Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that exploits moonlighting functions of its proteins to interfere with host cell functions. PE/PPE proteins utilize host inflammatory signaling and cell death pathways to promote pathogenesis. We report that M. tb PE6 protein (Rv0335c) is a secretory protein effector that interacts with innate immune toll-like receptor TLR4 on the macrophage cell surface and promotes activation of the canonical NFĸB signaling pathway to stimulate secretion of proinflammatory cytokines TNF-α, IL-12, and IL-6. Using mouse macrophage TLRs knockout cell lines, we demonstrate that PE6 induced secretion of proinflammatory cytokines dependent on TLR4 and adaptor Myd88. PE6 possesses nuclear and mitochondrial targeting sequences and displayed time-dependent differential localization into nucleus/nucleolus and mitochondria, and exhibited strong Nucleolin activation. PE6 strongly induces apoptosis via increased production of pro-apoptotic molecules Bax, Cytochrome C, and pcMyc. Mechanistic details revealed that PE6 activates Caspases 3 and 9 and induces endoplasmic reticulum-associated unfolded protein response pathways to induce apoptosis through increased production of ATF6, Chop, BIP, eIF2α, IRE1α, and Calnexin. Despite being a potent inducer of apoptosis, PE6 suppresses innate immune defense strategy autophagy by inducing inhibitory phosphorylation of autophagy initiating kinase ULK1. Inversely, PE6 induces activatory phosphorylation of autophagy master regulator MtorC1, which is reflected by lower conversion of autophagy markers LC3BI to LC3BII and increased accumulation of autophagy substrate p62 which is also dependent on innate immune receptor TLR4. The use of pharmacological agents, rapamycin and bafilomycin A1, confirms the inhibitory effect of PE6 on autophagy, evidenced by the reduced conversion of LC3BI to LC3BII and increased accumulation of p62 in the presence of rapamycin and bafilomycin A1. We also observed that PE6 binds DNA, which could have significant implications in virulence. Furthermore, our analyses reveal that PE6 efficiently binds iron to likely aid in intracellular survival. Recombinant Mycobacterium smegmatis (M. smegmatis) containing pe6 displayed robust growth in iron chelated media compared to vector alone transformed cells, which suggests a role of PE6 in iron acquisition. These findings unravel novel mechanisms exploited by PE6 protein to subdue host immunity, thereby providing insights relevant to a better understanding of host–pathogen interaction during M. tb infection. M. tb is the etiological agent of tuberculosis (TB) that remains the world’s most deadly infectious disease with the notorious distinction of being among the top ten causes of death worldwide. TB is a global pandemic, with the World Health Organization reporting 10 million infections and 1.4 million deaths in 2019 worldwide, with 21% (0.46 million) cases being drug-resistant TB  gene, family. These genes account for approximately 10% of the whole M. tb genome and are present preferentially in the pathogenic, slow-growing strains. Their name is derived from the conserved N-terminal amino acid sequence of Proline–Glutamic acid/Proline–Proline–Glutamic acid (PE/PPE) residues  TLR4, TLR2, TLR2 in combination with TLR1 or TLR6 and modulate downstream signaling cascade to generate and mount an effective immune response (via TLR2/ASK1/p38 or JNK-MAPK pathways (M. tb PE_PGRS47 has been shown to inhibit autophagy and Major Histocompatibility Complex II (MHC-II) antigen presentation. It thereby inhibits innate and adaptive immunity to prevent bacteria’s clearance by the host cell  as a starting material. E. coli and M. smegmatis strain and PCR primers used in the amplification of pe6 and PCR conditions are provided in pe6 cloned into pET-28a was used to express the recombinant protein in ClearColi® BL21(DE3), an endotoxin-free strain . The protein was purified as described earlier with minor modifications  affinity chromatography. The purified protein was dialyzed overnight against the dialysis buffer . The purity of the dialyzed protein was checked using SDS-PAGE and Western blot analysis  resources , USA and grown in DMEM media  supplemented with 10% Fetal Bovine Serum (Gibco) along with 1% antibiotic-antimycotic solution . Cells were grown in standard tissue culture conditions at 37°C in a 5% CO2 incubator.M. smegmatis  infected macrophage cells were performed from the supernatant collected after 24 and 48 h post-infection. Three million macrophage cells were seeded in 6-well culture plates and left for adherence overnight. Cells were infected with M. smegmatis containing pe6 and vector alone harboring cells for 24 and 48 h at Multiplicity of Infection) MOI 1:10). The supernatant was collected and stored at −80°C until used.RAW264.7, ΔTLR1, ΔTLR2, ΔTLR4, ΔMyd88, and ΔMyd88/Trif cells were seeded in a 12-well cell culture plate at a density of 1 million cells/well. Cells were kept at 37°C for 3 h for adherence. After adherence at the bottom of the culture plate, cells were treated with various concentration of the protein, i.e., heat-inactivated protein (HI) (2 µg/ml), 0, 0.5, 1 and 2 µg/ml for 24 h. Proteinase K  treated followed by the heat-inactivated protein at 2 µg/ml was used as a negative control. Lipopolysaccharide (LPS)  at 1 µg/ml was used as a positive control. Culture supernatant was collected and stored at −80°C until further use. Estimation of both proinflammatory  and anti-inflammatory cytokines (IL-10) were performed using a mouse ELISA kit provided by BD Biosciences . The detection limits of ELISA kits used for estimation of TNF-α, IL-6, IL-12, and IL-10 are 15.6, 15.6, 15.6, and 31.3 pg/ml. The protocol provided by the manufacturer was followed. Cytokine levels were determined by measuring absorbance at 450 nm using an ELISA plate reader. Cytokine estimation (TNF-α and IL-6) from recombinant RAW264.7 cells were seeded in 24-well plates at a density of 0.5 million cells/well. After 3 h of adherence, cells were treated with 5, 7.5, and 10 µg/ml of PE6 protein, and incubated for 24 h. Approximately 0.1 µM staurosporine  was used as a positive control. Approximately 20 µM pan-caspase inhibitor, ZVAD-FMK , was used as a negative control. Annexin V/PI staining was performed using a BD FACS apoptosis staining kit as per manufacturer protocol. Surface expression of TLR4 on Phorbol 12-myristate 13-acetate (PMA) differentiated (20 ng/ml of PMA for three days) THP1 cells was determined using (0.5 million) cells treated with different concentrations of PE6 (1 and 