregulation the expression of VEGFR2 and MMP-2/9, which may be one of the main reasons for the selective inhibition tumor, since angiogenesis is an effective tumor treatment strategy to provide oxygen and nutrients for tumour progression and metastasis. CQDs/Cu2O also exhibited stronger antiangiogenic effects than commercial antiangiogenic inhibitor (SU5416). Furthermore, CQDs/Cu2O induces alterations in the cytoskeleton of SKOV3 cells by disruption of F-actin cytoskeleton which is critical for numerous physical cellular processes, including cell adhesion, migration, division, and cancer cell apoptosis. It is worth noting that CQDs/Cu2O also regulated angiogenesis-related genes in SKOV3 cells, such as Maspin and TSP1 gene, to suppress angiogenesis. This is first time to elucidate selectively mediation of CQDs/Cu2O in SKOV3 cells via transcriptome analysis, providing significant insights into the development and clinical application of CQDs/Cu2O.Interestingly, CQDs/Cu2O has been proposed. CQDs/Cu2O selectively mediated of ovarian cancer SKOV3 cells death mainly through downregulation the expression of MMP-2, MMP-9, F-actin, and VEGFR2, meanwhile CQDs/Cu2O induced apoptosis of SKOV3 via S phase cell cycle arrest.From the above reasons, a probable mechanism for inhibition SKOV3 by CQDs/Cu2O in the selective and restrictive ovarian cancer SKOV3 cells. These findings could open a new avenue for using CQDs/Cu2O composite as potential therapeutics for cancer chemotherapy.Our findings reveal a new role of CQDs/CuAdditional file 1: Figure S1. The enriched GO terms in the DEGs of cells treated by CQDs/Cu2O . The green, red, and blue bars represent the terms of biological process, cellular component, and molecular function, respectively. Figure S2. The viability of HUVEC cells after treated with CQDs/Cu2O by the MTT assay for 12 h.


Smad4 gene in cardiac progenitors expressing Sfrp5 led to embryonic lethality with hypoplastic heart formation. Although the expression of Nkx2-5 is regulated by Bmp signaling, expression of Nkx2-5 was weakly detected in the mutant heart. However, the nuclear translocation of Nkx2-5 was impaired. Expression of CK2 or PP1, which could alter the phosphorylation status of the NLS of Nkx2-5, was not affected, but Nkx2-5 was found to bind to Smad4 by co-immunoprecipitation experiments. Introduction of Smad4 into cells derived from Smad4 conditional knockout embryonic hearts restored the nuclear localization of Nkx2-5, and exogenous Nkx2-5 failed to translocate into the nucleus of Smad4-depleted fibroblasts. These results suggest that Smad4 plays an essential role in cardiomyocyte differentiation by controlling not only transcription but also the nuclear localization of Nkx2-5.Bmp plays an important role in cardiomyocyte differentiation, but the function of Smad4 in Bmp signaling remains elusive. Here, we show that disruption of the  Understanding how signaling networks regulate heart development is critical for elucidating mechanisms underlying congenital heart diseases and for developing novel approaches in regenerative cardiovascular medicine.The heart is the first organ to establish a blood circulatory system required for the subsequent development of other organs. Decades of research have revealed that intricate signaling networks, including those involving members of the Wnt, Bmp, and Fgf pathways, play key roles in heart development because perturbation of these secreted factors has been shown to result in a wide range of defects in embryonic and adult hearts13. Bmp signaling has been reported to directly regulate transcription of the Nkx2-5 gene, which encodes one of the earliest and most important cardiac transcription factors15, given the presence of Smad binding sites in the enhancer region of the Nkx2-5 gene13. Smad4 is a common-mediator Smad (Co-Smad). Once the Bmp receptor is activated by ligand binding, Smad1/5/8 is phosphorylated and forms a complex with Smad4. This complex then translocates into the nucleus and regulates the transcription of target genes, suggesting that Smad4 plays a central role in Bmp signaling.Bmp signaling is involved in fate decisions of multiple cell lineages of cardiomyocytes through the transcriptional regulation of cardiac-specific transcription factors, such as GATA4, Mef2c, and Nkx2-5Smad4-deficient mouse embryos die by E7.5 due to impaired gastrulation, suggesting that Smad4 is required for mesodermal formation16. An analysis of cardiomyocyte-specific Smad4-KO mice revealed that Smad4 functions in the proliferation and differentiation of cardiomyocytes, as reflected in the thinner ventricles in both trabecular and compact hearts17. However, the function of Smad4 during the process of cardiomyocyte differentiation remains unknown.20. In a previous study, we reported that Sfrp5 (a decoy receptor of the Wnt ligand) is a marker of cardiac progenitor cells, which form the first heart field (FHF) and later contribute to the left ventricle (LV), both atria, sinus venous (SV), and a part of the outflow tract (OFT), but not to the right ventricle (RV)21. Thus, mice that express Cre in Sfrp5-expressing cells could serve as a tool to generate mice that specifically lack the Smad4 gene in cardiac progenitor cells.Wnt proteins are secreted proteins that play a key role in differentiation during the development of many organs, including the heartSmad4-conditional knock-out (Smad4-cKO) model by crossing Smad4floxed/floxed mice with Sfrp5-Cre mice. Smad4-cKO mice exhibited severe cardiac hypoplasia with inhibited myocardial cell differentiation due to the down-regulation of Nkx2-5 expression and loss of nuclear localization of Nkx2-5. These findings suggest that, in addition to a mediator of Bmp signaling, Smad4 could be responsible for cardiac differentiation by binding to Nkx2-5 and regulating its nuclear localization.In the present study, we provide evidence that Smad4 forms a complex with Nkx2-5 to control the nuclear localization of Nkx2-5, and that this in turn promotes the differentiation of cardiac progenitors and heart morphogenesis. To examine specific functions of Smad4 in cardiogenesis, we generated a Smad4 gene in Sfrp5-expressing cardiac progenitor cells. Specifically, Sfrp5 Cre/Cre; Smad4 del/+ mice were crossed with Smad4f./f mice21. Compared to the littermate controls , Smad4-cKO mice  were embryonic lethal at around E8.5, with a hypoplastic heart tube and edema of the pericardium , Mef2c, Hey2, Nppa (ANF), and Isl115. Since Smad4 is required for Nkx2-5 gene expression as it binds its upstream enhancer region in vivo13, we expected that Nkx2-5 expression would be low or absent in Smad4-cKO hearts. However, Nkx2-5 was expressed, albeit weakly   by Casein kinase 2 (CK2), a ubiquitously expressed and constitutively active Ser/Thr protein kinase25, we expected Mypt1 to be dominantly localized in the nuclei and thereby prevent Nkx2-5 nuclear localization. However, in both control and Smad4-cKO hearts, Mypt1 was robustly expressed outside the nuclei , a regulatory subunit of PP1 specifically expressed in the heart, might be increased