We perform an X-ray fragment screen on NSP14, revealing 72 hits bound to sites of inhibition in the ExoN and MTase domains. These fragments serve as excellent starting point tools for structure guided development of NSP14 inhibitors that may be used to treat COVID-19 and potentially other future viral threats.NSP14 is a dual function enzyme containing an N-terminal exonuclease domain (ExoN) and C-terminal Guanine-N7-methyltransferase (N7-MTase) domain. Both activities are essential for the viral life cycle and may be targeted for anti-viral therapeutics. NSP14 forms a complex with NSP10, and this interaction enhances the nuclease but not the methyltransferase activity. We have determined the structure of SARS-CoV-2 NSP14 in the absence of NSP10 to 1.7 Å resolution. Comparisons with NSP14/NSP10 complexes reveal significant conformational changes that occur within the NSP14 ExoN domain upon binding of NSP10, including helix to coil transitions that facilitate the formation of the ExoN active site and provide an explanation of the stimulation of nuclease activity by NSP10. We have determined the structure of NSP14 in complex with cap analogue  The global COVID-19 pandemic is caused by the SARS-CoV-2 virus and has infected over 200 million and caused over 4 million fatalities worldwide at the point of writing. Despite the successes with vaccines, there are currently a lack of effective drugs to treat people infected with SARS-CoV-2, and identification of such agents is a global priority. SARS-CoV-2 has a relatively large (∼30 kb) positive-sense single stranded RNA genome that consists of 12 functional open reading frames (ORF), encoding four structural proteins, six accessory proteins, and 16 non-structural proteins NSP1-16 (2) with the N-terminal region of NSP14 packing against two faces of NSP10. The metal active site of NSP14 lies close to the NSP10 interacting regions although a structure of NSP14 in the absence of NSP10 is not known and thus the molecular basis for the nuclease activity stimulation is not fully understood.The SARS-CoV-2 NSP14 is a bifunctional protein containing an N-terminal DEDDh-type exoribonuclease (ExoN) domain and a C-terminal SAM-dependent guanine-N7 methyltransferase (N7-MTase) domain . The ExoS-adenosyl methionine (SAM) dependent manner to form cap-0 structure (7MeGpppN) (7MeGpppN2′OMe) (in trans backtracking mechanism for proofreading.The NSP14 N7-MTase domain is located in the C-terminus of NSP14 (residues 288–527) and contributes an important methylation step in the formation of the viral 5′ RNA cap, which is essential for hijacking host translational machinery and suppressing innate immune responses. The viral 5′ RNA cap is formed by the sequential action of multiple enzymes that collectively assemble into a multi protein Replication and Transcription Complex (RTC). The helicase NSP13 is the RNA 5′ triphosphatase that cleaves the terminal triphosphate to a diphosphate . The SARMeGpppN) . The pro7MeGpppN product. NSP14 is one of the most conserved targets in the Coronavirus genome and thus is an excellent target for broad spectrum antivirals. We have used our crystals to perform a crystallographic fragment screen of NSP14 and find multiple fragments bound to sites of inhibition including the MTase and ExoN active sites and sites that may block the potential of NSP14 to interact with its partner NSP10. These fragments are important starting points for structure guided development or optimization of NSP14 inhibitors that may be useful as antiviral therapeutics with the potential for broad spectrum of action.In this study, we have determined the 1.7 Å crystal structure of SARS-CoV-2 NSP14 alone in the absence of its partner NSP10. Unexpectedly significant regions of the interaction interface retain some degree of folded structure. The predominantly hydrophobic interface, however, forms a collapsed self-association with significant rearrangement of the loops and residues that form the ExoN active site. The MTase domain also shows conformational differences when compared with previously solved NSP14/NSP10 structures. The MTase SAM binding loop occupies an alternative conformation that may be optimal for binding the 6 and Z-Basic tags at the N-terminal was synthesized in a pNIC-ZB vector with codon optimization for expression in Escherichia coli (E. coli. Cell cultures were grown in Terrific Broth media (Merck) supplemented with 20 μM zinc chloride at 37 °C, shaking at 180 rpm. Once OD600 reached 2.5, IPTG was added to the media at a final concentration of 300 μM. Cultures were incubated overnight at 18 °C, shaking at 180 rpm. Cells were harvested by centrifugation in a JLA-8.1000 rotor at 4400 × g for 25 min at 4°C.The plasmid for NSP14 with Hishia coli . The plaCells were re-suspended in lysis buffer  supplemented with protease inhibitor  and 1× benzonase nuclease. Cells were sonicated for 20 min . Cell debris was removed by centrifugation with Beckman JA-17 rotor at 39 000 × g at 4°C for 45 min. The supernatant was incubated with 7 ml pre-equilibrated Ni-IDA resin in tubes at 4°C for 2 h. After batch binding, the tubes were spun at 700 × g at 4 °C for 5 min and the supernatant was discarded. Resin was washed with 30 ml lysis buffer twice and transferred into a gravity column with 25 ml W20 buffer . Resin was washed again with 25 ml W30 buffer . Protein was eluted with 25 ml IMAC elution buffer  twice. Elution fractions were pooled and applied onto a pre-equilibrated 5 ml-HiTrap-SP High Performance column using a syringe. Column was washed with 50 ml SP wash buffer . Protein was eluted with 25 ml SP elution buffer . Elution fraction was diluted in 50 mM HEPES pH:7.5, 5% glycerol, 1 mM TCEP to reduce salt concentration to 500 mM before TEV cleavage. Diluted sample was treated with TEV protease (1:5 mass ratio) for 3.5 h at room temperature. To remove ZB tag, the cleavage sample was passed through a 5 ml-HiTrap-SP HP column and the flow-through was collected. The flow-through fraction was concentrated with 30 MWCO Amicon filters and loaded onto a HiLoad 16/600 Superdex 200 pg column pre-equilibrated with SEC buffer . Pure NSP14 fractions were collected concentrated and stored at –80°C for future experiments.E. coli (E. coli. Cell cultures were grown in Terrific Broth media (Merck) supplemented with 20 μM zinc chloride at 37 °C, shaking at 180 rpm. Once OD600 reached 2–3, IPTG was added to the media at a final concentration of 300 μM. Cultures were incubated overnight at 18 °C, shaking at 180 rpm. Cells were harvested by centrifugation at 4400 × g at 4°C for 25 min. Cells were re-suspended in lysis buffer  supplemented with protease inhibitor  and 1× benzonase nuclease. Cells were sonicated for 20 min . Cell debris was removed by centrifugation with Beckman JA-17 rotor at 39 000 × g at 4°C for 45 min. The supernatant was incubated with 10 ml pre-equilibrated GST resin in tubes at room temperature for 45 min. After batch binding, the tubes were spun at 700 × g at 4 °C for 10 min and the supernatant was discarded. Resin was washed with 25 ml lysis buffer thrice. Resin was transferred into a gravity column with 25 ml Elution buffer (pH 7.5) , incubated for 15 min at RT, and protein was eluted. Elution step was repeated twice. Elution fractions were pooled and treated with 3C-protease in 1:15 mass ratio, and dialysed in 50 mM HEPES pH: 7.5, 300 mM NaCl, 5% Glycerol, 1 mM TCEP overnight at 4°C. To remove GST tag and GST-tagged 3C-protease, the dialysed sample was passed through a gravity column with GST resin and the flow-through was collected. Resin was washed with 25 wash buffer  thrice. Fractions with cleaved protein were collected and concentrated with 10 MWCO Amicon filters. Sample was loaded onto a HiLoad 16/600 Superdex 75 pg column pre-equilibrated with SEC buffer . NSP10 fractions were collected concentrated and stored at –80°C.The plasmid for NSP10 with GST tag and 3C-protease cleavage site was synthesized in a pNIC-CTH0 vector with codon optimization for expression in E. coli . The plaet al., 2022). Vectors and sequences are available at: https://www.addgene.org/159613/ and  were prepared at a volume of 11 uL. NSP10 and NSP14 stock proteins were diluted to 1 mg/ml. NSP10/NSP14 stock was already prepared at 1 mg/ml during purification. 