2 µg/ml). Cells were seeded in a 24-well culture plate. After differentiation, cells were treated with PE6 and harvested after 24 h and incubated with anti-human PE-TLR4 antibody. The samples were processed as per protocol provided by the supplier . LPS (500 ng/ml) treated cells were used as a positive control for TLR4 expression. Fluorescence intensity was measured using the BD FACSVerse cytometer (BD Biosciences). Untreated and heat HI PE6 treated samples were taken as negative controls. The data were analyzed by Flow Jo software (Tree Star Inc. USA).The modeled PE6 was subjected to protein-protein docking with TLR4, PDB id:3vq2 using the ClusPro docking engine , 30. TheM. tb fractions. anti-Flag  and anti-His  antibodies were used to detect recombinant PE6.RAW264.7 cells and ΔTLR4 macrophage cells were seeded at a density of 3 million cells/well in a 6 well culture plate. Cells were treated with 1 and 2 µg/ml concentrations of PE6 protein. HI, protein-treated cells, and untreated cells were used as negative controls. After 24 h of treatment, cell culture media was removed, and cells were washed with 1× cold PBS . The cells were harvested in 2× SDS sample loading dye . Samples were heated at 95°C for 10 min, cooled, and pulsed centrifuged to remove cell debris. An equal amount of protein was loaded in SDS-PAGE gel and transferred onto the PVDF membrane. The transferred blot was blocked using BSA (5%) or skimmed milk (5%) in TBST and probed against NFκB1 (Cloud-Clone Corp), pP-65, , Caspase 3, Caspase 9, Bax, Cytochrome C, pcMyc, LC3BII, p62/SQSTM1, Beclin1, pULK1, MtorC1, Atf6, Chop, Calnexin, BIP, Eif2α, Ire1α, Nucleolin , GroEL2, Mpt32 , GAPDH and β-actin antibodies (Santa Cruz Biotechnology) as per manufacturer’s protocol. The anti-PE6 antibody was used to study the localization of PE6 in various Horse Radish peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit) were used for signal generation . In order to study the autophagy pathway, cells were treated with rapamycin (200 nM) and bafilomycin A1 (50 nM)  for 6 h along with protein treatment . Cells were harvested and processed, as described earlier, followed by Western blot analysis. BSA (5%) was used for blocking in the Western blot of phosphoproteins. Images were captured using the Bio-Rad-Chemi-Doc MP imaging system (Bio-Rad Laboratories India Pvt. Ltd.). The band intensity was quantified using ImageJ software and normalized to GAPDH or β-actin.Purified PE6 protein was used as an antigen to generate an anti-PE6 antibody. Approximately 500 µg of purified protein was mixed with an equal volume of Freund’s incomplete adjuvant (500 µl each) and emulsified. Produced protein adjuvant emulsion was used for injection into 90–120 days old New Zealand white rabbit. Three booster doses were given at an interval of 21 days. After the final booster dose rabbit was bled, and serum was collected and stored at −80°C until used. The specificity of the anti-PE6 antibody was validated using Western blot analysis  and intracellular survival, RAW264.7 cells were lysed in sterile 0.01% SDS prepared in 1× PBS and plated on 7H11 agar plates . Colonies were counted after four days of incubation at 37°C. For Western blot analysis, cells were washed with 1× cold PBS and harvested in 2× SDS sample loading dye. An equal amount of protein was loaded in SDS-PAGE, and protein was transferred onto the PVDF membrane, blocked, and probed with desired antibodies.RAW264.7 cells were seeded at a density of 2 million cells/well in a 6-well culture plate and incubated overnight for adhesion. Log phase growing HEK293T and macrophage cells were grown on coverslips in a 24-well plate for microscopic analysis. HEK293T cells were transiently transfected with pEGFPN1-PE6 (GFP-PE6) and pEGFPN1 (GFP), using lipofectamine 3000 . After 24, 48, and 72 h post-transfection, cells were either stained with Mitotracker Deep Red FM  as per manufacturer’s protocol or with an anti-Nucleolin antibody . Cells were fixed with 4% formaldehyde in 1× PBS, followed by permeabilization with 0.2% Triton-X100 in PBS. For nucleolar localization of PE6, manufacturers protocol was followed for staining the cells with the anti-Nucleolin antibody. RAW264.7 cells were treated with PE6 protein at a concentration of 1 and 2 µg/ml. After 24 h of treatment, cells were fixed and permeabilized with chilled methanol for 5 min, followed by washing with 1× PBS. Coverslips were incubated with primary antibodies  as per the manufacturer’s protocol. For studying the surface localization of PE6 in RAW264.7 cells, PE6 protein was treated at 2 µg/ml, after 2 h of treatment, cells were fixed using 4% formaldehyde, washed, and treated with anti-PE6 antibody (1:500) for overnight at 4°C. For studying the effect of PE6 on autophagy using RAW264.7 cells, a similar immunofluorescence protocol was followed as described. Anti-rabbit LC3 antibody  was used to detect membranous punctate foci. DAPI  was used to stain the nucleus. Cells were washed and incubated with Alexa fluor 488 (A488) or A547 conjugated secondary antibodies for 2 h. The coverslips were mounted using Prolong anti-fade mounting agent , viewed at 63× using a Carl Zeiss fluorescence microscope and a Olympus FLUOVIEW FV1000 confocal laser scanning microscope. Images were analyzed using Axio-vision and FLUOVIEW FV1000 software. Spatiotemporal localization of PE6 at 24, 48, and 72 h were presented from 100 positively transfected cells from three independent experiments. Punctate foci representing LC3BII were analyzed using Axio-vision software  from 10 different fields and the data presented are representative count from 50 cells.HEK293T (2 million) cells were cultured in 6-well tissue culture plate transfected with pEGFP-N1 and pEGFP-N1-PE6 for 24 h, re-suspended in 750 µl of lysis buffer , lysed by sonication [three cycles  at an amplitude of 20%], centrifuged at 13,000 rpm for 30 min. The supernatant was pre-cleared by adding pre-immune rabbit serum, along with protein-A agarose beads. Approximately 750 μl of pre-cleared samples  was used in Co-IP. To each pre-cleared sample, 3 μl of anti-PE6 and 1:50 dilution of anti-Nucleolin antibodies were added, incubated on a rotating rocker overnight at 4°C. Next, 25 μl of the packed volume of protein-A agarose beads were added to each sample, incubated for an additional 2 h, centrifuged at 3,000 rpm for 1 min, the supernatant was discarded, and the beads were washed with lysis buffer (5 min each washing on rotating shaker at 4°C). Bound protein complexes were released by boiling the beads in 2× SDS sample loading