in Smad4-cKO hearts. Here too, no difference was noted in the level of expression of Ppp1r3c by RT-PCR and ISH in Smad4-cKO and control hearts  might explain the impaired nuclear localization of Nkx2-5. Since the regulatory subunit of PP1β, myosin phosphatase targeting subunit 1 (Mypt1), has been reported to be responsible for the nuclear exclusion of Nkx2-5 through direct interactionSince phosphorylated Smad1/5/8 forms a complex with Smad4 to translocate into the nucleus and regulate the transcription of target genes, we hypothesize that a protein complex containing both Smad4 and Nkx2-5 might similarly be involved in the proper nuclear translocation of Nkx2-5. Immunoprecipitation (IP) experiments using COS-7 cells demonstrated an interaction between Smad4 and Nkx2-5 Fig. A, B.FiguSmad4 has two functional domains, MH1 and MH2, both of which bind to distinct proteins, such as importins and exportins. To gain insight into whether Nkx2-5 binds to either of these domains, we performed additional IP experiments with MH1- or MH2-deleted Smad4. As shown in Fig. We then tested subcellular localization of Smad4 and Nkx2-5 by western blot analysis of subfractionated cellular compartments of COS-7 cells, which were transfected with their expression vectors Fig. C. Smad4 Smad4-cKO heart cells, which express exogenous Flag-tagged Smad4 showed a significant increase in the ratio of cells with Nkx2-5 localized in the nucleus  via its linker regionSmad4-cKO embryonic hearts. This suggests that while Bmp/Smad signaling does not serve as a trigger for myocardial differentiation, it can promote cardiac differentiation by controlling the nuclear localization of Nkx2-5 via Smad4. Hence, Smad4 could function not only as a mediator of Bmp signaling for the transcriptional regulation of cardiac-specific genes but also as a regulator of the nuclear localization of Nkx2-5 to ensure proper cardiac differentiation.Our results provide evidence that Smad4 is required for the transcription of several cardiac-specific genes, despite the fact that initial tube formation occurs and residual Nkx2-5 expression remains in Sfrp5 Cre/+; Smad4 del/del mice with a C57Bl6 background were obtained by crossing Sfrp5 Cre/Cre; Smad4 del/+ mice with Smad4 floxed/floxed mice, which carry the Smad4 gene flanked by loxP recognition sites. Generation of the Sfrp5 Cre/+ line was described previously21. The Smad4 floxed/floxed line was a gift from Dr. CX Deng37. All procedures had local approval by the research ethics committee of Hiroshima University (A18-109–2) and conformed to the NIH Guide for the Care and Use of Laboratory Animals and all study carried out in compliance with the ARRIVE guidelines. Mouse tissues for all experiments were isolated after euthanizing mice by CO2 asphyxiation.The Smad4-cKO embryos at E9.5. Cardiomyocytes were harvested by trypsinization with 0.1% (w/v) Trypsin  for 2 min, seeded onto 24 mm × 24 mm glass plates, and cultured in 6-well tissue culture plates. A plasmid containing Smad4 cDNA (Flag-tagged) or an empty vector (pDEF) was transfected using 3.23% (w/v) Polyethylenimine  on the following day. The immunofluorescent analysis was performed 24 h after transfection to detect the cellular distribution of Nkx2-5.Hearts were dissected from Smad4f./f embryos using the same method described above and cultured in 6-well tissue culture plates. To delete Smad4, plasmids containing Cre cDNA were transfected into the fibroblasts twice using 3.23% (w/v) PEI.Fibroblast cells were obtained from 2.COS-7 cells were maintained in DMEM  containing 4.5 g/L D-glucose supplemented with 1 mM sodium pyruvate, 4 mM L-Glutamine, and 10% (v/v) fetal bovine serum  at 37 °C and 5% COSmad4, MH1, or MH2  and Myc-tagged Nkx2-5  using 3.23% (w/v) polyethyleneimine  following the same steps described above for the rescue experiment.COS-7 and fibroblast cells were transfected with expression plasmids, including Flag-tagged 21. Embryos were dissected at E9.5 and fixed with 4% paraformaldehyde in PBS for durations of 1 h to overnight. After dehydration using a methanol series, embryos were rehydrated with 0.1% Tween 20 in PBS (PBST), bleached with 6% H2O2 in PBS, treated with Protease K, and then hybridized with digoxygenin (DIG)-labeled probes at 68ºC overnight. Embryos were then washed with 5 × and 2 × SSC/50% formamide and PBST. To detect DIG-labelled probes, embryos were incubated with anti-DIG antibodies after treatment with blocking reagent for 1 h, and the signal was developed with BM purple AP substrate .Whole-mount in situ hybridization was performed as described previously21. Total RNA was isolated using TRIzol . Reverse transcription was performed with the ReverTra Ace qPCR RT Kit . Real-time PCR was conducted using SYBR Premix Ex Taq II . Intensities of PCR products were measured and analyzed using Mini-Opticon . Amplification conditions were: 5 s at 95 °C, 20 s at 60 °C, and 15 s at 72 °C for 49 cycles. G3pdh was amplified as an internal control. Primers used in the present study are shown in Supplementary Table Real-time PCR was performed as previously describedEmbryos were embedded in optimum cutting temperature (OCT) compound  after fixation and cut into 10–14 μm sections. After washing with PBS and blocking with 1% BSA in PBS for 1 h, sections were incubated with 500 × diluted primary antibodies, including anti-Smad4 , anti-Mypt1 , anti-Flag (DYKDDDDK)-Tag , anti-Myc Tag , anti-Nkx2-5 , and anti-TnT  at 4 °C overnight. Sections were then incubated with 500 × diluted secondary antibodies conjugated with Alexa Fluor 488 or 555 . Nuclei were stained with 4, 6-diamidino-2-phenylindole .COS-7 cells, transfected expression vectors, were collected after 24 h, and then their nuclei and cytoplasm fractions were separated. Their containing proteins were harvested by using a NE-PER Nuclear and Cytoplasmic Extraction Reagents  following the instruction protocols and stored at – 80 °C for further use.Cultured cells were washed with cold PBS and lysed on ice with Sample buffer  SDS). After centrifugation at 15,500 rpm for 10 min at 4 °C, supernatant fractions were collected and analyzed by SDS-PAGE and immunoblotting.Cultured cells were washed with cold PBS and lysed on ice with RIPA buffer  IGEPAL CA-630, 0.1% (w/v) SDS, and 0.5% (w/v) sodium deoxycholate) containing phenylmethylsulfonyl fluoride  for 30 min. After centrifugation at 15,000 rpm for 10 min at 4 °C, supernatants were collected and used for immunoprecipitation (IP). IP was performed with the Immunoprecipitation Starter Pack  according to the manufacturer’s instructions, using 100 μl column resin conjugated with 5 μg anti-Flag antibody, 5 μg anti-Myc antibody, 5 μg anti-Smad4 antibody, 5 μg anti-Nkx2-5 antibody, or 100 μg IgG, and then analyzed by immunoblotting after SDS-PAGE. Signal detection was performed with the LuminoGraphI  system.https://www.ibm.com/analytics/spss-statistics-software). *P < 0.05 was considered statistically significant.Data are expressed as mean ± SEM and were analyzed using one-way analysis of variance (ANOVA) with the Tukey–Kramer post hoc test for multiple comparisons or the nonparametric Mann–Whitney U test to compare two independent populations. Statistical analyses were performed using SPSS 20.0 software (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp. Supplementary Information.