5 ul of NSP10, NSP14 or NSP10/NSP14 (at 1 mg/ml) were separately added to the individual tryptic digest strips in the presence and absence of trypsin. Samples were incubated for 1 h and overnight on ice. After incubation, 6 μl of samples were mixed with 45 ul 0.1% formic acid and subjected to intact mass analysis on electrospray ionization quadrupole time of flight (ESI-QTOF MS), and the rest of the samples were used for SDS-PAGE analysis.Assays were performed on Agilent 6530 RapidFire QTOF mass spectrometer in 384-well polypropylene plates  with ∼0.1 mg/ml protein samples in 0.1% formic acid. Samples were aspirated under vacuum and adsorbed onto a C4 solid-phase extraction (SPE) cartridge (Agilent Technologies), equilibrated and washed with LC-MS grade water containing 0.1% formic acid to remove buffer components. Following the aqueous wash, analytes were eluted from the C4 SPE onto an Agilent 6530 accurate mass Q-TOF in an organic elution step containing 85% acetonitrile and 0.1% formic acid. Masses of fragments were determined using the Agilent Masshunter Qualitative Analysis software (Agilent).7MeGpppG crystals, m7GP3G monomethylated cap analogue  was dissolved in 10 mM HEPES pH 7.5 at 50 mM to make a 10× stock solution which was diluted with crystallization buffer . Mature NSP14 crystals  were loop mounted and transferred to a drop containing cap analogue in 96-well plates and incubated overnight at 4°C. Crystals were cryo-protected in a solution consisting of well solution supplemented with 20% glycerol, loop-mounted, and flash-frozen in liquid nitrogen.Crystallization was performed by sitting-drop vapor-diffusion method with SGC-HIN3 HT-96 screen (Molecular Dimensions). Crystal plates were set up with 10 mg/ml protein and HIN3 screen, using 150 nl drops (1:1 ratio). Rod shaped crystals were grown in condition containing 1.26 M sodium phosphate monobasic, 0.14 M potassium phosphate dibasic at 4°C. Further crystals were grown in this single condition from Molecular Dimensions. For NSP14-All data were collected at Diamond light source beamlines I03 and I04-1 processed using XDS . Data weA total of 634 fragments from the DSI poised fragment library (500 mM stock concentration dissolved in DMSO) were transferred directly to NSP14 crystallization drops using an ECHO liquid handler , and soaked for 1–3 h before being loop mounted and flash cooled in liquid nitrogen. A total of 588 datasets were collected at Diamond light source beamline I04-1 and processed using the automated XChem Explorer pipeline . StructuE. coli as a bicistronic construct, or reconstituted from components in vitro, failed to produce crystals of diffraction quality. We explored N-terminal truncations of NSP14 as a way to increase stability of NSP14. We found a construct omitting the first 7 amino acids (residues 8–527) gave significantly improved purification yields. Crystallization of this NSP14 construct alone in a condition containing 1.26 M sodium phosphate monobasic and 0.14 M potassium phosphate dibasic produced rod shaped crystals that diffracted to 1.7 Å with moderately anisotropic diffraction. We solved the structure by molecular replacement using NSP14 from SARS-CoV NSP14/NSP10 complex   with distinct conformational differences observed for both the ExoN and MTase domains Figure . The mos) Figure .th strand   . However, the fold of this region is distinctly different and bears almost no relation to the complex structure Figure , C. In parallel) . This moarallel) . A long arallel) . The potnd 1 μM)  despite 2+) bound in an octahedral environment coordinated with D90, E92, D273 and three water molecules  are in the N-terminal region, and undergo disorder to order conformational changes upon interaction with NSP10. Thus the cleavage of these loops in solution is a sensitive measure of the conformational solution state. A third loop (residues 457–462) is in the MTase domain and does not change upon complex formation and serves as an internal control. As would be expected given its compact nature NSP10 is not cleaved significantly by trypsin during the time course of the experiment Figure . In contS-adenosyl methionine (SAM), and with S-adenosyl homocysteine (SAH) and cap analogue GpppA(5) show both substrates binding close together within the same large active site with the SAM/SAH binding and making interactions with the extended loop between second and third strands, whilst the methionine/homocysteine moiety approaches the GpppA in a manner that appears compatible with transfer of the methyl group to the Guanine N7.The MTase domain of NSP14 is located in the C-terminal half and consists of a central five stranded predominantly parallel β-sheet that is capped at its C-terminal end by and additional small three stranded antiparallel β-sheet (inserted between the fourth and fifth strands of the main β-sheet) that has been described as a ‘hinge’ domain , as it s7MeGpppG which gave clear electron density despite a slightly lower overall resolution  although large differences (displacements of up to 25 Å) can be seen in the conformation of the extended SAM/SAH interacting loop (residues 356–378). In our NSP14 structure, this loop makes several polar interactions with residues in the ExoN domain  and hinge domain (S577 to H427 and K359 to D415). The loop continues on the opposite side of the central β-sheet, in comparison to the SARS-CoV structures Figure . As was n Figure . The 7Mebstrates .7MeGpppG binds with the 7MeG in the product state with the 7MeG stacking between F426 and N386. The electron density map shows a clear bulge in the position of the methylated N7  is significantly less well ordered in the electron density map  that was found to adopt a different conformation than in previous NSP14/10 complex structures. Fragments at this site make polar contacts to E365, L366 and N375 with a subset of the fragments  inducing the ordering of an otherwise disordered loop (residues 370 to 374) within the crystals, allowing the loop to be modelled into good quality electron density. Whilst we note that a significant proportion of the pocket and interactions formed with these fragments are provided by residues in a crystal contact -RTC(14). This complex is comprised of NSP12, NSP7, NSP9, two copies of NSP8 and NSP13 and a single copy of the NSP14/NSP10 sub-complex. In this model NSP14/NSP10 primarily contacts NSP9 and the NiRAN domain of NSP12 via residues in the NSP14 ExoN domain. A subset 15%) of the particles picked from micrographs belong to a dimeric form which has additional contacts formed between the NSP14 MTase domain and the Zn-binding domain of NSP13 and the ‘fingers’ and ‘thumb’ region of the NSP12 polymerase % of the , B. OutsCrystallographic coordinates and structure factors for all structures have been deposited in the Protein Data Bank with the following accessing codes: 7QGI, 7QGF, 5KSW, 5SKX, 5SKY, 5SKZ, 5SL0, 5SL1, 5SL2, 5SL3, 5SL4, 5SL5, 5SL6, 5SL7, 5SL8, 5SL9, 5SLA, 5SLB, 5SLC, 5SLD, 5SLE, 5SLF, 5SLG, 5SLH, 5SLI, 5SLJ, 5SLK, 5SLL, 5SLM, 5SLN, 5SLO, 5SLP, 5SLQ, 5SLR, 5SLS, 5SLT, 5SLU, 5SLV, 5SLW, 5SLX, 5SLY, 5SLZ, 5SM0, 5SM1, 5SM2, 5SM3, 5SM4, 5SM5, 5SM6, 5SM7, 5SM8, 5SM9, 5SMA, 5SMB, 5SMC, 5SMD, 5SME, 5SMF, 5SMG, 5SMH, 5SMI.gkac1207_Supplemental_FilesClick here for additional data file.


Mouse monoclonal antibody M4M was recently designed to block human TRPM4 channel. The polypeptide for generating M4M is composed of peptide A1 between the transmembrane segment 5 (S5) and the pore, and a second peptide A2 between the pore and the transmembrane segment 6 (S6). Using peptide microarray, a 4-amino acid sequence EPGF within the A2 was identified to be the binding epitope for M4M. Substitution of EPGF with other amino acids greatly reduced binding affinity. Structural analysis of human TRPM4 structure indicates that EPGF is located externally to the channel pore. A1 is close to the EPGF binding epitope in space, albeit separated by a 37-amino acid peptide. Electrophysiological study reveals that M4M could block human TRPM4, but with no effect on rodent TRPM4 which shares a different amino acid sequence ERGS for the binding motif. Our results demonstrate that M4M is a specific inhibitor for human TRPM4. Recently, TRPM4 has been found to play a key role in many diseases. TRPM4 mutations are known to cause cardiac conduction problems such as right bundle-branch block, tachycardia, and Brugada syndrome2. In cancers from prostate, liver, urinary bladder, cervix, colon, and large B cell, aberrant TRPM4 activity affects cancer cell growth and migration3. In nervous system, TRPM4 plays an important role in neurovascular cell death in diseases such as multiple sclerosis4, stroke5, head injury6, and spinal cord injury7. Under ischemia, elevated cytosolic Ca2+ level and ATP depletion greatly enhance TRPM4 activity. TRPM4 expression was found upregulated in neurological diseases with hypoxic insult. In together with an increased channel activity, an excessive Na+ influx via TRPM4 channel causes oncotic cell death in neurons and vascular endothelial cells8.Transient receptor potential melastatin member 4 (TRPM4) is a voltage-dependent, non-selective monovalent cation channel9. Glibenclamide is a widely prescribed antidiabetic medicine for treating type 2 diabetes. It regulates blood glucose level by inhibiting KATP channels via interacting with SUR1 subunit in pancreatic beta-cells10. As TRPM4 forms a channel complex with SUR1, glibenclamide exhibits an inhibitory effect on TRPM4 when SUR1 and TRPM4 are co-expressed at a certain ratio11. Without SUR1, glibenclamide has no effect on TRPM4 activity12. During the past years, we seek to inhibit TRPM4 function by targeting TRPM4 directly. siRNA was first used to suppress TRPM4 expression in both in vitro and in vivo studies. Following stroke induction, application of TRPM4 siRNA could protect neurons and vascular endothelial cells. Accordingly, functional improvement was identified in both permanent and transient stroke models14. As siRNA acts on transcriptional level, TRPM4 siRNA needs to be applied at very early stage of disease onset, prior to TRPM4 protein upregulation. Considering the limitation of siRNA, we seek to develop a specific blocker, directly acting on the TRPM4 protein.Antidiabetic drug glibenclamide has been reported to inhibit TRPM4 function and is currently under investigation in clinical trials for stroke15. M4P was shown to reduce TRPM4 current under both normoxic and hypoxic conditions, protecting vascular endothelial cells and neurons from oncotic cell death following hypoxia16. Application of M4P ameliorated reperfusion injury in an animal model of stroke15. Based on the success of M4P, we further developed a monoclonal antibody M4M against human TRPM417. The polypeptide targeted by M4M is a 21-amino acid sequence between S5 and S6 of human TRPM4 channel. This polypeptide is composed of two small peptides separated