buffer and then subjected to SDS-PAGE followed by Western blot analysis using the desired antibodies.M. smegmatis cells harboring pe6 and vector alone were grown in 7H9 media  supplemented with 10% OADC  till the O.D reached 0.6. The culture was then diluted to an O.D to 0.1. The media was supplemented with 150 µM 2,2’- Bipyridyl , used as an iron-chelating agent to deplete the iron. To supplement the iron 150 µM Fecl3 was added in the presence of BPS. The culture O.D was taken every 3 h till O.D reaches 1. Wild-type, pe6, and vector alone containing M. smegmatis cells were diluted to an O.D of 0.1. A 10-fold serial dilution was made and spotted onto a 7H11 agar plate containing no BPS, 150 µM BPS, and 150 µM BPS, or 150 µM BPS + 150 µM Fecl3. The growth pattern was observed after four days of incubation at 37°C.Recombinant https://ihg.gsf.de/ihg/mitoprot.html), TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) and PSORT II (https://psort.hgc.jp/form2.html). The presence of a nucleus targeting sequence in PE6 was analyzed using LocSigDB.Mitochondrial localization signal sequence in PE6 was predicted using MITOPROT . All experiments were performed in 10 mM Tris–HCl pH 7.4, 100 mM NaCl at 298 K in a Perkin Elmer LS55 spectrofluorometer. The excitation wavelength was set at 290 nm, while the emission spectrum was recorded between 300 and 450 nm. Buffer controls were recorded by titration of protein with buffer. The single site-specific binding module of GraphPad (v5.0) was used to determine Kd from fluorometric titrations data using the following equation:Full-length PE6 was modeled using i-Tasser . The reswhere Bmax is maximum binding upon addition of ligand X (dsDNA).To evaluate the thermal stability of PE6 in the presence of dsDNA, an intrinsic fluorescence-based thermal shift (FTS) assay was performed by monitoring change in fluorescence emission at 330 nm with gradual temperature ramping from 25 to 90°C (2°C step size). Excitation and emission slits were kept at 5/10. Before each reading, an equilibration time of 1 min was kept for both PE6 (4 μM) alone and 10 μM dsDNA. PE6 was pre-incubated with 10 μM of DNA for 30 min at 298K before experiments.Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons, two-way ANOVA with Sidak’s multiple comparisons, paired T-test in combination with Kruskal–Wallis post-test. The data were represented as the mean of replicates  for 24 h, and 48 h with recombinant M. smegmatis containing pe6 and vector alone transformed cells proinflammatory cytokines levels  were measured using ELISA. We observed that pe6 containing recombinant M. smegmatis induced enhanced secretion of TNF-α and IL-6  to the ΔTLR1, ΔTLR2, ΔTLR4, ΔMyd88, and ΔMyd88/Trif knockout cells and measured proinflammatory cytokines  levels using sandwich ELISA. We found that PE6 robustly induces the production of proinflammatory cytokines by ΔTLR2 and ΔTLR1 cells. In contrast, ΔTLR4, ΔMyd88, and ΔMyd88/Trif cells produced negligible amounts of these cytokines, indicating that PE6 interacts and activates TLR4 and downstream adaptor protein Myd88 for the production of proinflammatory cytokines (Pathogen-associated molecular patterns (PAMPs) are recognized by various TLRs present on the host cell surface. To further validate the innate host receptor involved in recognizing PE6 and the downstream signaling, we treated wild-type RAW264.7 cells and ΔTLR4 knockout macrophage cells with recombinant purified PE6 protein (2 µg/ml). After treatment, recombinant PE6 protein was fixed on the cell surface together with its interacting receptor. Its localization was observed using fluorescence microscopic analysis. We found that PE6 is localized on the cell surface in RAW264.7 macrophage cells, whereas no signal was observed in ΔTLR4 knockout cells indicating that PE6 interacts with TLR4 on the macrophage cell surface Table S3TLR4 receptor activation on the macrophages’ surface leads to the recruitment of Myd88 and activation of the NFκB pathway . NFκB isin-silico analysis revealed that PE6 harbors an N-terminal signal peptide (1–24 aa), a mitochondrial targeting sequence, and a nuclear localization signal (LocSig Database). PE6 also harbors the Spt5 C-terminal nonapeptide repeat binding Spt4 domain. Spt4 and Spt5 together control transcription elongation by RNA polymerase II  to hijack or subdue innate host defenses  (m) was determined for PE6 alone and dsDNA. Interestingly, during the thermal ramping of PE6 alone, we observed an increase in fluorescence intensity, whereas, in the presence of dsDNA, the characteristic sigmoidal curve was observed with a Tm of 56.2 ± 1.1°C  pathway plays a critical role in the induction of apoptosis. We accordingly investigated whether PE6-induced apoptosis is through the activation of the ER linked to the UPR pathway. RAW264.7 cells were infected with recombinant M. smegmatis to RAW264.7 cells and analyzed the activatory phosphorylation of MtorC1 and inhibitory phosphorylation of ULK1 using Western blot analysis. Infection with pe6 containing M. smegmatis induced the activatory phosphorylation of MtorC1 (Ser2448) and increased inhibitory phosphorylation of ULK1 (Ser757), whereas no change in the Beclin1 level was observed  and analyzed the levels of autophagy markers utilizing Western blot analysis. Cells treated with PE6 protein decreased the conversion of LC3BI to LC3BII and the increased accumulation of autophagy substrate p62  and downstream signaling emanating from TLRs , 56. NFĸM. tb. Apoptosis plays an essential role in defense against intracellular pathogens by preventing the release of intracellular pathogens and, thus, the spread of the M. tb infection  are potent inducers of apoptosis in macrophages compared to virulent strains such as H37Rv . New Zealand white female rabbit was maintained at Central Animal Facility of National Institute of Pathology as approved by the Institutional Animal Ethics Committee. After completing experimental procedures, the rabbit was returned to Central Animal Facility, AIIMS, for rehabilitation.Conceptualize the project: NE and SH. Data curation and analysis: NS, MS, NQ, JS, NE, and SH. Funding acquisition and investigation: NE and SH. Writing manuscript draft: NS, MS, NE, and SH. Manuscript review and editing: MS, JAS, NE, and SH. Project administration and supervision: NE and SH. All authors contributed to the article and approved the submitted version.JAS is funded by the Start-up Research Grant from UGC and DST-SERB. SH and NE are supported by DBT North-East Grants BT/PR23099/NER/95/632/2017 and BT/PR23155/NER/95/634/2017 by the Department of Biotechnology, Ministry of Science and Technology (MoS & T), Government of India (GoI).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.