Their structural and physical properties were examined by X-ray diffraction, X-ray absorption, transmission electron microscopy, and electrical resistivity. All of the Cex1−PrxOBiS2 single crystals with 0.1 ≤ x ≤ 0.9 exhibited tetragonal phase. With increasing Pr content, the a-axis and c-axis lattice parameters decreased and increased, respectively. Transmission electron microscope analysis of Ce0.1Pr0.9OBiS2 (x = 0.9) single crystal showed no stacking faults. Atomic-resolution energy dispersive X-ray spectrometry mapping revealed that Bi, Ce/Pr, O, and S occupied different crystallographic sites, while Ce and Pr randomly occupied the same sites. X-ray absorption spectra showed that an increase of the Pr ratio increased the ratio of Ce4+/Ce3+. All of the Cex1−PrxOBiS2 crystals showed superconducting transition, with a maximum transition temperature of ~4 K at x = 0.9.Ce The flux growth of OBiS2 single crystals with various Ce/Pr ratios would provide an opportunity for discovering new superconductors.To present, various ROBiSx1−PrxOBiS2 superconducting single crystals with different Ce/Pr ratios  using a CsCl flux. The crystals with high Pr contents showed superconducting properties with a maximum transition temperature of ~4 K, which could not be observed in powder samples synthesized without using the flux  were grown using CsCl flux , Pr2S3 , Bi2O3 , Bi2S3  were weighed to have a nominal composition of Cex1−PrxOBiS2 (0.1 ≤ x ≤ 0.9). The mixture of the raw materials (0.8 g) and CsCl flux (5.0 g) were ground by using a mortar, and then sealed into an evacuated quartz tube. The quartz tube was heated at 950°C for 10 h, followed by cooling to 650°C at a rate of 1°C/h, then the sample was cooled down to room temperature in the furnace. The heated quartz tube was opened in air, and the obtained materials were washed and filtered by distilled water for removing the CsCl flux.Single crystals of Ce2 single crystals were evaluated from synchrotron powder X-ray diffraction measurements using crushed single crystals powder. Synchrotron powder X-ray diffraction measurements were performed at 150 K in SPring-8 using the BL02B2 beamline with the approval of 2018A0074. Local structure and elemental distribution of Ce0.1Pr0.9OBiS2 (x = 0.9) single crystal along the c-axis were observed by scanning transmission electron microscope with energy dispersive X-ray spectrometry (STEM-EDS) (JEOL JEM-ARM200F).The compositional ratio of the single crystals was evaluated by energy dispersive X-ray spectrometry (EDS)  associated with the observation of the microstructure by using scanning electron microscope (SEM) . The obtained compositional values were normalized using S = 2.00, with Ce, Pr, and Bi measured to a precision of two decimal places. After that, the Pr composition is normalized by the total Pr and Ce content. Identification and orientation of the grown crystals were performed by X-ray diffraction (XRD) using Rigaku MultiFlex with CuKα radiation. Crystal system and lattice parameters of OBiSL3 and Pr-L3 edges using an Aichi XAS beamline with a synchrotron X-ray radiation . For XAFS sample, the obtained single crystals were grinded and mixed with boron nitride (BN) powder, pressed into a pellet of 4 mm diameter.The valence state of the Cerium and Praseodymium component in the obtained single crystals was estimated by X-ray absorption fine spectroscopy (XAFS) analysis of Ce-T) characteristics of the obtained single crystals were measured by the standard four-probe method with a constant current density (J) mode using physical property measurement system . The electrical terminals were fabricated by silver paste. And, ρ–T characteristics in the temperature range of 0.25–15 K were measured with an adiabatic demagnetization refrigerator (ADR) option for PPMS. The magnetic field applied for operating the ADR was 3 T at 1.9 K; subsequently, it was removed. Consequently, the temperature of sample decreased to ~0.25 K. The measurement of ρ–T characteristics was started at the lowest temperature (~0.25 K), which was spontaneously increased to ~15 K. The superconducting transition temperature (Tc) was estimated from the ρ–T characteristics. The transition temperature corresponding to the onset of superconductivity (T characteristics. The zero resistivity (T characteristics of Ce0.1Pr0.9OBiS2 (x = 0.9) single crystal under a magnetic field (H) parallel to the c-plane with range of 0.1–9.0 T and the c-axis with range of 0.1–0.3 T were measured in the temperature range of 2.0–10.0 K. We measured the angular (θ) dependence of resistivity (ρ) in the flux liquid state under various magnetic fields (H) and calculated superconducting anisotropy (γs) using the effective mass model  are shown as x1−PrxOBiS2 (0.1 ≤ x ≤ 0.9) single crystals. The appearance of only 00l diffraction peaks indicates that the c-plane is well-developed. This is a typical feature of BiS2-based single crystals , but increased slightly at x = 0.9. The lattice parameter of the c-axis increased with increased Pr contents (x).c-axis was performed by energy dispersive X-ray spectrometry mapping with a scanning transmission electron microscope (STEM-EDS). 2 with x = 0.9 single crystal and also shows the corresponding elemental maps obtained using Ce-L, Pr-L, Bi-M, O-K, and S-K in the same area. No stacking faults were observed in the STEM image for a wide area. Atomic-resolution EDS mapping revealed that Bi, Ce/Pr, O, and S occupied different crystallographic sites, whereas Ce and Pr randomly occupied the same sites.Direct observation of the stacking layers along the 2 single crystals by XAFS analysis. L3- and (b) Pr L3-edge absorption spectra of OBiS2 single crystals with 0 ≤ x ≤ 1.0 obtained by XAFS analysis at room temperature. The Ce L3-edge of the OBiS2 single crystals showed a peak at around 5,727 eV, assigned as Ce3+, with is consistent with the other XAFS result for the trivalent electronic configuration (Ce3+) , and these peaks were assigned tetravalent electronic configuration (Ce4+) ] increased with increase of Pr content (x), suggesting that the valence state of Ce fluctuated in the OBiS2 single crystals. On the other hand, the Pr L3-edge of the OBiS2 single crystals showed a peak at ~5,968 eV, which can be assigned as Pr3+. This is consistent with the other XAFS results for the trivalent electronic configuration (Pr3+)  shows a signature peak at ~5,980 eV OBiS2 with 0.1 ≤ x ≤ 0.9 single crystals was a mixed state consisting of both trivalent (Ce3+) and tetravalent (Ce4+) valences.We evaluated the valence state of Ce and Pr in the obtained OBiST) dependence of resistivity normalized at 15 K [ρ/ρ(15 K)] for the OBiS2 single crystals with 0 ≤ x ≤ 0.9 in the temperature range of 0.25–15 K. Above 5 K, decrease in temperature increases relative resistivity. The increases in relative resistivity