by the hydrophobic channel pore18. It is unclear what is the binding epitope, and how the location of individual amino acid affects M4M action. In this study, we performed epitope mapping, amino acid substitution, structural analysis and electrophysiological study to examine the binding epitope for M4M, and the specificity of M4M to human TRPM4 channel.Around 12 years ago, we started to generate a polyclonal antibody M4P against a 28-amino acid polypeptide between the transmembrane segment 5 (S5) and 6 (S6) of rat TRPM4 channelMouse monoclonal antibody M4M was designed to target an extracellular polypeptide of human TRPM4 Fig. a. This p1SNCSSEPGFWAHP13 was further examined by substitution of individual amino acid for the 20 standard amino acids. The heat map, the substitution matrix and the amino acid plot  between the transmembrane segment 5 (S5) and the pore of the channels. When we started to develop TRPM4 blocking antibody22, we found that the E3 domain of TRPM4 is short and contains mostly hydrophobic amino acids. Therefore, a longer sequence covering the whole sequence linking S5 and S6 was taken into consideration for designing the antigen. The first TRPM4 blocking antibody that we developed is a polyclonal antibody against the rat TRPM4 channel15. Recently, we produced a monoclonal antibody M4M targeting a similar peptide of human TRPM417. M4M was revealed to bind to human TRPM4 on the surface of cell membrane. Electrophysiological study further proved that M4M could inhibit human TRPM4 activity.More than 20 years ago, a TRPC1 antibody has been developed by targeting an extracellular domain to inhibit the channel activityThe antigenic sequence for producing M4M contains an A1 peptide of 4-amino acid sequence RDSD, and an A2 peptide of 17-amino acid sequence SNCSSEPGFWAHPPGAQ from human TRPM4 channel  which is close to the channel pore of human TRPM4 was used for antibody production. Among the several clones produced, M4M was selected to own the best binding affinity and inhibitory effect on TRPM4 channel activity.The production of mouse monoclonal antibody M4M has been described previouslyPeptide microarrays were performed by PEPperPRINT . The sequence of peptide RDSDSNCSSEPGFWAHPPGAQ was elongated with neutral GSGSGSG linkers at the C- and N-terminus to avoid truncated peptides. The elongated peptide sequence was converted into 7, 10 and 13 amino acid peptides with peptide-peptide overlaps of 6, 9 and 12 amino acids. After on-chip peptide synthesis, all peptides were cyclized via a thioether linkage between a C-terminal cysteine and an appropriately modified N-terminus. The resulting conformational peptide microarrays contained 99 different peptides printed in duplicate (198 peptide spots) and were framed by additional HA  control peptides. The corresponding peptide microarrays were incubated with M4M at a concentration of 0.1 μg/ml in incubation buffer, followed by staining with the secondary goat anti-mouse IgG (H+L) DyLight680 antibody . The control antibody is mouse monoclonal anti-HA (12CA5) DyLight800 (0.2 µg/ml). Read-out was performed with an Innopsys InnoScan 710-IR Microarray Scanner at scanning gains of 50/10 (red/green). The additional HA control peptides framing the peptide microarrays were subsequently stained with the control antibody as internal quality control to confirm assay performance and peptide microarray integrity. Microarray image analysis was done with PepSlide Analyzer (Sicasys GmbH).https://arriveguidelines.org). The experimental protocol was approved by the Institutional Animal Care and Use Committees (IACUC No. A19107). All rats were housed in Animal Research Facility, Lee Kong Chian School of Medicine, Nanyang Technological University.Using CRISPR/Cas9 technology, we generated a transgenic rat model carrying human TRPM4 sequence. The binding motif, which is located between exon 19 and 20, was replaced with the corresponding human TRPM4 sequence (mutation sequence is from rat QDRSLPSILRRVFYRPYLQIFGQIPQEEMDVALMNPSNCSAERGSWAHPEGPV to human RDSDFPSILRRVFYRPYLQIFGQIPQEDMDVALMEHSNCSSEPGFWAHPPGAQ). The remaining sequence of TRPM4 is still of rat origin. Genotyping and DNA sequencing confirmed the presence of human TRPM4 sequence. All animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the National Neuroscience Institute and international ARRIVE guideline  pregnant transgenic rats were obtained and digested for 40 min in Earle's Balanced Salt Solution  containing 20 U/ml papain . Dissociated cells were seeded on 12 mm round glass coverslips coated with poly-L-lysine and laminin with neuron culture medium containing Neurobasal Medium, 2% B27 supplement, 1% GlutaMAX supplement . Medium was replaced 1 day after plating, and half of the medium was changed every 3 days. The cells were treated with 4 µM cytosine arabinoside from days in vitro (DIV) 3–6 to restrict mitotic cell proliferation and maintained for 10–21 days in neuron culture medium at 37 °C.3 and 10 mM 2-deoxyglucose to reduce glycolytic capacity and interfere with cellular respiration in energy metabolism.For acute oxygen–glucose deprivation (OGD) during patch clamp recording, the cells were perfused with an anoxic artificial cerebrospinal fluid (aCSF) containing 5 mM NaN2, 5% CO2 and 94% N2 for 5 min to purge the ambient air from the chamber. Following that, the hypoxia chamber was tightly sealed, and placed in a 37 °C incubator for 24 h.For 24-h OGD, the cells were grown in low glucose media  and placed in a polycarbonate hypoxia induction chamber . The chamber was first flushed with a gas mixture containing 1% OFollowing fixation with 4% paraformaldehyde, cells was incubated in blocking serum  for 1 h. Primary antibodies include M4M (0.4 µg/ml), anti-Myc , anti-WGA , IgG from human serum , anti-MAP2 . Secondary antibodies are conjugated with FITC or Alexa Fluor 594, Alexa Fluor 647.17 or mouse TRPM415 using lipofectamine 2000 transfection reagent . 24 h after transfection, whole-cell currents were recorded at room temperature using a patch clamp amplifier . Patch electrodes were pulled using a Flaming/Brown micropipette puller  and polished with a microforge . The bath solution contained (in millimole/litre) NaCl 140, CaCl2 2, KCl 2, MgCl2 1, glucose 20, and HEPES 20 at pH 7.4. The internal solution contained (in millimole/litre) CsCl 156, MgCl2 1, EGTA 10, and HEPES 10 at pH 7.2 adjusted with CsOH. Additional Ca2+ was added in the pipette solution to get 7.4 μM free Ca2+, calculated using WEBMAXC v2.10. Monoclonal antibody M4M, or mouse control IgG (subclass IgG1) was added into bath solution 30 min before recording at a concentration of 20.8 μg/ml. The current–voltage relations were measured by applying voltage ramps for 250 ms from − 80 to + 80 mV at a holding potential of 0 mV. The sampling rate was 20 kHz and the filter setting was 1 kHz. Data were analysed using pClamp10, version10.2 . Baseline currents were recorded on non-transfected HEK293 cells. Hypoxia was induced by applying a bath solution containing 5 mM NaN3 and 10 mM 2-deoxyglucose (2-DG) continuously through a MicroFil  around 10 μm away from the recording cells. The flow rate was set at 200 μL/min. Normoxic currents were recorded at 0 min and compared with hypoxic currents recorded 7 min after hypoxic incubation. Human and mouse TRPM4 currents can be obtained by subtracting baseline currents from non-transfected cells. Neuron recordings were performed in the presence of antagonists: 10 μM picrotoxin, 10 μM CNQX , 50 μM D-APV (D(-)-2-amino-5-phosphonovaleric acid), 1 μM TTX (Tetrodotoxin), 60 μM cadmium and 100 μM 4AP (4-aminopyridine).Whole-cell patch clamp was used to compare the effect of M4M on human and mouse TRPM4. In brief, HEK293 cells were transfected with 1 μg human TRPM41SNCSSEPGFWAHP13 was based on an exchange of all amino acid positions with the 20 main amino acids. Peptides further away from binding epitope EPGF were not included due to the peptide length limitation of the assay. After on-chip peptide synthesis, all peptides were cyclized via a thioether linkage between a C-terminal cysteine and an appropriately modified N-terminus. The corresponding peptide microarrays contained 260 different peptide variants and were framed by additional HA (YPYDVPDYAG) control peptides. Microarrays were incubated with the M4M antibody at a concentration of 1 μg/ml for 16 h at 4 °C and orbital shaking at 140 rpm. Subsequent staining with secondary goat anti-mouse (H+L) DyLight680 antibody (Thermo Fisher) was followed by readout with a Innopsys InnoScan 710-IR Microarray Scanner and quantification of spot intensities, as well as peptide annotation with PepSlide Analyzer (Sicasys GmbH).To investigate the proposed epitopes and the target specificity of M4M, Epitope Substitution Scans were performed by PEPperPrint GmbH. The substitution scan of wild-type peptide 19 were downloaded from the Protein Data Bank (PDB) (http://www.rcsb.org/)27. Channel pore radii for dots visualization were calculated using the program HOLE28 and a sample size of 0.225 Å. Electrostatic surface potentials were calculated with the APBS web suite29 using default parameters. Channel pores, electrostatic surfaces and protein structures were visualized using the PyMOL Molecular Graphics System, Version 1.8. 2015. Schrodinger, LLC.Coordinate files for the calcium-free (PDB ID: 6BQR) and calcium-bound human TRPM4 tetramers (PDB ID: 6BQV)P < 0.05 was considered significant.Data are expressed as the mean ± s.e.m. Statistical analyses were performed using GraphPad Prism version 6.0. Student’s t test was used to compare two means.