Asf1 is a well-conserved histone chaperone that regulates multiple cellular processes in different species. Two paralogous genes, Asf1a and Asf1b exist in mammals, but their role during fertilization and early embryogenesis remains to be investigated further.We analyzed the dynamics of histone chaperone Asf1a and Asf1b in oocytes and pre-implantation embryos in mice by immunofluorescence and real-time quantitative PCR, and further investigated the role of Asf1a and Asf1b during fertilization and pre-implantation development by specific Morpholino oligos-mediated knock down approach.Immunofluorescence with specific antibodies revealed that both Asf1a and Asf1b were deposited in the nuclei of fully grown oocytes, accumulated abundantly in zygote and 2-cell embryonic nuclei, but turned low at 4-cell stage embryos. In contrast to the weak but definite nuclear deposition of Asf1a, Asf1b disappeared from embryonic nuclei at morula and blastocyst stages. The knockdown of Asf1a and Asf1b by specific Morpholino oligos revealed that Asf1a but not Asf1b was required for the histone H3.3 assembly in paternal pronucleus. However, knockdown of either Asf1a or Asf1b expression decreased developmental potential of pre-implantation embryos. Furthermore, while Asf1a KD severely reduced H3K56 acetylation level and the expression of Oct4 in blastocyst stage embryos, Asf1b KD almost eliminated nuclear accumulation of proliferating cell marker-PCNA in morula stage embryos. These results suggested that histone chaperone Asf1a and Asf1b play distinct roles during fertilization and pre-implantation development in mice.Our data suggested that both Asf1a and Asf1b are required for pre-implantation embryonic development. Asf1a regulates H3K56ac levels and Oct4 expression, while Asf1b safeguards pre-implantation embryo development by regulating cell proliferation. We also showed that Asf1a, but not Asf1b, was necessary for the assembly of histone H3.3 in paternal pronuclei after fertilization.The online version contains supplementary material available at 10.1186/s13072-021-00430-7. The basic structural unit of chromatin is the nucleosome, which consists of 146 bp DNA wrapped around a histone octamer containing two molecules each of histones H2A, H2B, H3, and H4 [Drosophila and human cells [Asf1 is a histone H3–H4 chaperone that is well conserved in eukaryotes . The hisan cells –13 and sIn most vertebrates, there are two distinct Asf1 paralogous genes, Asf1a and Asf1b , 14, 15.Drosophila oocytes leads to failure in decondensation of the male pronucleus after fertilization [In mammals, the sperm genome is packaged by protamine into a highly condensed chromatin. Soon after fertilization, the sperm is gradually de-condensed and protamine is replaced by maternal histone to form the male pronucleus , 23. Hislization . HoweverIn this study, we characterized dynamic changes in nuclear accumulation of histone chaperone Asf1a and Asf1b in mouse oocytes and early embryos. Moreover, taking advantage of Morpholino oligos to silence the expression of Asf1a or Asf1b, we showed that both Asf1a and Asf1b were required for pre-implantation embryonic development. Furthermore, while Asf1a is required for the deposition of histone H3.3 in the paternal genome after fertilization and regulates embryonic H3K56ac and Oct4 level, Asf1b mainly regulates cell proliferation during early embryogenesis.We first examined the dynamics of the histone chaperone Asf1a and Asf1b in oocytes and pre-implantation embryos using specific antibodies  provides better specificity, low toxicity and long-time efficiency –29, we uTo investigate the effect of Asf1a or Asf1b knockdown on pre-implantation development, zygotes (2 hpi) were microinjected with specific MO against Asf1a or Asf1b, respectively, and examined their development rates. In agreement with previous studies , knockdoIn order to verify that the decreased developmental potential was due to the knockdown effect of the two histone chaperones, we examined nuclear accumulation of Asf1a and Asf1b in 4-cell, morula and blastocyst stage embryos Fig. E, H. ImmDrosophila, is required for H3K56ac [It has been shown that the histone chaperone Asf1a in yeast and human, Asf1 in  H3K56ac , 13. H3K H3K56ac  and prom H3K56ac . However H3K56ac , H3K56acIt was reported that H3K56ac correlates positively with Oct4 binding on its target gene promoters, and their interaction promotes pluripotency both in human and mouse embryonic stem cells (ESC) , 37. In Previous studies have shown that Asf1b expression is abnormally high in cancer cells and closely related to cell proliferation –21, 38. Mammalian reproduction begins with the fusion of sperm and oocyte, followed by successive mitotic division to form a multicellular embryo. Epigenetic reprogramming is a key feature of this developmental process and nucleosome dynamic is critically required for efficient epigenetic reprogramming and normal development . Since hWe found that nuclear enrichment of both Asf1a and Asf1b were restricted to fully grown oocyte and early cleavage stage embryos Fig. H. IntereDrosophila embryos in which Asf1 does not localize to the decondensing sperm nucleus [Xenopus egg extracts prevents de novo nucleosome assembly on prepared mouse sperm nuclei [Drosophila results in limited and insufficient paternal nucleosome assembly [In contrast to  nucleus , both Asm nuclei  and sileassembly . Hence, assembly . Since Aassembly , 15, 48,assembly  and nuclassembly . Howeverassembly , represeassembly –52. Therassembly .H3K56ac is a core histone acetylation which has been indicated to play important role during early embryonic development . In agreAsf1b is related to meiosis onset of germ cells and loss of Asf1b reduces female reproductive capacity in mice . Asf1b aIn this study, we found that both Asf1a and Asf1b were abundantly accumulated in 1-cell and 2-cell nuclei, but decreased or disappeared in embryonic nuclei at morula and blastocyst stages. In addition, we suggested that Asf1a, but not Asf1b, was necessary for the assembly of histone H3.3 in paternal pronuclei after fertilization in mice and the knockdown of either Asf1a or Asf1b expression compromised developmental potential of pre-implantation embryos. Moreover, we provided further evidences that reduction of H3K56ac through Asf1a KD results in decreased Oct4 expression and accumulation of DNA damage in blastocyst stage embryos. In contrast, Asf1b mainly regulates cell proliferation in early embryos.nd polar body extrusion and clear parental pronuclei formation. Embryos were cultured in KSOM  covered by mineral oil under condition of 37 °C, 5% CO2, and saturated humidity. All studies referring to experimental animals were performed according to the experimental protocols and standards, and approved by the Institutional Animal Care and Use Committee at the Inner Mongolia University.ICR mice were purchased from the Vital River Laboratories , and maintained in a special pathogen-free facility at the Inner Mongolia University. The mice were kept in a constant light and temperature controlled environment , and had free access to sufficient chow and water. Female ICR mice aged 8 weeks were superovulated by intraperitoneal injection of 10 IU pregnant mare serum gonadotropin . 