are suppressed by higher Pr content. x) dependence of x) increased the transition temperatures up to x = 0.9. In particular, OBiS2 with x = 0.9 showed a highly superconducting transition temperature of ~4 K. The Pr content (x) dependence of the Ce4+ ratio [Ce4+/(Ce3+ + Ce4+)] in OBiS2 single crystals with 0 ≤ x ≤ 1.0 showed similar tendency of the transition temperatures OBiS2 single crystal with x = 0.9. The resistivity at normal state shows almost metallic behavior although it shows an anomaly at 80–120 K. Similar anomalies of NdBiS2 and LaBiSe2 single crystal are reported in 60–140 K  superconductors although further investigation is required to reveal the origin of these anomalies.2 single crystal with x = 0.9 below 10 K under the magnetic field (H) parallel to the c-plane  and to the c-axis . The superconducting transition was monotonically suppressed with increasing magnetic fields parallel to the c-plane. The magnetic fields dependence of the H) parallel to the c-plane (H//c-plane) is plotted in the inset of H//c-plane approaches 16.3 T, which is estimated to be the maximum of upper critical field on the c-plane (H//c−planec2). In a conventional (BCS-like) superconductor in the weak-coupling limit, the Pauli limit is calculated to be 9.22 T, derived from Hp = 1.84 Tc, (c-plane (H//c−planec2 = 16.3 T) is significantly higher than the Pauli limit (Hp = 9.22 T), indicating the possibility of an unconventional superconductor. In contrast to the superconductivity in the c-plane, its superconductivity in the applying magnetic fields perpendicular to the c-axis almost disappeared at 2 K with only 0.3 T  and the superconducting anisotropy (γs) could not be estimated from ly 0.3 T , suggests) of the OBiS2 single crystal with x = 0.9 by an effective-mass model  in the flux liquid state to estimate the superconducting anisotropy (γs). The reduced field (Hred) is calculated using the following equation for an effective mass model:We also evaluated the superconducting anisotropy  at 3.0 K in the flux liquid state for OBiS2 single crystal with x = 0.9. The ρ–θ curve exhibited an almost two-fold symmetry. Hred scaling obtained from the ρ–θ curves in s = 31. In contrast, the superconducting anisotropy (γs) of OBiS2 with other Pr concentrations  single crystals using Equation (1) were estimated to be 7.5–13. These results suggest that the superconducting anisotropy of OBiS2 single crystal with x = 0.9 is highest.where θ is the angle between the 2 single crystals with various Ce/Pr ratios were synthesized and characterized. All of the crystals exhibited tetragonal phase, and the superconducting transition temperature increased to 4 K with increasing ratio of Pr. With increasing Pr content, the lattice parameters of the a-axis decreased and those of the c-axis increased. TEM analysis revealed mixed occupancy of Ce and Pr without stacking faults.OBiS0.1Pr0.9OBiS2 (x = 0.9), the corresponding powder sample with mainly monoclinic structure did not show zero resistivity   showed the highest superconducting transition temperature of ~4 K and also showed high superconducting anisotropy. When compared with Ce0.3Pr0.7OBiS2 (x = 0.7), the lattice parameter of the a-axis was slightly longer and that of the c-axis was significantly longer. The elongated c-axis could be the reason for the high superconducting anisotropy. The ratio of Ce4+/Ce3+ was the highest in the series, but this cannot explain the increase in the lattice parameters considering the ionic radii. TEM analysis did not show the ordering of Ce and Pr. Structural analysis using single crystal X-ray diffraction via a highly monochromatic synchrotron source would facilitate better understanding of the relationship between structure and superconductivity.Cex1−PrxOBiS2 (0.1 ≤ x ≤ 0.9) platelet single crystals were grown using a CsCl flux. The crystal structures of Cex1−PrxOBiS2 single crystals with 0.1 ≤ x ≤ 0.9 maintained the tetragonal phase. Increase in Pr content up to x = 0.7 decreased the lattice parameter of the a-axis and increased the parameter of the c-axis. Ce0.1Pr0.9OBiS2 single crystal showed a slightly elongated a-axis lattice parameter but a significantly elongated c-axis parameter. Atomic-resolution TEM analysis of the Ce0.1Pr0.9OBiS2 single crystal showed no stacking faults and no ordering of Ce and Pr. The superconducting transition temperature of Cex1−PrxOBiS2 single crystals increased with increasing Pr content, showing a trend similar to that of the ratio of Ce4+/(Ce3+ + Ce4+). High superconducting transition temperature of ~4 K and high superconducting anisotropy appeared in the Ce0.1Pr0.9OBiS2 (x = 0.9) single crystal. While the Ce0.1Pr0.9OBiS2 (x = 0.9) single crystal with tetragonal structure showed the highest transition temperature, the corresponding powder samples of mainly monoclinic phase did not show zero resistivity (Miura et al., CeThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.MN, AM, KT, and IT designed the research. MN and YMa prepared the samples. MN and SW measured electronic conductivity. MN and YT measured magnetic properties. MN and AM measured X-ray absorption. YG, YMi, CM, and YK measured SXRD. YW performed preliminary TEM analysis. DU and TA performed atomic-resolution TEM analysis. MN and AM wrote the draft. All the authors checked the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These receptors are the target proteins for the therapy for several neurologic and psychiatric disorders, including Parkinson’s disease and schizophrenia. The dansyl-labeled ligands exhibit binding affinities up to 0.44 nM and 0.29 nM at D2R and D3R, respectively. When the dansyl label was exchanged for sterically more demanding xanthene or cyanine dyes, fluorescent ligands 10a-c retained excellent binding properties and, as expected from their indanylamine pharmacophore, acted as agonists at D2R. While the Cy3B-labeled ligand 10b was used to visualize D2R and D3R on the surface of living cells by total internal reflection microscopy, ligand 10a comprising a rhodamine label showed excellent properties in a NanoBRET binding assay at D3R.Fluorescent ligands are versatile tools for the study of G protein-coupled receptors. Depending on the fluorophore, they can be used for a range of different applications, including fluorescence microscopy and bioluminescence or fluorescence resonance energy transfer (BRET or FRET) assays. Starting from phenylpiperazines and indanylamines, privileged scaffolds for dopamine D D2-like receptors represent the main targets for the therapy of severe neurological and psychiatric disorders including Parkinson’s disease, schizophrenia, restless legs syndrome, and addiction1. Over the past years, high-resolution X-ray crystal structures or cryo-electron microscopy maps have become available for the entire subfamily , enabling structure-based drug design7 as an alternative to ligand-based drug development. Independent from the employed design strategy, drug discovery campaigns require fast and reliable in vitro assays to determine target affinity, selectivity, kinetics and functional