Clusiaceae plants contain a wide range of biologically active metabolites that have gotten a lot of interest in recent decades. The chemical compositions of these plants have been demonstrated to have positive effects on a variety of ailments. The species has been studied for over 70 years, and many bioactive compounds with antioxidant, anti-proliferative, and anti-inflammatory properties have been identified, including xanthones, polycyclic polyprenylated acylphloroglucinols (PPAPs), benzophenones, and biphenyls. Prenylated side chains have been discovered in many of these bioactive substances. To date, there have been numerous studies on PPAPs and xanthones, while no comprehensive review article on biphenyls from Clusiaceae has been published. The unique chemical architectures and growing biological importance of biphenyl compounds have triggered a flurry of research and interest in their isolation, biological evaluation, and mechanistic studies. In particular, the FDA-approved drugs such as sonidegib, tazemetostat, daclatasvir, sacubitril and trifarotene are closely related to their biphenyl-containing moiety. In this review, we summarize the progress and development in the chemistry and biological activity of biphenyls in Clusiaceae, providing an in-depth discussion of their structural diversity and medicinal potential. We also present a preliminary discussion of the biological effects with or without prenyl groups on the biphenyls. Clusia L., Garcinia L., Pentadesma Sabine and so on. Among them, Garcinia is a representative species comprising of about 400 recognized plants and is extensively dispersed throughout tropical and subtropical Asia, Africa, and America (2022). The chemical compositions of these plants contain a variety of valuable natural products with associated beneficial and healing properties such as anti-tumor  showed relatively good potency against the cells tested, with IC50 values below 10 μM. 3, 6, and 45 performed well in the anti-microbial test, with MIC values of roughly 20 μg/mL against corresponding bacteria. In terms of the capacity to inhibit tobacco mosaic virus, two-thirds of the compounds examined exhibited good activity, with inhibition rates greater than 20%, while the positive control ningnanmycin generally showed inhibition rates in the range of 30%–35% -3-methoxy-4.4′,5,6-tetraol (1)  and ferric reducing antioxidant power (FRAP) tests   (Rukacharaol (1) . The soundonesia . The n-hP) tests . In the  > 50 µM    . By utill lines.    , a constucuparia , also co species . Althougtifungal , anti-TMtifungal , anti-intifungal . Given t3 showed different levels of cytotoxicity against P-388 , HT-29 39 μg/mL ), XF498, > 30 µM ), KB, He> 100 µM ) cells (> 100 µM .3 (3a) and phenylboronic acid (3b) were used as the starting materials to make 3c, which underwent quantitative methylation by reacting with MeI and K2CO3 in acetonitrile to give trimethoxybiphenyl 3d. After selective demethylation, biphenyl 3 was accomplished in 82% yield.For the synthesis of oxygenated biphenyls, the tandem Michael addition reaction followed by aromatization reaction was developed by Chittimalla group. They successfully applied this methodology in the synthesis of 3    was produced from 2,6-dimethoxyphenol (4a) via the bromination of 4a (4b was reacted with (4-hydroxyphenyl) boronic acid (4c) in the presence of Pd/C catalyst in water to give 4 in 52% yield. In addition, this research reported that it could get access to diverse biphenyls via Suzuki−Miyaura cross coupling (yl C (4) , by way yl C (4) . Biphenyse cells     were obt in 2005 . The cytnd so on  (Ito et nd so on . Bipheny 0.36 µM .Garcinia nuntasaenii Ngerns. and Suddee attained the isolation of garcinuntabiphenyls A-C (7–9) (Garcinia nuntasaenii is distributed in northeastern Thailand and commonly named “Chang-nga-ek”. It is a dioecious shrub growing with white flowers and green fruits. Its roots could be used for relieving muscle pain in Thai folk medicine (7–9) against a panel of cultured mammalian cancer cell lines, including P-388, KB, HT-29, MCF-7, A549, ASK, were generally weak. Only the cytotoxicity of 8 to P-388 (IC50 = 37.87 µM) and KB (IC50 = 41.94 µM), as well as 9 to KB (IC50 = 39.04 µM) were detected and quantified  , which w-C (7–9) . Garcinimedicine . The cytantified .10) (Garcinia schomburgkiana Pierre and determined as [1.1′-biphenyl]-2-(3-hydroxy-3-methylbutyl)-3-methoxy-4.4′,5-triol  , was obt,5-triol . Its souoperties . Biphenythe test   (11) was screened for the ability to repress the growth of several leukemia cell lines containing Jurkat, NALM-6, K562 and HPB-ALL. It was found that biphenyl 11 showed limited cytotoxicity against NALM-6 cells, with cell viability ranging from 73% to 80% at the concentration of 50 µM (Investigating the EtOAc extract of the stems of l B (11) , as coloof 50 µM  (Ito et of 50 µM .12–13)  were examined with cytotoxic properties against a panel of cancer cell lines . The activity of 12–13 to restrain these tumor cells was not prominent. The IC50 value of 12 against KB cells was 89.05 ± 3.11 µM, and the IC50 value of 13 against P388 cells was 18.48 µM  , were isanalysis . Garcinind so on . Garcios18.48 µM  (Pailee 18.48 µM .14) and B (15)   from the02.52 μM    (Mixed forests of of China . To sear (16–18) , were exctively)  (Li et actively) .Garcinia mckeaniana Craib, the constituents belonging to the flower and twig extracts of this plant were investigated by Auranwiwat and others and a biphenyl (19) was found  was denominated as mckeaninabiphenyl and had been synthesized previously   were evaluated (20–22) were moderate against the five cells with the IC50 values ranging from 12.4 to > 40 μg/mL (20) could make compound 20 more active in inhibiting tumor cells.Yuan group systematically discovered and structurally characterized Garmultines A-C (20–22)  from Gar Bentham , which c Bentham  and othe Bentham . Compounvaluated . Their c40 μg/mL . It conc23) and multiflorabiphenyl D (24)  were evaluated with IC50 values > 10 µM   were obt > 10 µM    , were acnd HL-60 . It turnin vitro . Whereas40 μg/mL .29)  was measured for its cytotoxic effects against NB4, A549, SHSY5Y, PC-3 and MCF-7 tumor cells. The results showed 29 could modestly exert the repression toward tumor proliferation with IC50 values of 7.1, 6.2 and 4.8 µM against SHSY5Y, A549 and MCF-7 cells, respectively (The new biphenyl (29)  was obta Merrill . Garcini Vietnam . The pla Vietnam . The bipectively  (Liu et ectively .30) (Garcinia schomburgkiana at the same time with Schomburgbiphenyl B (11). Like compound 11, 30 was accessed for the cytotoxicity against a series of leukemia cell lines containing Jurkat, NALM-6, K562 and HPB-ALL cells. It (30) was not very effective to repress the proliferation of these tumor cells in study (Schomburgbiphenyl A (30)  was alsoin study  (Ito et in study .Biphenyls with bis-hydroxyl substituted at the C2 and C4 positions and their cytotoxic testing results are outlined in this section.31–33) . Compound 33 was observed to be inactive. Looking into their structures, the ring generated by cyclization involving the prenyl and hydroxyl groups distinguishes compounds 31 and 32. And compound 33 lacks the catechol and geranyl moiety in comparison to compounds 31 and 32. As a result, it is possible that the 3,3-dimethylallyl group and the hydroxyl group of compound 31 have no effect on DNA strand scission activity, because there was no significant difference in DNA strand scission activity between compounds 31 and 32. Furthermore, based on previous work by Wall and Wani’s group   were obtG. Mariz , which iG. Mariz . These tmodestly . The rep’s group , it is c31 was successfully finished in 2002 by Fukuyama’s team (31) since it is not entirely symmetrical. Beginning with O-dimethylphloroglusinol 31a, the preparation of left part was undertaken. After the protection, bromination, C-alkylation, removal of the TBDMS and subsequent triflation, the left part of 31g with a triflate group was provided quantitatively. Starting with the commercially available 3,4-dihydroxybenzaldehyde 31h, the protection of the catechol group, Baeyer–Villiger oxidation of the aldehyde, and reduction were performed to afford compound 31i. Then, the generated phenolic group was protected as an allyl ether, and subsequent bromination, deallylation, palladium-catalyzed Stille reaction yielded compound 31l. Next, it was successful to convert the bromide 31l into a pinacol boronic ester 31m using the Suzuki-Miyaura protocol. Following that, Suzuki coupling between 31g and 31m was carried out smoothly, yielding the coupling product 31n in 90% yield. At last, following the deprotection of all MOM groups and separation by HPLC, the desired biphenyl 31 was synthesized (The first synthesis of biphenyl a’s team . The desthesized .34) was found by Tip-pyang and others during the investigation of exploring Garcinia cylindrocarpa (34) cytotoxicity against KB, HeLa S3, MCF-7, Hep G2, or HT-29 cancer cell lines was assayed by MTT in vitro. But it was inactive to all tested cells (Cylindrobiphenyl A (drocarpa . Its (34ed cells  (Sukandaed cells .35–36)   were sep in 2004 . Compounectively  (Chen etectively .37)   was founen group . The ED5 therapy  (Chen et therapy .38) (24), was obtained from the leaves of Garcinia multifora. The cytotoxic activity of compound 38 against five human cancer cell lines  was evaluated, and the results showed that its IC50 values were all above 10 µM (Multiflorabiphenyl C (38) , an isomve 10 µM  (Fu et ave 10 µM .39) (Garcinia speciose by extensive spectroscopic methods in 2018 (39) was examined on a panel of cancer cell lines . In fact, biphenyl 39 cannot inhibit tumor cells effectively. The IC50 value of 39 against P388 cell was 32.36 µM. In addition, it was inactive against the other cell lines (Garciosine C (39)  was anot in 2018 . The cytll lines   had a better influence on the bioactivity compared to the hydroxyl substitution. From the structures and their cytotoxicity of compounds 7 and 8, 7 and 9 (6 (C10) or the R4 (C5) positions may improve the cytotoxic effect. Although the cytotoxic effects of compounds 14 and 15 were not greatly outstanding, it suggested that compound 14 was slightly superior to compound 15  were listed in the cytotoxicity section above. Except for compound 43  (Staphylococcus aureus (MRSA) with MIC values of 64 μg/mL . Meanwhile, penicillin-resistant S. aureus (PRSA) strains (MIC = 6.25 μg/mL) seemed to be more sensitive to 3 than penicillin-susceptible S. aureus (PSSA) strains (MIC = 12.5 μg/mL), which was advantageous for clinical drug development. The observation that anti-bactericidal activity of 3 functionalized in a dose-dependent manner was proved by investigating the kinetics of bactericidal activity against S. aureus at six concentrations of 3 . It was surprising that there was no regrowth of bacteria after monitoring for up to 24 h    exhibite64 μg/mL  Table 21    were recof China . CompounISA-Mu50 . As it ractivity .42 , a dimeric dihydrobenzopyran with highly symmetrical structure, was found and identified from the stembarks of Garcinia cowa -Garcnia cowa . The antanalyzed  and the 43)  and S. aureus (MIC = 50 μg/mL−1). However, it was weaker against Bacillus subtilis and Streptococcus mutans with MIC values at 100 μg/mL−1. Additionally, it had no effect on Gram-negative bacteria chromane (43)  was extrbacteria  (Ribeirobacteria .44–45) (4) from Garcinia linii by Chen group in 2006   were dis in 2006 . Regardiectively  (Chen etectively .S)-3-hydroxygarcibenzopyran (46)  ((ran (46)  was founia linii . It is a0 μg/mL)  (Chen et0 μg/mL) .15 (40 (15 and 40 were more active than compound 41 (para-position of the hydroxyl group (C1) or cyclized with the hydroxyl group rather than at the ortho-position of hydroxyl group (C5). Conclusions could also be obtained by comparing the activity of compounds 44 and 45, 44 and 46   , they we (14–15)  (Shang e (14–15) .47)   , found i, 33.5%)   .48–50)  showed the strongest activity among these biphenyls (48–50) with an inhibition rate of 31.1 ± 3.5% at the concentration of 20 μM, while the inhibition rates of 48 and 49 were 21.5 ± 2.6%, 22.8 ± 2.8% respectively  , were isttention . To evalectively  (Hu et aectively .16–18 (18 showed similar inhibition rates compared with ningnanmycin (30.2%). In addition, this study also tested the anti-TMV activity of biphenyl 25 (25 could inhibit tobacco mosaic virus moderately with the inhibition rate of 21.0 ± 2.5% at the concentration of 20 µM (51)   was anothenyl 47 , it was henyl 47  (Xu et ahenyl 47 .5 had higher anti-tobacco mosaic virus activity than compound 6 (5 (C9) position may be helpful to increase the anti-TMV activity. Since compound 3 and compound 5 exhibited similar activity, it presumed that the adjacent double hydroxyl group at C9 and C10 had no impact on the compounds’ potential to inhibit the tobacco mosaic virus. The results of the anti-TMV activity tests for compounds 14 and 15 were also in conflict with the results of the cytotoxic activity tests. Since biphenyl 15 was shown to have a higher rate of inhibition than biphenyl 14 in both studies  . It was evident that 52 and 53 were potential for medicinal development against rotavirus with TI values above 10  (is plant . Garcila (52–54) , which aactivity  of 52–54ctively) .55–57) were three new biphenyls isolated from Garcinia multiflora Champion ex Bentham  (Multibiphenyls A-C ( Bentham  (Gao et  Bentham . The dis (55–57) .58–59) -dihydrobenzofuran, which can be known as the first biphenyl substituted with 2-isopropenyl dihydrobenzofuran. Another one (59) was recorded as [1.1′-biphenyl]-3-methoxy-4.4′,5-triol following the extensive analysis of spectroscopy techniques. The compounds (58–59) were screened to figure out their anti-rotavirus activity in MA104 cells. Both of them exhibited good anti-rotavirus potential with SI values were above 10 (Two new biphenyls (58–59)  were yieabove 10   was 1.88 in the model of 1A2 cells infected by ΔTat/RevMC99 virus (50 was not determined in the anti-HIV-1 RT assay (Probing the anti-HIV ability of 4  was prac99 virus . HoweverRT assay .7–9)  , they prectively .12 and 13 (12–13) anti-HIV effects, the tests were also performed by utilizing syncytium inhibition assay and the RT assay. They all demonstrated activity in the syncytium inhibition assay proceeding with the ΔTat/RevMC99 virus and 1A2 cell line system, with SI values all above one and were 1.91, >4.76 respectively  SI value was above one and was 7.51 in the anti-syncytium test (39) was inactive in the HIV-1 RT assay as the inhibition rate at the concentration of 200 μg/ml was 7.84%. The IC50 value was not determined. (Biphenyl 39  was isolium test . Howeverermined. .9 (7 (4(C5) site of biphenyl 9 favored to enhance inhibition ability.It was clear from the aforementioned and 9  demonstr9  (ABTS) radical cation decolorization assay, the anti-oxidant property of biphenyls 20–22  and biph2  is a predecessor of other reactive oxygen species (ROS) and could be generated by activated neutrophils in response to stimuli (2− is a major cause for the development of inflammation and the scavenging of excess O2− is helpful for anti-inflammatory (3 (2− generation induced by chemotactic peptide N-Formyl-L-methionyl (Met)-L-leucyl (Leu)-l-phenylalanine (Phe) (fMLP). The results shown that 3 presented effective inhibitory activity (IC50 = 17.0 ± 2.8 μM) on O2− generation compared with positive control Ibuprofen (IC50 = 27.5 ± 3.5 μM) (3 were probed through measuring nitric oxide (NO) inhibition in lipopolysaccharide (LPS)-stimulated murine microglia BV2 cell and determining the secretion of nerve growth factor (NGF) in C6 glial cells, respectively , and the potency of 3 in offering more NGF for neuroprotection was moderate with NGF secretion levels of 125.76 ± 4.52% . In 2021, Kim and Yoon group presented a plausible mechanism of how 3 inhibited idiopathic pulmonary fibrosis (IPF), a lung disease generating lung scarring, in a bleomycin (BLM)-induced lung fibrosis mouse model. The research found out that 3 could depress the genes of inflammation and decrease macrophage activation marker genes, along with raising the expression level of anti-fibrotic markers in vivo. More significantly, it revealed that 3 could hamper the production of inflammatory cytokine-induced by transforming growth factor-β (TGF-β) from macrophages and the collagen synthesis from fibroblasts. These results demonstrated that 3 could play a protective role in lung fibrosis via its anti-inflammatory ability and it might be a promising therapy for IPF   . Furtherectively . It indi for IPF . Meanwhi IPF (15  could sl0.12 μM) .1 . However, when 10 μM of 31 was administered to neural cells, all neurons were killed during 6 days culture period. The morphology and viability of cortical neurons at a lower dosage of 0.1 μM were not affected by 31. It was suggested that biphenyl 31 with a biaryl moiety containing alkenyl groups, would have some beneficial effects on the growth and survival of neurons (A study examined the impact of biphenyl 31  on neuri neurons .60)   is a new effects .Garcinia kola Heckel, a plant that is native throughout West and West Central Africa, has been intensively studied for many years due to its edible seeds, usually known as bitter kola or male kola, and medicinal potential in the treatment of a variety of infectious disorders (61) from Garcinia Kola. (isorders . In 1994ia Kola.  (Niwa etia Kola. .62–63)  and bromo-malonic ester, followed by hydrolysis, and subsequent decarboxylation according to the literature  , from th in 1994 . 62 and  in 1997 . The benterature . Next, a3% yield .64)  (Garcinia mangostana L (mangosteen) is a tropical tree used as medicine in Southeast Asia over the past decades and has been well-known because of its fruit, which contains a great deal of polyphenolic xanthones exhibiting various biological effects in vitro (66) (Garcinia mangostana by Dharmaratne and co-workers (67) (Garcinia multiflora in 2013 (68) (69)  , a novel article . Furtherene (65)  (Teixeirene (65) . To the in vitro . The novtro (66)  was sepa-workers . Meanwhiers (67)  was foun in 2013 . And 3.4013 (68)  and 3,5-68) (69)  were extnse Warb .30–31) (70)  , clusiap31) (70)  was isol in 2002 .. The chemical structure and biological activity of 70 biphenyls were discussed in this review. Among these biphenyls, 52 compounds are found to present prenylated segment or mutative structures. Since the discovery of biphenyl 68–69 in Pentaphafangium Sofomonse, a total of 24 other Garcinia plants have been identified as sources of various biphenyls, in which Garcinia multiflora and Garcinia linii are the top two plants with the most biphenyls ascertained. The discovery of compounds 68–69 can be traced back to 1974 (3, which was obtained from other species (Garcinia.This review summarized the research progress of the biphenyls from Clusiaceae to 1974 . And com species , could b1–70 have been subjected to extensive bioactive studies, primarily involving cytotoxicity, antibacterial and antivirus activity. Compound 6, a derivative of chemical 3 with one additional hydroxyl and methoxyl substitution, showed especially potential in repressing the proliferation of P388 cell line with IC50 values of 0.36 μM (5, 16–18, 29, 31 and 37 also displayed promising cytotoxic effects, with IC50 values below 10 μM (3 is effective at inhibiting Bacillus subtilis with MIC values of 3.12 μg/mL (44 and 45 were tested for antimicrobial activity. The MIC values for 45 were nearly half of those for 44 (44 would have a positive influence on their antibacterial activity. Furthermore, the structure-activity relationship of compounds 16–18 based on the anti-tobacco mosaic virus activities (50 is the most promising compound to resist tobacco mosaic virus with the inhibition rates of 31.1 ± 3.5% (52 exhibiting great potential against rotavirus with TI values of 17.65 (Biphenyls  0.36 μM . Therefoow 10 μM . With re12 μg/mL . Both coe for 44 . The restivities  indicate1 ± 3.5% . Moreoveof 17.65 .12 had no cytotoxicity against the P-388 cell line, whereas compound 13 with a protected C2 hydroxyl group had moderate cytotoxicity. Similarly, compound 37 with a tetrahydropyran ring had better cytotoxicity against P-388 and HT-29 compared to compound 36 with same core structure. To avoid the metabolic deactivation, the free hydroxyl group on the phenyl may be replaced by its bio-isosterism in the drug design. In addition, biphenyls are known for atropisomerism and have been exploited in drug discovery. It should be noted that compounds 42 and 60 with bulky groups next to C6 and C7 may have this structural feature.Most of the biphenyls collected in this review have free hydroxyl groups, which may undergo metabolic deactivation by forming sulfates or glucuronides. This could be one of the reasons that some of the biphenyls in this review have moderate or no bioactivity. For example, compound 6, 50 and 52. Evaluation of the action mechanism of these promising biphenyls should be noted and endeavored in the future.Although biphenyl is a kind of compound with simple structure, it is worth to be studied because of its easy modification and diverse bioactivity. Several biphenyls from Clusiaceae do possess outstanding activity in multiple fields, such as compounds