46–48 h after PMSG injection, each female mouse was injected with 10 IU human chorionic gonadotropin . Metaphase (M) II-stage oocytes were collected from the ampullae 14 h after HCG injection and treated with 1% hyaluronidase  to remove the surrounding cumulus cells. Sperm cells were obtained from the cauda epididymis of male ICR mice and placed in HTF for capacitation. Gametes were co-incubated in HTF for 2 h for in vitro fertilization. For assessment of embryonic development, the fertilized oocytes, 2-cell, 4-cell, morula and blastocyst stage embryos were elevated morphologically at 10 hpi, 24 hpi, 48 hpi, 72 hpi and 96 hpi under a dissection microscope, and the corresponding developmental ratio was determined relative to the number of fertilized oocytes confirmed by 2Specific Morpholino oligos (MO) against Asf1a (Asf1a-MO), Asf1b (Asf1b-MO) and Control Morpholino (Control-MO) were designed and synthesized by Gene Tools . All Morpholino oligos were diluted with enzyme-free water to the final concentration of 1 mM and stored at room temperature. The sequences of Asf1a, Asf1b and standard Control morpholino oligos are listed as below: Asf1a-MO, 5ʹ-GTG CAT CAG CCT AGA GTA ATT CAG A-3ʹ; Asf1b-MO, 5ʹ-ATT CAG CAC CGA CAC CTT GGC CAT C- 3ʹ; Control-MO, 5ʹ-CCT CTT ACC TCA GTT ACA ATT TAT A -3ʹ. Asf1a/b-MO and Control-MO were microinjected into the cytoplasm of fertilized oocytes at 2 hpi (hour post insemination) in a droplet of M2 medium using Transfer Man 4R micromanipulator and microinjector . The microinjected zygotes were cultured till blastocysts in KSOM medium .®Premix EX Taq TM . 12.5 μL reaction solution composed of 1 μL cDNA, 6.25 μL TB Green Premix TaqII (2×), 0.4 μL of each primer and 4.45 μL ddH2O was incubated in LightCycler480 real-time PCR system  and amplified using two-step conditions: 95 °C for 10 min; 50 cycles of 95 °C for 5 s and 60 °C for 20 s. All amplifications were done in biological triplicate with technical duplicate and the relative gene expression was normalized to embryo number (n = 20). Data were analyzed using LightCycler 96 SW 1.1 software and relative expression levels of the target genes were calculated by 2−ΔΔCT method. The primer sequences for the Asf1a and Asf1b are listed as below: Asf1a sense: 5′-AAT GCA GGA CTC ATC CCA GAT-3ʹ; Asf1a anti-sense: 5′-TTC TTG ACC TCG GTA GGT GC-3ʹ; Asf1b sense: 5′-CTC ATT CCT GAG ACG GAC GC-3ʹ; Asf1b anti-sense: 5′-TAG CCC ACA CGG ATG AAC TC-3′.20 fully grown GV oocytes or embryos collected at different developmental stages were dissolved in RNAiso Plus  and stored at – 80 °C till use. Total RNA was extracted using RNAiso Plus according to the manufacturer’s instructions. Total RNA was reversely transcribed to cDNA using PrimeScript™ RT Reagent Kit with gDNA Eraser . cDNA was used as template for quantitative PCR with TB Green Oocytes and embryos were fixed in PBS containing 4% paraformaldehyde (PFA) for 20 min at room temperature, and washed three times with 1% BSA/PBS. The cells were permeabilized for 20 min using 1% BSA/PBS, 0.5% Triton X-100 and blocked for 30 min using 1% BSA/PBS, 0.1% Triton X-100. Primary antibody staining was carried out in the same buffer at 4℃ overnight. The cells were subsequently washed three times in 1% BSA/PBS (5 min each wash), incubated with secondary antibodies (Molecular Probes) for 1 h at room temperature in dark. Then, washed in 1% BSA/PBS for 5 min and stained with 4,6-diamidimo-2-phenylindole (DAPI) for 10 min. The images were taken under confocal microscopes (Zeiss). Quantification of fluorescence intensity was carried out using Image J, ROI Manager tool. First, the cytoplasmic pixel value of the protein was subtracted from the nucleus pixel value of the protein and the cytoplasmic pixel value of the DNA was subtracted from the nucleus pixel value of the DNA. Then, the ratio between the nucleus pixel value of the target protein and the nucleus pixel value of DNA was calculated as the fluorescence intensity value. The straight tool of the Image J was used to measure the diameter of male pronucleus.Primary antibodies and their dilution: Asf1a (Protein tech) 1:400; Asf1b (Protein tech) 1:400; H3K56ac (Epigentek) 1:100; H3F3B (Abnova) 1:200; Oct4 (BD Biosciences) 1:200; PCNA (Santa Cruz) 1:200. Secondary antibodies and their dilution: anti-rabbit IgG Fab2 Alexa Fluor (R) 488 Molecular Probes  1:600; DyLight 594-conjugated AffiniPure Goat Anti-Rabbit IgG (Jackson IR) 1:100; Alexa Fluor 488-AffiniPure Goat Anti-Mouse IgG (H + L)(Jackson IR) 1:200.P < 0.05 was used to determine significant difference. * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001.Data were presented as mean ± SEM (standard error of the mean). Comparison of means was performed using the independent-samples Student’s t-test. In order to compare the differences of quantitative data between groups, normal distribution of data was verified and statistical analysis was carried out by analysis of variance (ANOVA). GraphPad Prism version 7  was used for data analysis. Additional file 1: Figure S1. Validation of the specificity of the Asf1a and Asf1b antibodies.  Representative IF staining of the Asf1a (A) and Asf1b (B) in the Asf1a-KD zygotes at 8 hpi.  Quantification of the fluorescence intensity of Asf1a  and Asf1b  in the Asf1a-KD zygotes.  Representative IF staining of the Asf1b (E) and Asf1a (F) in the Asf1b-KD zygotes at 8 hpi.  Quantification of the fluorescence intensity of Asf1b  and Asf1a  in the Asf1b-KD zygotes. Data were presented as mean ± SEM, and analyzed using Student’s t-test, **P < 0.01. Figure S2. Quantification of dynamic changes of Asf1a and Asf1b in GV oocytes and pre-implantation embryos.  