activity of novel lead structures. Whereas numerous assay technologies, such as enzyme fragment complementation, fluorescence or bioluminescence resonance energy transfer (FRET or BRET), proximity-based assays and even label-free methods have been employed for the determination of functional activity at GPCRs8, the characterization of a ligand’s affinity towards a given receptor is mostly based on radioligand competition. This binding assay has been proven useful due to the high chemical similarity of unlabeled and labeled probe molecules, the highly specific detection of radioactive labels, and the relatively easy assay format9. Nevertheless, radioligand-based methods have several disadvantages. Besides the costs and problems associated with the disposal of radioactive waste and regulatory requirements, high-affinity radioactive probe molecules have to be available for the receptor of interest9.Dopamine receptors belong to the large family of G protein-coupled receptors (GPCRs). With their transmembrane architecture, these proteins are responsible for signal transduction from the extracellular environment to intracellular compartments. Dopamine receptors respond to the binding of the neurotransmitter/hormone dopamine and are divided into two sub-groups, the D12. Similar to radioactive probes, fluorescent molecules can be detected at very low concentration with high specificity. Although the synthesis of small-molecule fluorescent ligands is far from trivial, it requires lower level regulatory and safety precautions compared to the preparation of radioligands. Moreover, several fluorescence-based technologies like resonance energy transfer (RET) and fluorescence anisotropy  do not require separation of unbound ligands and assays can thus be run in a homogenous, fast “mix-and-measure” setup13. In combination with sophisticated techniques like total internal reflection fluorescence (TIRF) microscopy, fluorescent ligands allow to detect and track GPCRs with single molecule resolution16. Fluorescent ligands have also been used to monitor receptor internalization17, and to study receptor oligomerization in native tissue18. Very recently, the development of a small and bright luciferase variant 19 has greatly facilitated the implementation of BRET-based ligand binding assays11. In contrast to Renilla luciferase, which is frequently used to study intracellular protein–protein interactions, N-terminal Nluc fusion proteins can be easily targeted to the cell surface11. NanoBRET between fluorescently labeled ligands and Nluc-receptor fusion proteins has already been used to study ligand binding at a number of therapeutically relevant GRCRs, including β-adrenergic11, adenosine11, histamine20, free fatty acid21, and chemokine22 receptors. Despite the high therapeutic relevance of the D2-like receptor family, only a small number of fluorescent ligands targeting these receptors have been reported so far. Whereas a fluorescently labeled agonist (2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin scaffold, PPHT-red)24 has been employed in FRET ligand binding studies at D2R , NanoBRET binding assays have not yet been established for this receptor family. Besides the agonistic PPHT derivatives, different fluorescent ligands comprising an antagonistic N-(p-aminophenethyl)spiperone (NAPS) scaffold have also been described28. For example, a Cy3B-labeled NAPS derivative has been used for microscopic analyses of ligand binding at D3R29. In our group, we have developed Cy3B-labeled fluorescent ligands with an N-propylamino-5-hydroxytetraline or phenylpiperazine substructure for TIRF microscopy studies of D2R and D3R homodimerization in living cells14.Over recent years, fluorescence-based technologies have emerged as exciting alternative to study ligand affinity2-like receptor subfamily. Starting from well-known dopamine receptor recognition elements, we developed dansyl-labeled fluorescent ligands that possess excellent binding affinity at D2R and D3R receptors. We demonstrate that our design strategy is suitable for accommodating larger xanthene and cyanine fluorophores, while maintaining high-affinity dopamine receptor binding. Depending on the fluorophore, our novel ligands can be used to label D2R and D3R in living cells (TIRF microscopy) or in a newly established D3R-NanoBRET ligand binding assay that will open up the path for future drug discovery campaigns.Taking advantage of this expertise, we describe the design, molecular docking, chemical synthesis and pharmacological characterization of novel fluorescent ligands targeting the D2-like receptor family, we made use of four different privileged scaffolds that are frequently found in dopamine receptor ligands30. The N-propyl substituted 2-amino-dihydro-1H-indene  selectively targets D3R33. Previous studies have revealed the importance of a second lipophilic moiety for high-affinity binding to dopamine receptors34. Thus, we envisioned connection of the primary pharmacophores to a triazole-benzylamine or triazole-benzoic acid moiety through a flexible four-carbon aliphatic linker. This linker size was chosen so that ligands should possess high affinity for both D2R and D3R, while a considerably shorter linker would have been beneficial for the D4R subtype30. For an initial set of fluorescent ligands, we focused on the incorporation of a dansyl label, a naphthalene-derived fluorophore emitting green light. The dansyl fluorophore has a relatively low molecular weight and is commercially available as reactive sulfonyl chloride (dansyl chloride). The dansyl dye is widely used to label proteins for fluorescence polarization measurements due to its favorable lifetime or as an acceptor for resonance energy transfer from protein tryptophan residues. It is also known for its large Stokes shift, resulting in a good signal-to-noise ratio35. Since we have previously found that the binding pockets of D2R and D3R can accommodate flexible linkers14 but also sterically more demanding substituents in their extended binding pocket37, installation of the dansyl fluorophore was envisioned through sulfonamide-formation either directly with the primary benzylamine (ligands 4a-d) or through addition of a short 1,3-diaminopropane spacer to the benzoic acid precursors . The latter strategy has already been successful for the development of Cy3B-labeled dopamine receptor ligands in the context of TIRF microscopy17.For the development of fluorescent probes targeting the DN-propyl-2,3-dihydro-1H-inden-2-amine, 2,3-dichlorophenylpiperazine, 2-methoxyphenylpiperazine or 2-(tert-butyl)-4-(piperazin-1-yl)-6-(trifluoromethyl)pyrimidine -catalyzed azide-alkyne cycloaddition with 4-azido-benzonitrile41, giving access to the 1,4-disubstituted triazoles 2a-d in excellent yield. Subsequent reduction of the benzonitriles with LiAlH4 afforded the primary amines 3a-d, which were used in the next step without further purification. In the final step, dansyl-labeled title compounds 4a-d were obtained by reaction with dansyl chloride in presence of triethylamine. Fluorescent ligands 8a,b comprising