By knocking down lncRNA expression in in vitro fertilization (IVF) embryos, we investigated its function and mechanism of regulating embryonic development. The results showed that the expression pattern of lncRNA was consistent with the time of gene activation. The lncRNAs were highly expressed in the 4-cell to blastocyst stage but barely expressed in the oocytes and 2-cell stage. So we speculated this part of lncRNAs may regulate gene expression. The lncRNA LOC102165808 (named lncT because the gene near this lncRNA is TFAP2C) was one of them. The knockdown (KD) of lncT inhibited embryonic development, resulting in decreased H3K4me3, H3K4me2, and H3K9me3, and increased DNA methylation. Meanwhile, RNAseq showed SIN3A was the top decreased gene in lncT-KD embryos. There was a severe blastocyst formation defect in SIN3A-KD embryos. Both lncT and SIN3A could affect NANOG and induce more cell apoptosis. In conclusion, the knockdown of lncT inhibits embryonic development by regulating H3K4me3, H3K4me2, DNA methylation, pluripotency gene, and apoptosis, and SIN3A is one of the downstream genes of lncT in regulating embryonic development.The study of preimplantation development is of great significance to reproductive biology and regenerative medicine. With the development of high-throughput deep sequencing technology, it has been found that lncRNAs play a very important role in the regulation of embryonic development. In this study, key lncRNAs that regulate embryonic development were screened by analyzing the expression pattern of lncRNAs in porcine  MALAT-1 plays a key role in the embryonic nervous system development of zebrafish. GET is essential for correct ZGA processes and further promotes the cleavage of two-cell embryos via regulating transcription and RNA alternative splicing in mice. In the goat embryos, the knockdown of the lncRNA TCONS_00460156 significantly decreases the developmental rate , under accession IDs: MII_1 (SRR17041081), MII_2 (SRR17041080), MII_3 (SRR17041074), IVV_2C_1 (SRR17041073), IVV_2C_2 (SRR17041072), IVV_2C_3 (SRR17041071), IVV_4C_1 (SRR17041070), IVV_4C _2 (SRR17041069), IVV_4C_3 (SRR17041068), IVV_8C_1 (SRR17041067), IVV _8C_2 (SRR17041079), IVV_8C_3 (SRR17041078), IVV_Bla_1 (SRR17041077), IVV_Bla_2 (SRR17041076), and IVV_Bla_3 (SRR17041075). LncRNAs were isolated according to non-coding annotations of pigs in the ALDB database ; the specific data are shown in http://www.webgestalt.org/option.php). The bubble plot of pathway enrichment was constructed by BioLadder (https://www.bioladder.cn/web/#/pro/cloud); the specific data are shown in The RNAseq data on IVV embryos we used are the data we have uploaded to NCBI previously , H3K4me3 , H3K9me3 , 5mC , 5hmC , DNMT1 , Alexa Fluor 488 goat anti-rabbit , and/or Alexa Fluor 488/594 goat anti-mouse  antibodies. All the chemicals used to culture were purchased from Sigma-Aldrich , unless otherwise stated.The pig ovaries we used were taken from the same local slaughterhouse ; the pigs are the cross of Landrace boar and Large White sow. The ovaries were transported to the laboratory within 2 h. They were kept in 0.9% NaCl supplemented with 200 IU/ml penicillin and streptomycin at 35–36.5°C.2, and 95% air. Then, they were transferred to a hormone-free maturation medium  for 20 h at 38.5°C, 5% CO2, and 95% air. Then, cumulus cells were removed from oocytes with a manipulation fluid supplemented with 0.2% hyaluronidase. The oocytes with polar body 1 (PB1) were considered matured and used for the following experiments.The follicular fluid containing cumulus–oocyte complexes (COCs) from 3–6 mm ovarian follicles was aspirated using an 18-gauge needle. COCs with at least three layers of cumulus cells were selected, washed three times in a manipulation fluid , and then cultured in the media of IVM. Approximately, every 200 COCs were cultured in a 1 ml drop of maturation medium  for 22–24 h at 38.5°C, 5% COlncT siRNA, SIN3A siRNA, or NC siRNA was injected into MⅡ oocytes, and then, the oocytes were incubated with siemens in PGM . The lncT siRNA, SIN3A siRNA, and NC siRNA were designed and synthesized by Sangon Biotech . The sequences were listed as follows:lncT: sense: CCAGAUGAGAUGGUGAUAATT,anti-sense: UUAUCACCAUCUCAUCUGGTT;SIN3A: sense: CCAAGUGAAGCUACAGUUUTT,anti-sense: AAACUGUAGCUUCACUUGGTT;NC: sense: UUCUCCGAACGUGUCACGUTT,anti-sense: ACGUGACACGUUCGGAGAATT.siRNA was injected using the microinjection meter . A measure of 5–10 pL of 25 nM 5—5.0×105 sperm/ml, at 38.5°C and 5% CO2 for 5–6 h. After washing off the adherent sperm, the fertilized oocytes were transferred to PZM-3 . Genes with false discovery rates (FDRs) ≤ 0.05, |log2FC| ≥ 1.5, and p-value ≤ 0.01 were selected as candidate genes, and the specific data are shown in The smart-seq2 method was used to amplify each sample (6–8 embryos for each group), according to the manufacturer’s instructions. The RNA concentration of library was measured by using a Qubit 2.0 Fluorometer . The Agilent Bioanalyzer 2100 system was used to assess the insert size, and the quality of the amplified products was evaluated according to the detection results. The amplified product cDNA was used as the input for the library construction of transcriptomes. After the library construction, the Agilent Bioanalyzer 2100 system was used to assess the insertion size, and the TaqMan fluorescence probe of an AB Step One Plus Real-Time PCR system  was used to quantify the accurate insertion size. Clustering of the index-coded samples was performed using a cBot cluster generation system and the HiSeq PE Cluster Kit v4-cBot-HS (Illumina). Then, the libraries were sequenced by Zhejiang Annoroad Biotechnology  on an Illumina platform, and 150-bp paired-end reads were generated. STAR was used to compare transcriptome data and Cufflinks for quantitative splicing. DEmRNAs were identified by the IDEP website  was synthesized with the SuperScript™ IV CellsDirect™ cDNA Synthesis kit , following the manufacturer’s instructions. The qPCR was performed with FastStart Essential DNA Green Master  CT−ΔΔ method. The qPCRs were all repeated three times.The results were analyzed by the 2A total of 5–8 embryos of IVF were washed with PBS containing 0.1% polyvinylpyrrolidone (PVP). The zona pellucida of embryos was dissolved in an acidic Tyrode solution (pH 2.5). After washing in PBS-PVP, embryos were fixed with 4% paraformaldehyde for 30 min in the dark. After being washed in PBS-PVP, embryos were permeabilized with 0.2% Triton X-100/PBS (v/v) for 20 min and then blocked with 2% BSA/PBS for 1 h. For 5mC/5hmC staining, the embryos were treated with 4N-HCl and Tris-HCl for 30 min each before BSA blocking. Embryos were incubated with primary antibodies at 4°C overnight. After being washed in PBS-PVP, embryos were stained with a secondary antibody at 37°C for 2 h in dark. Then, DNA was stained with 10 μg/ml DAPI for 15 min. All samples (DNMT1 excepted) were observed under a Nikon Eclipse Ti-U microscope equipped with appropriate filters  after mounting. Color images were captured using a DS-Ri2 CCD camera  and an analysis software application . The same exposure times and microscope settings were used for all captured images. Evaluation of the fluorescence intensity of individual images was performed by ImageJ software . The cytoplasmic background fluorescence intensity was measured as an average intensity level within the cytoplasmic area. Thereafter, the correction for the cytoplasmic background was carried out, and the background-subtracted images were used for further analysis . The DNMIn Situ Cell Death Detection Kit  at 37°C for 1 h in darkness. The blastocysts were washed with PBS-PVP three times and stained with DAPI (10 μg/ml) for 15 min. The stained blastocysts were mounted between a cover slip and a glass slide and observed under a fluorescence microscope , with 5–8 blastocysts per group.After the removal of the zona pellucida, fixation by 4% paraformaldehyde, permeabilization by 1% Triton-X 100, and blocked by 1% BSA, the blastocysts were incubated with a term deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) solution from the SIN3A KD embryo data between the two groups. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.Data are presented as the means ± SEMs. The experiments were repeated at least two times in triplicate. Statistical analysis was performed by GraphPad Prism 6.01 . A chi-squared test was performed to analyze the ratio of the developmental capacity and efficiency of the control and To explore the differentially expressed lncRNAs in IVV embryos, a heatmap was structured. As shown in lncT is 2038bp, and the whole sequence is shown in lncT was upregulated in 4-cell and 8-cell stages. Meanwhile, to explore the expression characteristics of lncT, we detected its expression in other organs, as shown in lncT, we knocked down the expression of lncT in embryos of IVF via the injection of small interfering RNAs (siRNAs) against it. As shown in lncT-KD group (13.65 ± 0.82%) was significantly decreased when compared to the control group (**p < 0.01). As expected, lncT was decreased in lncT-KD embryos compared to NC or the control group (***p < 0.001) (p < 0.01) (lncT inhibited porcine embryonic development.Since lncT (ALDBSSCT0000005403 in < 0.001) . Meanwhi < 0.01) . These dlncT in embryonic development, we collected embryos at the 4-8-cell stage, according to the arrest period of cleavage for transcriptome sequencing. As shown in lncT-KD group embryos , while KDM5C, ESC O 1, SETD2, and KDM5B were significantly upregulated (*p < 0.05) in lncT-KD embryos. Thus, we detected the expression of H3K4me3, H3K4me2, H3K9me3, and 5mC/5hmC during embryonic development. As shown in lncT siRNA injection was injected in 2-cell and 4-cell stages (*p < 0.05). Also, H3K4me2 had a similar expression trend after lncT siRNA injection (***p < 0.001)  (lncT-KD embryos (*p < 0.05)  . The exp < 0.01) . When we < 0.05) . Taken tlncT regulated in embryonic development, we evaluated the top changes in genes between the two groups. As shown in SIN3A was observed as the significantly downregulated gene after lncT siRNA injection (*p < 0.05) . Next, we tested if the embryos had a phenotype as lncT depletion by knocking down SIN3A. As shown in SIN3A (**p < 0.01). Meanwhile, we detected the expression of 5mC/5hmC, as shown in SIN3A (*p < 0.05); although there was no significant difference in 5mC at the blastocyst stage, the modification of 5hmC was decreased in the SIN3A-KD blastocyst (*p < 0.05). The previous study reported the KD of SIN3A-induced DNMT1 in the nucleus  and SIN3A (***p < 0.001)  . Thus, w nucleus . Thus, wKD group . In addi< 0.001) . Also, i< 0.001) . These rlncGET and lin28 are critical for mouse embryonic development beyond the 2-cell stage. Lnc-137 regulates zygotic genome activation by regulating histone methylation, which is essential for early embryonic development in goats  embryos . Many stlncT-KD embryos, which was different from the results in SCNT embryos, while the increase in the DNA methylation level certainly would be the barrier to zygotic genes and cellular reprogramming, which is consistent with the phenotype we observed.DNA methylation is associated with histone modifications, particularly the absence of histone H3 lysine 4 methylation (H3K4me0) and the presence of H3K9 methylation . A previSIN3A is a scaffold component of the chromatin repressive complex Sin3/histone deacetylase (HDAC)  . It is re (HDAC)  (Icardi e (HDAC) , while lof Hdac1 . Luo et  reports . MeanwhiSIN3A in regulating the pluripotent embryonic cell cycle, and MYC and E2F targets in SIN3A-null ICMs are downregulated (SIN3A in MEFs results in a profound growth defect, significant G2/M accumulation, and increased apoptosis (NANOG was decreased in both siRNA groups, suggesting the regulation function of lncT and SIN3A on NANOG. In addition, the number of apoptotic cells was increased, to some extent, in blastocysts after siRNA injection. Our results indicated that the knockdown of lncT and SIN3A affected embryo development via inhibiting the expression of NANOG and inducing apoptosis.The expression of pluripotency genes is crucial for the development of preimplantation embryos . A previegulated . Patrickpoptosis , while ZlncT is one of the key lncRNAs regulating embryonic development. It may affect embryonic development by regulating epigenetic modifications, such as H3K4me3, H3K4me2, H3K9me3, and DNA methylation modification. In addition, SIN3A is one of the key genes affected by lncT, and both of them can affect the pluripotency gene NANOG and induce more cell apoptosis. These findings fill in the deficiency of lncRNA research in porcine embryonic development and provide theoretical support for biomedical and animal husbandry production.Our results first demonstrate that