Quantification of nuclear accumulation of Asf1a (A) and Aaf1b (B) in GV oocytes and pre-implantation embryos by IF stainging. Data were presented as mean ± SEM. Significant difference  was determined by the one-way ANOVA and Student’s t-test. Figure S3. mRNA expression of the Asf1a/b in 1-cell, 2-cell, 4-cell, morula and blastocyst stage embryos derived from published RNA-seq data. Figure S4. Knockdown of the Asf1a/b throughout the pre-implantation embryonic development.  Representative immunofluorescence staining of the Asf1a (A) and Asf1b (B) in the pre-implantation embryos developed from zygotes injected with control or KD Morpholino oligos.  Quantification of the knockdown efficiency for Asf1a (C) and Asf1b (D) in the pre-implantation embryos. Data were presented as mean ± SEM, and analyzed using One-way ANOVA, **P < 0.01, ***P < 0.001. Figure S5. Nuclear localization of histone H3.3 detected by specific antibody. Fully grown GV oocytes and in vitro fertilized zygotes were stained with specific antibody against histone H3.3. Zygotes were collected at 2, 4, 6, 8, and 10 hpi, respectively. DNA was stained with DAPI (blue); Scale bar, 20 µm. Figure S6. Quantification of nuclear H3K56ac accumulation in GV oocytes and pre-implantation embryos. Data presented as mean ± SEM. Significant difference  was determined by the one-way ANOVA and Student's t-test. Figure S7 Nuclear accumulation of Oct4 in the Asf1a or Asf1b knockdown embryos.  Immunofluorescence staining of Oct4 in morula (A) after knocking down Asf1a, and the quantification in morula . Immunofluorescence staining of Oct4 in morula (C) after knocking down Asf1b, and the quantification in morula .  Immunofluorescence staining of Oct4 in blastocyst (E) after knocking down Asf1b, and the quantification in blastocyst . Data were presented as mean ± SEM, and analyzed using Student’s t-test. Figure S8. Knockdown of Asf1a leads to increased γH2A.X in blastocyst stage embryos (A) Confocal images of γH2A.X staining at blastocyst stage. DNA was stained with DAPI (blue). Scale bar, 20 µm. (B) Quantification of the γH2A.X fluorescence intensity in blastocyst. . Data were presented as mean ± SEM, and analyzed using Student’s t-test, ***P < 0.001.


The Golgi matrix protein giantin is essential for normal skeletal formation. Here, Stevenson et al. show that it also has a role in fracture repair and is required for intracellular processing of the N-terminal propeptide of type I procollagen. Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.  The golgins are a family of coiled-coil domain proteins that extend out from the surface of the Golgi apparatus to tether transport vesicles and other Golgi membranes . The larO-glycosylation have been reported following knockout (KO) of the GOLGB1 gene encoding giantin in cells  and one pro-α2(I) chain (encoded by COL1A2). Structurally, each chain is made up of a helical domain, composed of Gly–X-Y repeats, flanked by globular N- and C-propeptide domains  chains  data, which show a reduction in COL1A1 mRNA in GOLGB1 KO human telomerase-immortalized retinal pigment epithelial (hTERT-RPE1) cells relative to WT . Imaging of tissue from four individuals of each genotype failed to show any consistent differences in fibril structure or abundance ; howeverGOLGB1 KO hTERT-RPE1 cell line as a more tractable system for studying intracellular events –GFP-COL1A1  antibody identified these as collagen fibrils, indicating that the GFP-tag is incorporated into the ECM . No GFP-In our reporter construct, GFP is inserted upstream of the N-propeptide cleavage site and is expected to be removed during procollagen processing . The preTo verify our interpretation of these observations with respect to the N-propeptide, we performed immunoblots using antibodies specifically targeting the N- and C-propeptide domains of pro-α1(I). C-propeptide cleavage was comparable in WT and KO cells , with a We also used the N-propeptide–specific antibody to immunolabel the ECM of WT and KO cells. While this antibody did not label the cell-derived matrix of WT cells, it did label fibrils in the KO cell matrix where it colocalized with the extracellular GFP signal . TogetheSecretion of unprocessed procollagen has been reported in the absence of Hsp47 ; howeverThe detection of free N-propeptide in WT cell lysates is consistent with intracellular cleavage. N-propeptide abundance was also sensitive to proteasomal and lysosomal inhibitors in these assays, consistent with an intracellular pool . It is, First, a cell surface trypsin digest was performed to see whether the N-propeptide is in fact at the plasma membrane. As expected, treatment of cell cultures with trypsin for increasing amounts of time resulted in the gradual degradation of EGFR, a known resident of the plasma membrane . Both HSSecreted proteins can be prevented from leaving the Golgi using a temperature block at 20°C. In our second assay, we therefore performed secretion assays at 20°C to see whether procollagen can still be processed if trapped in the ER and Golgi. This would localize cleavage activity to one of these compartments. To ensure we only assayed procollagen affected by the temperature block, we first flushed out all the preexisting procollagen by treating cells with ascorbate to encourage secretion and cycloheximide to prevent synthesis of new procollagen. This removed most of the N-propeptide from the cell layer . The cycGOLGB1 KO cells, we generated new KO cell lines in which to validate our findings. This time we targeted exon 13 of the GOLGB1 gene (instead of exon 7) and used the lentiCRISPRv2 system  did not detect any ADAMTS2 transcript, even in WT cells; however, ADAMTS3 and ADAMTS14 mRNA was present, and interestingly, their expression was down-regulated following giantin KO  type VII, a disease characterized by joint hypermobility, skin hyperextensibility, and dislocations  or skin fragility /pro-α2(I), respectively, directly impact N-propeptide cleavage regardless of cellular location or tissue environment. We propose that giantin acts more indirectly to regulate an intracellular processing pathway, which works in concert with other complementary mechanisms, such as extracellular cleavage, for coordinated and efficient processing under different conditions. This raises the intriguing possibility of specific pools of collagen that require intracellular processing for specific functions, such as fracture repair. It is also possible that the observed phenotypes have a different underlying mechanism. RNA-seq data showed that the expression of many matrix components is altered following loss of giantin , which mbsl077golgb1 (henceforth X3078golgb1) allele