an additional aminopropane spacer were prepared in a very similar reaction sequence. To this end, the terminal alkynes 1a,b were first reacted with 4-azido-benzoic acid42 in a copper(I)-catalyzed azide-alkyne cycloaddition41 yielding the triazoles 5a,b. The benzoic acid moiety was then coupled to mono Boc-protected 1,3-diaminopropane43 using TBTU as the coupling reagent, yielding intermediates 6a,b. Boc-deprotection was carried out using hydrochloric acid in dioxane, before the resulting primary amines 7a,b were reacted with dansyl chloride to afford the title compounds 8a and 8b.Synthesis of the dansyl-labeled ligands started from the secondary amines max ≈ 350 nm)35 prevents usage in NanoBRET assays. In recent years, many organic fluorophores with emission maxima in the red spectral range and enhanced photochemical properties have been developed. Chemically, these fluorophores often belong to the xanthene or cyanine dye families, but differ in their substituents, leading to a variety of net charges and different degrees of hydrophilicity44. We selected three different fluorophores, the commercially available dyes Alexa488 and Cy3B and a recently described bis-trifluoroethyl substituted rhodamine38 for the synthesis of our fluorescent dopamine receptor probes. Alexa488 and Cy3B, but also tetramethylrhodamine dyes (TAMRA), have already been shown to be suitable for characterizing ligand binding to GPCRs45, and especially Cy3B-labeled ligands have proven useful for fluorescence microscopy29. For the synthesis of these ligands, we focused on the N-propyl substituted 2-amino-dihydro-1H-indene scaffold. 5 ´-Carboxy-N,N ´-bisrhodamine (9) was prepared as described previously38 and connected directly to the benzylamine 3a using TBTU as the coupling reagent to give the desired fluorescent ligand 10a in a yield of 28%. Alexa488 and Cy3B were purchased as tetrafluorophenyl (TFP) or N-hydroxysuccinimid (NHS) esters, respectively, and reacted with the 1,3-dipropylamino-substituted benzoic acid 7a under basic conditions in DMF, affording the fluorescent ligands 10b (Cy3B) and 10c (Alexa488).Despite the advantages of the dansyl label, it is not ideally suited for all popular fluorescence-based technologies, for example bioluminescence resonance energy transfer. For instance, its fluorescence properties are highly sensitive to solvent polarity and the fluorescence excitation maximum  or D3R . As expected from the ligand design, typical receptor-ligand interactions in the orthosteric binding pocket5 including the salt bridge formation with Asp3.32 were observed for the indanylamine moiety of 8a and 10b in both receptors , while the affinities for the D1R were substantially lower . The direct comparison of the dansyl-labeled ligands 4a-c with their benzonitrile precursors 2a-c clearly demonstrates that the insertion of the naphthyl-derived fluorophore is well tolerated by D2LR, D2SR, D3R and D4R, as highly similar binding affinities were determined in the presence and absence of the fluorescent moiety. As expected, ligands 2d and 4d, containing a pyrimidylpiperazine as primary dopamine receptor recognition element, were found to be selective for the D3R subtype. The introduction of the dansyl moiety further increased the D3R-selectivity, as it had no major influence on the D3R affinity (9.5 vs. 11 nM), but decreased the affinity for the D2LR and D2SR subtypes by a factor of ~ 20. Since 4d is also selective for D3R over the investigated serotonin and α1 receptors, it could potentially be used in fluorescence microscopy for D3R localization studies in tissues. Connection of the dansyl label and the lipophilic moiety through a short 1,3-diaminopropylene-spacer did not substantially improve binding affinities at the D2R and D3R, indicating that this additional spacer is not strictly required for the small dansyl fluorophore. However, with a Ki value of 0.44 nM, ligand 8a displayed the highest D2SR affinity of the entire series. When the small dansyl fluorophore was exchanged for the sterically more demanding xanthene or cyanine dyes, binding affinities slightly decreased for the D2S and D2L receptors (4.8–46 nM). Similar to their dansyl analogs, fluorescent ligands 10a–c displayed the strongest binding affinity for the D3R subtype (0.76–0.97 nM). With the exception of the D3R-selective ligands 2d and 4d, the series of fluorescent ligands and precursors also showed affinity for 5-HT1AR (13–260 nM) and α1-AR (1.1–150 nM). This is not unexpected, since the 2-methoxyphenylpiperazine is a common motif in α1-AR antagonists46. At 5-HT2R, observed affinities ranged from 220 to 2,200 nM, only.Affinities for the dopamine receptor subtypes Des Table . In gene2SR employing an assay based on enzyme fragment complementation . Since the presence of GRK2 is known to be important for a sensitive detection of ligand effects in HEK293 cells, GRK2 was coexpressed together with the D2SR fused to the enzyme donor. When the cells were incubated with the fluorescent ligands 8a and 10a–c, substantial stimulation of β-arrestin-2 recruitment was observed, confirming the agonistic nature of the N-propyl-2-aminoidane dopamine-isostere  and displayed a potency (EC50 51 ± 11 nM) that is only slightly lower compared to the reference agonist quinpirole . Incorporation of the larger trifluoroethyl-rhodamine, Cy3B or Alexa488 fluorophores led to a two- to eightfold reduction in ligand potency alongside with slightly reduced ligand efficacy for the fluorescent agonists 10a , 10b  and 10c , respectively. In agreement with our docking studies, these results indicate that D2-like receptors can accommodate the small dansyl- but also larger xanthene or cyanine-derived fluorescent ligands. However, not only the size but also the type of the fluorophore can have an impact on binding affinity, intrinsic activity and potency. This is illustrated in particular by the Cy3B-labeled ligand 10b, which has threefold higher affinity but fourfold lower potency for β-arrestin-2 recruitment at D2SR compared to 10c that only differs from 10b in terms of the fluorophore.In order to evaluate the impact of the fluorophores on the functional activity of the ligands, we determined their capacity to elicit β-arrestin-2 recruitment to the D48. We employed our previously developed protocol for the imaging of dopamine receptors14 to verify that our newly developed fluorescent ligands are generally suitable for fluorescence imaging. For TIRF microscopy, we focused on the Cy3B-labeled ligand 10b, as the same fluorophore was previously used for the imaging of D2R and D3R17. Indeed, when CHO cells stably expressing D2SR or D3R were labeled with 10b in 10 nM (D2SR) or 1 nM (D3R) concentration, receptors were detected in the cytoplasmic membrane as discrete fluorescent spots . Analysis of the absorption spectra confirmed the expected spectral properties of the employed fluorophores. While absorption occurred only in the UV range for the dansyl-labeled ligand 8a, compounds 10a-c showed significant absorption in the area of 450–550 nm (up to 600 nm for 10b). Thus, ligands 