Changes in size, shape, and markers (CD206 and MHCII) documented during this interval align with the understanding that F4/80+ cells have different origins during embryonic life. CD3+ cells and F4/80−/MHCII+ were absent to rare until PND0. Ly6G+ cells were scarce at E13.5 but increased robustly by PND0 to represent half of the CD45+ cells. These immunofluorescence data were in accord with transcript analysis, which showed that immune marker mRNAs increased with testis age. F4/80+ and Ly6G+ cells were frequently inside cords adjacent to germ cells at E13.5 and E15.5. F4/80+ cells were often in clusters next to other immune cells. Macrophages inside cords at E13.5 and E15.5 (F4/80Hi/CD206+) were different from macrophages at PND0 (F4/80Dim/CD206−), indicating that they have distinct origins. This histological quantification coupled with transcript information identifies new cellular interactions for immune cells in fetal testis morphogenesis, and highlights new avenues for studies of their functional significance.Fetal testis growth involves cell influx and extensive remodeling. Immediately after sex determination in mouse, macrophages enable normal cord formation and removal of inappropriately positioned cells. This study provides new information about macrophages and other immune cells after cord formation in fetal testes, including their density, distribution, and close cellular contacts. C57BL6J mouse testes from embryonic day (E) 13.5 to birth , were examined using immunofluorescence, immunohistochemistry, and RT-qPCR to identify macrophages , T cells (CD3), granulocytes/neutrophils (Ly6G), and germ cells (DDX4). F4/80The online version contains supplementary material available at 10.1007/s00418-022-02129-6. SRY at embryonic day (E) 10.5 in Sertoli cells , is present on both immune and non-immune cells  can have either inflammatory or immune tolerogenic functions, depending on environmental cues . They are relatively common in the adult testis, representing approximately 25% of all immune cells in the adult rat testis . The frequency, localization, and close cell contacts of macrophages (F4/80+ cells), T cells (CD3+ cells), and neutrophils (Ly6G+ cells) were documented following immunofluorescence (IF) and immunohistochemistry (IHC), including in relationship to gonocytes (DDX4+ cells). This histological approach avoids the potential for under-reporting of cell types that can arise following tissue-dissociation for flow cytometry  Monash Animal Research Platform or at the SPF Monash Monash Medical Centre Animal Facility in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, with a 12-h light and 12-h dark cycle, with food and water available ad libitum. All investigations conformed to the National Health and Medical Research Council/Commonwealth Scientific and Industrial Research Organisation/Animal Advisory Committee Code of Practice for Care and Use of Animals for Experimental Purposes, and were approved by the Monash University Standing Committee on Ethics in Animal Experimentation. Timed matings of C57BL/6 J mice were performed, with the day of plug identified as embryonic day (E) 0.5. Pregnant mice were killed by cervical dislocation, and the embryos  were immediately removed from the uterus, killed, and the testes collected. Embryonic age was verified using forelimb and hindlimb development stages. At least four pups per age  were analyzed from more than one litter for each age. Testes from three individual C57BL/6 J mice were examined on PNDs 2, 7, 10, 15, 20, and adult to visualize Ly6GTwo different fixation and embedding approaches were employed. For IHC experiments, one testis from each mouse was immersed at room temperature (RT) in Bouin’s solution , followed by washing in 70% ethanol (3 × 1 h). Fixed tissues were processed, embedded in paraffin, and sequential sections of 5 μm thickness were collected onto Superfrost Plus microscope slides . For IF analyses, one testis from each mouse was fixed in 4% paraformaldehyde overnight at 4 °C, then cryoprotected in phosphate-buffered saline (PBS) first with 15% (w/v) sucrose overnight at 4 °C, then with 30% sucrose solution the following day. Tissues were embedded in Tissue Tek OCT compound  then stored at − 80 °C. Cryosections (5 μm) were placed onto Superfrost Plus microscope slides for immediate use or stored at − 80 °C.Each testis was entirely serially sectioned, with three sections placed on each slide. Four slides corresponding to the centre of the testis were examined with each antibody, ensuring that replicate sections for each antibody were spaced at 15–30 µm to prevent double-counting of individual cells. Section cross-sectional area was measured using ImageJ software ; this did not differ significantly between samples within each age group . Tissue embedding and sectioning were performed by staff at the Monash Health Research Precinct node of the Monash Histology Platform, Monash University.n = 3/age) were dissected away from the mesonephros, snap-frozen immediately on dry ice, and stored at – 80 °C.For RNA extraction, testes at each age  to detect all immune cells (anti-CD45), macrophages , T cells (anti-CD3), neutrophils (anti-Ly6G), laminin (anti-laminin), and germ cells (anti-DDX4). Adult mouse spleen was used as a positive control for antibodies to immune cell markers, and adult and newborn mouse testis were used for antibodies to laminin and germ cells. Each directly conjugated antibody bound as expected to cells in appropriate locations and with appropriate morphology . Negative controls for unconjugated primary antibodies lacked primary antibodies and were used to establish background binding of secondary antibodies.For IHC, sections were deparaffinized and rehydrated through a series of 5-min washes: twice each in histolene  and 100% ethanol, then once each in 95, 80, 70, 50, and 30% ethanol and deionised water. Heat‐induced antigen retrieval was performed by immersing slides in Tris–EDTA buffer , pH 9.0); the container with slides was microwaved for 4–5 min at 800 W, then 9 min at 450 W, followed by cooling at RT for 30 min. Sections were incubated for 20 min at RT with 3% hydrogen peroxide (Merck Millipore) to quench endogenous peroxidase, followed by washes with Tris-buffered saline . Sections were next incubated in blocking solution, consisting of 5% normal serum from the animal, in which the secondary antibody was raised (5% in TBS) in a humid chamber at RT for 1 h. Primary antibodies diluted in TBS with 1% bovine serum albumin  were applied overnight at 4 °C.Slides were washed three times, 3 min each, between incubations at RT using TBS. Controls for non‐specific secondary antibody binding in all experiments lacked primary antibody; these consistently showed no signal. Primary antibody binding was detected using a biotinylated secondary antibody in a humid chamber for 1 h at RT. After consecutive washes (3 × 3 min), Vectastain Elite ABC kit reagents were added  according to the manufacturer’s instructions. Antibody binding was detected as a brown precipitate following development with 3, 3‐diaminobenzidine tetrahydrochloride , then sections were counterstained with Harris hematoxylin (Sigma-Aldrich). Stained slides were then dehydrated through a graded ethanol series , plus twice in 100% ethanol and histosol (5 min). Finally, the slides were mounted under glass coverslips using DPX mounting media (Sigma‐Aldrich) and left flat overnight to air-dry before imaging.Slides for indirect and direct IF stored at − 80 °C were air-dried for 20 min at RT and rehydrated in PBS for 10 min. To permeabilize the sections, 0.1% Triton X-100 (Sigma-Aldrich) in PBS was applied for 5 min at RT. Slides were washed in PBS (×3), followed by a two-step blocking protocol in a humid chamber at RT. First, 5% Mouse on Mouse Blocking Reagent (Vector Laboratories) diluted in TBS was applied to sections for 30 min, then the slides were washed in PBS (3 min), and, finally, sections were exposed to 5% normal serum (from the species of secondary antibody origin)/2% BSA/PBS for 1 h. Blocking solution was replaced with unconjugated primary antibodies (for indirect IF) diluted in 2% BSA/0.1% Triton X-100/PBS, and the slides were incubated overnight in a humid chamber at 4 °C. Negative control sections lacked primary antibody. The next day, slides were washed once in 0.1% Triton X-100/PBS (3 min), twice in PBS (3 min each), and appropriate conjugated secondary antibodies were applied for 1 h at RT, with the slides protected from light from this point onwards. Secondary antibodies were removed, and the slides were washed as above. For direct immunofluorescence, after the blocking step, conjugated primary antibodies were added overnight at 4 °C, the slides were washed as above, and the sections were mounted under a glass coverslip using ProLong Gold Mountant  and stored at 4 °C until scanning for image analysis (within 24 h).Immunostained sections were scanned using an Olympus VS120 Slide Scanner  and analyzed using OlyVIA Software . The minimum detection level at which no fluorescence could be seen on the negative control was established as the baseline, and signals visible above this were considered genuine; detection levels were set identically for each section treated with the same primary and secondary antibody in an individual experiment to ensure consistency. Cells with a distinct signal evident at 6000 or higher on the intensity scale were labeled as ‘Hi’, cells with a signal between 3000 and 6000 were identified as ‘Int’, and a signal less than 3000 was recorded as ‘Dim’.2, measured using ImageJ software. Analysis was initially performed by blinded counting of three slides by two independent operators, with a variation of 5.3% for total macrophage numbers. The entire series was subsequently counted blinded, by a