10a-c could serve as BRET acceptors in combination with the Nluc enzyme as BRET donor, which shows luminescence in the range of 400–550 nm (maximum at 460 nm)19.To further take advantage of the newly developed ligands as fluorescent probes, we planned to establish a NanoBRET assay for the detection of ligand binding at dopaminergic receptors. In this assay, ligand binding is detected through RET between the bioluminescent Nluc3R, since our fluorescent ligands showed the highest affinity for this receptor subtype. To this end, a membrane targeted Nluc enzyme11  and D3R were fused in frame by polymerase chain reaction and cloned into pcDNA3.1 for mammalian expression. A second construct carrying an N-terminal HA export sequence and a FLAG-tag50 in front of the Nluc was generated, which allowed the detection of cell surface expression by ELISA17. Upon transient transfection into HEK293T cells, both variants of the Nluc-D3R fusion protein were well expressed, as determined by radioligand saturation , respectively), and both constructs showed the expected Nluc emission spectra in the presence of the substrate furimazine , which are likely a result of their higher expression level.For the development of the assay, we focused on the D10a-c are indeed suitable for BRET-based ligand binding assays, we performed saturation binding assays with live HEK293T cells expressing secNluc-D3R. For all three ligands, typical saturation hyperbolas were observed , that were in good agreement with their Ki values obtained by radioligand competition , which was surprising, given that its radioligand Ki value (0.90 ± 0.26 nM) was similar to those of 10a and 10c. Almost identical results were obtained, when additional saturation experiments with 10a  and 10b  were performed with membranes from HEK293T cells expressing secNluc-D3R instead of whole live-cells , obtained binding affinities of the four antipsychotics showed nearly the same rank order and were only slightly lower 19, that is fused to the extracellular part of the investigated GPCR. In both cases, binding of the fluorescent ligand is detected based on RET between the receptor as light emitting donor and the fluorescent ligand serving as the acceptor10. Both RET-assays have been proven extremely useful for the characterization of ligand binding at GPCRs, especially if high affinity radioligands are not available or if binding kinetics24 are central to the investigated research question. Typical saturation hyperbolas were obtained for all three ligands when we performed NanoBRET assays with living HEK293T cells expressing an Nluc-D3R fusion protein. For two of the ligands, 10a and 10c, observed binding affinities were almost identical to those derived from classical radioligand binding studies. This was also true, when NanoBRET experiments were performed with cell membranes instead of whole cells. For the Cy3B-labled ligand 10b, the NanoBRET KD was approximately tenfold higher. On the other hand, 10b showed and excellent behavior in TIRF microscopy studies with CHO cells expressing D2R and D3R. This illustrates that despite similar absorption and emission spectra, the choice of the fluorophores may be critical not only for ligand binding to the target but has to be tailored to the desired application of the fluorescent ligand.Taking advantage of our newly developed fluorescent ligands, we sought to establish a fluorescent ligand binding assay with the D2R and D3R receptors, which are the main targets in the treatment of severe neurological and psychiatric diseases. Depending on the employed fluorophore, the ligands can be used in high-resolution TIRF microscopy or to study ligand binding by resonance energy transfer. The established D3R-NanoBRET assay can be equally performed in whole cells and membranes and can be used for ligand binding screenings and characterization of novel ligands for D3R in the future.In summary, we have developed a set of high affinity fluorescent ligands for DSynthesis of the fluorescent ligands was achieved employing the following general procedures. Detailed protocols and analytical data for the individual compounds are provided as Supplementary Methods and Supplementary Figs. 2CO3 (2 eq) and KI (1 eq) in CH3CN was added 6-chlorohex-1-yne at room temperature and the reaction mixture was heated to reflux overnight. After addition of H2O, the aqueous phase was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4 and evaporated under reduced pressure to give the crude product.To a mixture of a secondary amine, Ktert.-BuOH-H2O-CH2Cl2 (1:1:1) was added CuSO4·5H2O (5 mol %) and sodium ascorbate (10 mol %) and the suspension was stirred at room temperature. After the completion of the reaction, the suspension was diluted by the addition of H2O, extracted with CH2Cl2, and the combined organic layers were dried over Na2SO4. After evaporation, the crude residue was purified by silica-gel column chromatography with CH2Cl2 followed by 95:5 CH2Cl2-MeOH.To a mixture of corresponding alkynes and aromatic azides in a solvent system of 2Cl2 was added dansyl chloride at 0 °C. The reaction mixture was stirred overnight at room temperature and the product was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4 and concentrated in vacuo to obtain the crude product. Target compounds were isolated by silica-gel column chromatography with CH2Cl2 followed by 98:2 CH2Cl2-MeOH.To a mixture of the crude primary amine and triethylamine (1:1) in CH2Cl2 at 0 °C was added TBTU in anhydrous DMF and the mixture was stirred for 30 min before a solution of tert.-butyl 3-aminopropylcarbamate in CH2Cl2 was added. After stirring for 2–3 h, saturated NaHCO3 solution was added and the mixture was extracted with CH2Cl2. The combined organic layers were dried over Na2SO4 and concentrated in vacuo to obtain the crude product. The compounds were isolated by silica-gel column chromatography with 95:5 CH2Cl2-MeOH.To a solution of the benzoic acid derivatives and DIPEA in CH8a and 10b was performed analogously to previously described protocols55. Ligands were geometry optimized by means of Gaussian1656 at the B3LYP/6–31  level of theory  and subsequently docked into the crystal structures of the D2R (PDB-ID: 6CM4) and the D3R (PDB-ID: 3PBL) using AutoDock Vina57. We applied a search space of 40 Å × 40 Å × 40 Å due to the large Cy3B moiety of 10b and to ensure a complete coverage of the binding pocket. Based on the scoring function and experimental data, four ligand-receptor complexes were selected, which were subsequently submitted to energy minimization using the PMEMD module of the AMBER 18 program package58. The all-atom force field ff14SB and the general AMBER force field (GAFF) were used for the receptors and ligands, respectively. Parameters for 8a and 10b were assigned using antechamber, and charges were calculated using Gaussian16 at the HF/6–31  level of theory and the RESP procedure according to the literature59. A formal charge of + 1 was defined for the ligands.Docking of 2LR, D2SR, D3R, D4R60, and porcine D1R, α1-AR, 5-HT1AR and 5-HT2R61 were determined as described previously. For D2LR, D2SR, D3R or D4R, membranes from CHO cells stably expressing these receptors and the radioligand [3H]spiperone were used. Binding studies with D1R were carried out with homogenates obtained from porcine striatum and [3H]SCH23390. For α1R, 5-HT1AR and 5-HT2R homogenates of porcine cerebral cortex and the radioligands [3H]prazosin for α1R, [3H]WAY100,635 for 5-HT1AR, and [3H]ketanserin in the presence of 10 µM prazosin for 5-HT2R were used. Detailed concentrations, KD values, etc. are summarized in Supplementary Table 1R-D4R), WAY100,635 (5-HT1AR), ketanserin (5-HT2R) or prazosin (α1R). Data analysis was performed using the algorithms for non-linear regression in PRISM6.0 to provide an IC50 value which was transformed into the Ki value employing the equation of Cheng and Prusoff52. Radioligand competition with Nluc-D3R membranes was performed in an analogous manner.Affinities of the fluorescent ligands and precursors towards the human D14. In brief, CHO-K1 cells stably expressing D2SR or D3R were seeded on 18 mm glass slides coated with fibronectin in phenol red-free DMEM/F12 supplemented with 10% FBS and were allowed to adhere over night at 37 °C, 5% CO2. Cells were washed twice with phenol red-free DMEM/F12 containing 10% FBS and incubated with 10b  for 1 h at 37 °C, before they were washed another three times. Specific labeling was confirmed by preincubation with 10 μM spiperone for 2 h, followed by incubation with the fluorescent ligand 10b (10 nM). Glass slides were mounted into a custom-built imaging chamber (500 µL) and washed with imaging buffer  twice, before the imaging chamber was placed on the microscope stage. TIRF imaging was performed at 24.0 ± 0.3 °C on a Nikon TI-Eclipse inverted microscope equipped with a 100x, 1.49 NA oil-immersion objective. For excitation, a Nikon D-Eclipse C1 laser box (561 nm), a 561/14 nm excitation filter and a dichroic long-pass mirror (561 nm) were used. Emitted light was passed through an emission filter 609/54 nm (Semrock Rochester) and projected onto a water-cooled EM-CCD camera at -98 °C . To ensure homogenous illumination, only the central quarter of the chip (300 × 300 pixel) was used for analysis. Microscope control and image acquisition were performed with the NIS Elements software (Nikon Instruments).TIRF microscopy was performed as previously described8a) or after dilution to 10 µM in PBS (10a-c). Emission spectra were collected employing an excitation wavelength of 335/10, 470/10 or 520/10 nm, respectively. Spectra were background corrected and normalized to the maximum absorbance/emission of each sample.Absorption spectra were recorded on a CLARIOstar  microplate reader. 1 mM solutions of the ligands were prepared in DMSO and measured either directly  and D3R  were amplified by polymerase chain reaction and fused in frame with a 4 AA linker (GSSG) by Gibson Assembly62 (New England Biolabs). To achieve surface expression, the fusion protein was either N-terminally tagged with an HA-signal sequence and a FLAG-tag50 or the secretory version of the enzyme19 (pNLF1-secN) was used. Sequence integrity was verified by DNA sequencing (Eurofins Genomics).For the generation of Nluc-D3R plasmids using polyethyleneimine (PEI) as the transfection reagent (PEI/DNA ratio 3:1). After incubation in DMEM/F12 with 10% FBS at 37 °C, 5% CO2 for 48 h, cells were harvested and cell membranes were prepared as described previously34. The protein concentration was determined using the method of Lowry and bovine serum albumin as standard63. Membranes (protein concentration 5–20 µg·mL−1) were incubated with the radioligand [3H]spiperone  for 1 h at 37 °C in binding buffer . Non-specific binding was determined in the presence of 10 µM haloperidol. Reactions were terminated by filtration through GF/B filters soaked with 0.3% PEI solution. Dried filters were sealed with scintillation wax and bound radioactivity was determined with a Microbeta Counter (Perkin Elmer). Data were analyzed with the one-site saturation binding model implemented in PRISM8.0  to determine the equilibrium dissociation constant (KD) and the receptor expression level (Bmax).HEK293T cells were grown to a confluence of 70–80% and transfected with the Nluc-D3R employing Mirus TransIT-293 (3:1 reagent to DNA ratio). After 24 h at 37 °C, 5% CO2, cells were detached using Versene and transferred to white, F-bottom 384-well plates at a density of 2,500 cells/well and incubated for another 24 h at 37 °C, 5% CO2. Cells were washed with PBS and incubated for 30 min at 37 °C with 5% FBS in DMEM without phenol red, before the fluorescent ligands  were added. Non-specific binding was determined in the presence of 10 µM haloperidol. After 1 h incubation with the ligands at 37 °C, furimazine  was added, followed by a 5 min incubation at room temperature in the dark. BRET was measured as the ratio of acceptor fluorescence and donor luminescence employing a CLARIOstar microplate reader equipped with 475/30 nm and 535/30 or 580/30 emission filters, respectively. Total, non-specific and specific binding were analyzed employing the algorithms for one-site saturation binding implemented in PRISM6.0 or 8.0.For whole cell NanoBRET saturation assays, HEK293T cells were transfected with secNluc-D3R membranes were resuspended in assay buffer20 , diluted to a total protein concentration of 0.5–3 μg/well in 384-well plates and incubated with the fluorescent ligands 10a or 10b (0–100 nM) at 37 °C for 90 min. Non-specific binding was determined in the presence of 10 μM haloperidol. After addition of furimazine , and 5 min incubation at room temperature in the dark, BRET readings were obtained using a CLARIOstar plate reader equipped with a 475/30 nm donor together with a 535/30 nm (for 10a) or 620/30 nm (for 10b) acceptor emission filter. Data were analyzed as described for live-cell NanoBRET. For association kinetics, secNluc-D3R membranes were added to various concentrations of 10a (1–100 nM) in the presence or absence of 10 μM haloperidol and furimazine . For dissociation, membranes were preincubated with 10a for 1 h, before furimazine was added and dissociation was initiated with 10 μM haloperidol. Kinetic assays were monitored at room temperature and minimal possible cycle time of the CLARIOstar was used for each measurement. Data was fitted to one phase association and dissociation equations in PRISM6.0 to determine the kinetic constants. In competition binding experiments, membranes were incubated with serial dilutions of unlabeled ligands and the fluorescent ligand 10a (10 or 100 nM) for 90 min at 37 °C. Addition of furimazine  and BRET measurements were carried out as described above. Data analysis was performed using the one site—fit Ki equation in PRISM6.0 to determine the inhibition constants (Ki) of the unlabeled ligands.SecNluc-Dwww.informatics.perkinelmer.com), PyMOL Molecular Graphics System, version 2.2.1 , NIS-Elements AR, version 3.22.13 , and GraphPad Prism version 8.0.0 for Windows, .Figures were prepared using ChemDraw, version 18.0.0 (Fig. Supplementary Information.