single operator.Immune cells were identified based on the presence of a well-defined nucleus visualized using DAPI . The position of each positive macrophage within an individual section was scored as being in the ‘perimeter’ area (the region between the capsule and cords) or ‘interior’ area (inside cords and interstitium) . Macrophage density was reported by adding the number of macrophages detected in the perimeter and interior areas of a section, divided by the section cross-sectional area (µm)Transcripts were measured by qRT-PCR in C57BL6J mouse testes at E13.5, E15.5, and PND0, from 3 independent samples per age. RNA extraction and DNase treatment using the RNeasy Mini Kit  was performed according to manufacturer's instructions. RNA purity and concentrations were quantified on a NanoDrop™ OneC . Complementary DNA (cDNA) was synthesized from 100 ng RNA with 500 ng oligo dTs , 50 ng random primers (Promega), and 200 units of SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) per sample, according to the enzyme manufacturer’s protocol. Primers designed to span exon–exon junctions in target genes  are listed in Supplementary Table S2. qRT-PCR was performed using SYBR-green and specific primer pairs  measuring each sample in triplicate; negative controls lacking Superscript III (− RT) were included for each sample. Runs conducted on Applied Biosystems QuantStudio 6 (Thermo Fisher Scientific) in the Genomics Centre, Monash Health Translation Precinct, Clayton, AU) used the following cycling conditions: denaturation at 95 °C for 10 min, followed by amplification for 40 cycles of 95 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s.The RPLP0 housekeeping gene transcript was used to normalize transcript loading. Data were analyzed using the standard curve method generated by 6 serial dilutions with 3 technical replicates using QuantStudio 6 software . Relative quantification of each transcript is presented as the quantity mean level. A single amplification product was detected for each target.t test with Welch’s correction were employed to analyze differences between age groups, as indicated. Values were considered significantly different if p < 0.05.GraphPad Software  was used for statistical analysis and graphing. A one-way ANOVA and Tukey’s multiple comparison post hoc and unpaired + cells), macrophages (F4/80+ cells), T cells (CD3+ cells), and granulocytes (Ly6G+ cells), including in relationship to male germ cells (DDX4+ cells) in sections of E13.5 and E15.5 and newborn mouse testes (PND0) . The predominant macrophage population at E13.5 consisted of relatively large and elongated cells, greater than 10 µm in diameter and featuring a round nucleus and intense F4/80 signal . A population of smaller (< 10 µm) macrophages displayed a lesser F4/80 signal; these were first detected at E15.5, and consisted of rounded cells with either a rounded or polymorphic nucleus . We speculated that these small, rounded F4/80Dim cells with either a band-shaped or segmented nucleus could be granulocytes.Testicular F4/80+ cell density increased 1.74-fold from E13.5 to PND0, while F4/80+ cells increased 1.34-fold, and Ly6G+ cells a remarkable 114-fold. No CD3+ cells (T cells) were detected in the E13.5 testis; they were rare at E15.5 (0–1/section), and more frequent at PND0 (10–20/section) . This matched the profile of the Cd3 transcript , Cd3 (encoding CD3), and Ly6g (encoding Ly6G) over this period of testis development . This difference in the F4/80 marker level was aligned with qRT-PCR data; despite a significant increase in macrophage number from E13.5 to PND0, the Adgre1 transcript level (encoding F4/80) did not change  significantly decreased: from 90% at E13.5, to 84% at E15.5, and to 65% at PND0  of CD45+ cells in the newborn testis were granulocytes  and CD45+/Ly6G+ cells (55%) could be explained by a small proportion of small, rounded CD45+ cells which expressed both F4/80 and Ly6G markers.The density of each immune cell type per testis cross-section increased significantly as the testis grew, indicating a numerical increase over time Fig. A. CD45+ + cells (germ cells) and the levels of the Ddx4 transcript decreased from E13.5 to PND0  was a consistent observation, with 2–6 cells typically identified within an individual cluster, and between 2 and 6 clusters present in each section from the E15.5 and PND0 samples . However, the absolute number of CD206+ macrophages per section increased during this interval. All F4/80+ cells inside cords at E13.5 were CD206+ . The most common macrophage population in the perimeter area and in the mesonephros/epididymis of fetal and newborn testes was F4/80+/CD206+ . Cd206 transcript levels elevated modestly (1.9-fold) from E13.5 to PND0  increased 3.9-fold from E13.5 to PND0 , and significantly increased by PND0 (8–16 per section). H2-eb1 transcript (encoding MHCII) levels roughly doubled from each 13.5 to E15.5 and from E15.5 to PND0 (1.9-fold)  F4/80+/CD206+/MHCII+ cells at PND0  at E15.5 . In the newborn testis, however, CD3+ cells were present in the perimeter region, interstitium, and positioned adjacent to cords   and had increased substantially by E15.5 (27–67 per section) . This contrasts with the macrophage distribution, which shifts from the perimeter to the interior between E13.5 and PND0  and/or with other immune cells (CD45+ cells lacking either F4/80 or Ly6G), including as clusters adjacent to cords and/or in interstitial areas was also regularly observed  and the other directly conjugated , leading to our conclusion that two distinct Ly6G+ cell populations are present, one lacking F4/80 and one with a low level of this protein , highlighting changes in the relative contributions of macrophages (F4/80+), T cells (CD3+), and neutrophils (Ly6G+)  Fig. . While eHi macrophages detectable before E14.5 are known to be yolk sac-derived, while F4/80Int are fetal liver-derived and emerge by E16.5  at PND0 , because almost all macrophages at PND0 appeared to lack this marker. Most of the small number of MHCII+ cells in PND0 testes were F4/80−, indicating either that another antigen presenting molecule is present on testicular macrophages, or that antigen presentation through MHCII is not the function of T cell–macrophage interactions at PND0. The finding of T cells in the fetal testis, both adjacent to cords and in interstitial regions at PND0, suggests that, similar to adult macrophages  at E12.5  of mouse testis sections at E13.5, E15.5 and PND0. Each data point represents the average cross-sectional area of 4 sections from the testis of an individual animal, shown with mean and SD. B and C. Delineation of perimeter and internal areas of fetal mouse testis cross sections. Two different locations of F4/80+ cells are shown using (B) immunohistochemistry (brown stain) in PND0 testis and (C) immunofluorescence in E15.5 testis (macrophages (green), germ cells (yellow) and cord boundary (red)). Dotted lines denote the division between the section ‘perimeter’, between the testis capsule and edge of the outermost cord, and the section interior or ‘internal’ area. F4/80: pan-macrophage marker, DDX4: pan-germ cell marker, laminin: marks cord basement membrane. Insets in B and C show nuclear staining with hematoxylin and DAPI, respectively. Insets show lack of signal in negative control sections lacking primary antibodies. (JPG 516 KB)Supplementary file2 Supplementary Figure S2. Two distinct F4/80+ cell populations at E15.5, each exhibiting a distinctive overall size, nuclear shape and F4/80 IF signal level in E15.5 mouse testis sections. A1 and B1: Small rounded F4/80Dim cells. A2: Co-localisation of two small rounded F4/80+ cells with band shaped and rounded nuclei. A3 and A4: Small F4/80+ cells with segmented and band shaped nuclei. A5, B2 and B3: Large and elongated F4/80Hi. Cells within numbered white boxes in A and B are shown in corresponding images in A.1 - A.5 and B.1 - B.3. Marker colours are indicated on image. (JPG 603 KB)Supplementary file3 Supplementary Figure S3. A marked increase in CD3+ (T cell) abundance occurs between E15.5 and PND0 in the mouse testis. Arrows in A1 and A2 highlight the detection of a single CD3+ cell E15.5 testis sections from two individual animals. B1 and B2 illustrated detection of multiple CD3+ cells in two individual PND0 sections. CD3: pan-T cell marker, DAPI: nuclear stain. Insets at higher magnification show the size and shape of CD3+ cells. Marker colours are indicated on the figure. (JPG 821 KB)Supplementary file4 Supplementary Figure S4. A marked redistribution of macrophages (F4/80+) from the testis perimeter to the interior occurs from E13.5 to PND0. Dotted lines designate the border of the testis with the mesonephros or epididymis regions. The rete testis is designated between two dotted lines on the E15.5 section. Marker colours are indicated on figure. (JPG 477 KB)Supplementary file5 Supplementary Figure S5. F4/80+ cells inside E15.5 testis cords. 1, 2: adjacent to basement membrane, and 3: in the cord centre. Numbered white boxes on left hand low magnification image are enlarged on right hand side. Marker colours are indicated on image. (JPG 576 KB)Supplementary file6 Supplementary Figure S6. F4/80+/CD206+ interactions, shape and cell distribution patterns in PND0 mouse testis sections. A: Contact between two large and elongated F4/80Hi/CD206+ cells. B: An elongated F4/80+CD206+ cell. C: Large and elongated CD206+ macrophages are the predominant macrophage phenotype in the testis section perimeter. Marker colours are indicated beside each panel set. (JPG 264 KB)Supplementary file7 Supplementary Figure S7. F4/80+/CD206+ and F4/80-/MHC Class II+ cell distribution patterns in E15.5 and PND0 mouse testis sections. A, A′, A″: This section illustrates the significantly higher number of CD206+ macrophages in the mesonephros/epididymis compared to the testis at E15.5. B and B´: The distribution of F4/80+/CD206+ cells across the whole testis section at PND0. C1-3: A low number of small, rounded F4/80-/MHC Class II+ cells are widely distributed in the PND0 mouse testis. Each panel corresponds to an individual animal. C3. Numbered white boxes (1-3) in the low magnification image are shown at high magnification in the right-hand panels. Marker colours are indicated on the figure. (JPG 1161 KB)Supplementary file8 Supplementary Figure S8. Distribution pattern of Ly6G+ cells. A: This E13.5 testis section displayed a single Ly6G+ cell, with a band-shaped nucleus. B1 - B3: Asymmetric distribution of neutrophils in the testis interior at E15.5; each corresponds to an individual animal. A′, B3′: The white box on the low magnification image on the left is shown on the right in higher magnification. C and C′: Ly6G+ cells were frequently detected inside cords (white rectangle) and in the interstitium at PND0. Marker colours are shown above. (JPG 948 KB)′Supplementary file9 (DOCX 24 KB)Supplementary file10 (DOCX 14 KB)Below is the link to the electronic supplementary material.