is described in X3078golgb1 allele was crossed with fish carrying the zf195tgTg(col1a1a:EGFP) (henceforth col1a1a:GFP) transgenic reporter of the 1.4-kb col1a1a promoter region  and 5 mM Hepes dissolved in Danieau’s buffer. Calcein green staining solution contained 40 µM calcein powder (Sigma-Aldrich) dissolved in Danieau’s buffer and was pH calibrated to pH 7.4. Live fish were immersed in either ARS or calcein green for 2 h, then immersed in fresh system water for 15 min before imaging to clear excess stain from the fish.Fluorescence intensity at fracture sites was measured using ImageJ; lines were drawn along the fracture callous length, and then the line width was expanded to encompass the whole fracture. The mean intensity was then measured across this area, before using the same region of interest to measure the mean intensity of adjacent, uninjured hemirays. Fracture intensity was then normalized against the healthy bone to account for differences in baseline expression caused by reporter integration number.wt/X3078;col1a1a:GFP+golgb1 and X3078/X3078;col1a1a:GFP+golgb1 fish were anesthetized in MS222 and placed in a petri dish with the head on a bed of tissue soaked in anesthetic and the tail splayed on the dish. With a fresh scalpel, the distal third of the caudal fin was amputated. After recovery in fresh water, the fish were then returned to their tanks for 7 d. The regenerated fin tissue was then amputated as above, cutting as close to the original dissection line as possible. The excised tissue was then cut in half to separate the immature tip of the fin and the more mature tissue. The tissue was fixed in 2.5% glutaraldehyde, 4% PFA, 0.05 M cacodylate, 1 mM MgSO4, and 1% sucrose for 4 h at room temperature with gentle agitation. Fins were washed 3× in 0.1 M cacodylate buffer and incubated in decalcification buffer  at 4°C for 2 wk, refreshing the buffer after 1 wk. Fins were then postfixed in a freshly made mixture of 2% osmium tetroxide and 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 1 h. Fix was rinsed off with 3× 5-min washes with water and then incubated with 1% tannic acid in 0.1 M cacodylate buffer for 2 h. Tissue was then washed again as before incubating in 2% osmium tetroxide in distilled water for 40 min at room temperature. After a further three washes in distilled water, tissue was stained with 1% uranyl acetate overnight before washing again. The fins were then subjected to a graded dehydration, incubating them in 50%, 70%, and 90% ethanol, then 100% ethanol four times for 10 min each. This was followed by a 15-min incubation in propylene oxide. Samples were gradually infiltrated with graded TAAB 812 premix kit–Hard-1 (TAAB) in acetone at room temperature using the following series: 25% TAAB overnight, 50% TAAB through the day, 75% TAAB overnight, and 100% TAAB for 6 h. Samples were then embedded in fresh 100% TAAB 812 Hard in a labeled mold at 60°C for 48 h. The more mature pieces of regenerated fins were then sectioned and imaged using an FEI Tecnai 12 BioTwin transmission electron microscope.For caudal fin lysates, two female and two male 8-mo-old X3078/X3078;col1a1a:GFPgolgb1 homozygotes were out-crossed with EEK strain WT fish to generate heterozygous larvae and also in-crossed for homozygotes. At 5 dpf, larvae were killed by overdose with MS222, and 20 larvae were transferred to an Eppendorf for lysis. Larvae were incubated in Ringers solution  for 30 min and then pipetted up and down to remove yolk. Tubes were centrifuged at 13,000 ×g for 5 min, the supernatant was discarded, and 100 µl RIPA buffer + 10% protease inhibitor cocktail was added per tube. Larvae were homogenized using a pellet pestle to agitate tissue for 1 min per sample and then left on ice for 30 min, mixing halfway by flicking the tube. Lysates were then centrifuged for 30 min at 4°C and 13,000 ×g. The supernatant was analyzed by SDS-PAGE, and the pellet was discarded.To collect larval lysates, wt//X3078−col1a1a:GFPgolgb and x3078/X3078;col1a1a:GFPgolgb1 were anesthetized, and the distal third of the caudal fin was removed as above for TEM and placed into 200 µl ice-cold PBS. After recovery in fresh water, the fish were returned to their tanks for 4 d. The fish were then culled by overdose in MS222, and the caudal fin was cut again, this time dissecting ∼2 mm proximal to the original cut site to collect the regenerated tissue plus a small amount of mature tissue for lysis.For caudal fin lysates, 13-mo-old g for 4 min at room temperature. Tubes were put on ice, the PBS was removed, and 100 µl RIPA buffer + 10% protease inhibitor cocktail was added per tube. Tissue was homogenized for 40 s with a pellet pestle and then incubated for 10 min at 4°C on a shaker. Samples were spun at 13,000 ×g for 10 min at 4°C, and the supernatant was collected for SDS-PAGE conducted as detailed below.The dissected fins were macerated in the PBS using scissors, then spun at 13,000 ×GOLGB1 KO hTERT-RPE1 cell line used is described in hTERT-RPE1 cells (American Type Culture Collection) were grown in DMEM-F12 (Life Technologies) supplemented with 10% decomplemented FCS (Gibco). Cell lines were not authenticated after purchase other than confirming the absence of mycoplasma contamination. The main GOLGB1 KO cell lines with mutations in exon 13 were generated using the lentiCRISPRv2 system . gRNA sequences were designed using Benchling (http://benchling.com) and inserted into the lentiCRISPRv2 construct as described in GOLGB1 KO by immunofluorescence and immunoblot. To identify the mutations, genomic DNA was extracted from each clone using the Purelink genomic DNA mini kit (Invitrogen), and the region targeted by the gRNAs was amplified by PCR . PCR products were cloned into the pGEM-T Easy vector according to the manufacturer’s instructions (Promega) and sequenced using predesigned primers against the T7 promoter (MWG Eurofins).New hTERT-RPE1 Stable cell lines were made by lentiviral transduction as described above using the Lenti X Packaging Single Shots  in combination with the lentiviral vector pLVXpuro-proSBP–GFP-COL1A1  described in Transient transfections were performed using Lipofectamine 2000 according to the manufacturer’s instructions using 2 µg DNA for a 35-mm well . Plasmid: Str-KDEL-IRES-ST-mCherry is described in The ALT-R CRISPR-Cas9 system from IDT was used to generate giantin KO MC3T3 cell lines. Guide RNA1 targeting exon 8 (KO1) was designed using Benchling (5′-GGAAAAGGTAGAACTCGAAG-3′), and gRNA2 (KO2) was ordered as a predesigned guide from IDT targeting exon 2 (5′-AATAATGGAATCCACGCAAG-3′ Mm.Cas9.GOLGB1.1.AB). The gRNAs and Cas9 RNPs were assembled and transfected in a 96-well plate using Cas9 working buffer , CRISPRMAX transfection reagent , and 20,000 cells/well according to the IDT published protocols. Cells were then expanded, and single cells were sorted into a 96-well dish to generate clones. Again, these were expanded, and giantin KO clones were identified by immunofluorescence and immunoblotting. From 275 cells seeded per gRNA, one KO was obtained per target. Genetic mutations were determined as for RPE1 cells using primers 5′-GCCTGCCTTCCTTCCTTTAC-3′ and 5′-GCGGAACCAAGCAACAATAC-3′ for exon 2 mutations and primers 5′-AGTGTTTCAGTGTGCTCCC-3′ and 5′-GTCTTCTCCATCTCTGTC-3′ for exon 8 mutations.Rabbit anti-COL1A1 , mouse anti-DIC74 , mouse anti-HSP47 , mouse anti-GM130 , rabbit anti-giantin , rabbit anti-giantin  before fixation with 4% PFA/PBS for 10 min, permeablization with 1% Triton X-100/PBS for 10 min, and blocking with 3% BSA/PBS for 30 min. Sequential incubations with primary and secondary antibodies diluted in block were performed for 1 h, washing in PBS in between. Nuclei were labeled with DAPI  for 3 min. Coverslips were mounted in MOWIOL 4–88 (Merck-Millipore). All steps were conducted at room temperature and in the dark after secondary antibody addition. When labeling for ECM, the Triton permeability step was excluded from the protocol.For widefield microscopy, an Olympus IX-71 inverted microscope was used with a 60× 1.42 NA oil-immersion lens, Exfo Excite xenon lamp illumination, and single-pass excitation, emission, and multipass dichroic (Semrock) filters. Images were captured on an Orca-ER charge-coupled device (Hamamatsu). The system was controlled using Volocity . Chromatic shifts in images were registration corrected using TetraSpeck fluorescent beads (Thermo Fisher Scientific). Fixed-cell imaging by confocal microscopy was performed using a Leica SP5II system at 1024 × 1024 x-y resolution with a 63× HCX PL APO CS oil immersion objective, photomultiplier tube detectors, and LAS X software. Tile scans were performed to capture larger areas of the cell-derived matrix using a Märzhäuser scanning stage. On both systems, image stacks were taken with Δz of 0.2 µm, and unless indicated, maximum projections are shown. Image processing was performed using ImageJ software .4OH and 0.5% Triton X-100 in PBS; 3 ml per 6-cm plate for 2 min). After three water washes, residual DNA was digested with 10 µg/ml DNase I (Roche) for 30 min at 37°C, and then extracts were washed again. The matrix was fixed in 4% PFA and stained as above.To prepare the cell-derived matrix, cells were grown for 3 d on coverslips until they reached confluence, and then 50 µg/ml L-ascorbic acid-2-phosphate (Sigma-Aldrich) was added to cultures. Cells were left for a further 7 d. To prepare samples, cells were washed in PBS and extracted using prewarmed extraction buffer  was added to the dish  to induce procollagen folding and synchronous release of the pro-SBP-GFP-COL1A1 from the ER hook. This is T = 0. Cells were then imaged live, taking a single confocal plane image every 20 s. Fluorophores were excited using a 65-mW Argon and a 20-mW solid-state yellow laser and detected using hybrid gallium arsenide phosphide detectors. Imaging conditions for movies were 1,024 × 1,024 x-y resolution, scanning speed 600 Hz, 2× zoom, three-line average, and imaging sequentially between frames.To image, growth media was replaced with 1 ml prewarmed Fluorobrite DMEM , and the dishes were mounted on a Leica SP8 confocal laser scanning microscope system with a 63× HC OL APO CS2 1.42 NA glycerol lens and an environmental chamber at 37°C with COTo immediately fix during live imaging, 2 ml 8% PFA was added to the 2 ml Fluorobrite already present at the desired time point. Cells were fixed for 10 min, and the above immunofluorescence protocol was applied.2. Medium was then collected, and the cell layer was rinsed with PBS, lysed in RIPA buffer  for 30 min rocking on ice and collected without scraping. Media and lysate fractions were spun at 13,000 ×g at 4°C for 10 min and the supernatants used for SDS-PAGE. For collection of lysates in other experiments, cells were scraped in RIPA buffer, collected in tubes, and incubated on a rotator at 4°C for 30 min. Bicinchoninic acid assays were performed where necessary to determine protein concentration according to the manufacturer’s instructions (Bio-Rad). Samples were boiled in 1× lithium dodecyl sulphate sample buffer  containing sample-reducing agent  for 10 min at 95°C and then analyzed by SDS-PAGE as below.To perform secretion assays, medium was aspirated from confluent cells and replaced with a minimal volume of serum-free DMEM-F12 supplemented with 50 µg/ml L-ascorbic acid-2-phosphate . Cells were left overnight at 37°C and 5% COFor inhibitor experiments, confluent cells were treated with 1 ml serum-free medium containing either DMSO (control), 10 µM MG132, or 200 nM bafilomycin overnight before media and lysate collection.g for 5 min to remove dead cells. The cell layer was washed twice with ice-cold PBS and lysed in ice-cold buffer  for 30 min with agitation on a rotator. Lysate supernatant was collected by centrifuging at 20,000 ×g for 10 min, and 66 µl was removed to be used as an “input” reference sample. Media and lysate supernatants were then incubated with equilibrated GFP nano-trap beads (Chromotek) on a rotator for 2 h. Beads were pelleted by centrifugation at 2000 ×g for 2 min. The supernatant was removed, but 66 µl was kept as an “unbound” reference sample. The beads were then washed three times with 500 µl dilution buffer , pelleting them each time by centrifugation at 2000 ×g for 2 min. All steps to this point were performed on ice/at 4°C. Finally, beads were resuspended in 88 µl 1× LDS sample buffer  containing sample reducing agent (Life Technologies) Inbound and unbound samples were mixed with 22 µl 4× LDS sample buffer + reducing agent (Life Technologies). Samples were boiled at 95°C for 10 min and analyzed by SDS-PAGE as outlined below.For GFP-trap immunoprecipitation, confluent cells were serum starved for 24 h in the presence of 50 µg/µl L-ascorbic acid-2-phosphate . At the time of assay, medium was collected and spun at 2,700 ×2 to stimulate procollagen trafficking and then incubated in PBS ± 30 µg/ml digitonin for 2 min at room temperature to permeabilize the cells. Cells were washed twice in PBS and twice in sucrose buffer  before incubating with 0.05% trypsin-EDTA for the indicated time at 37°C/5% CO2. As a control, one well was incubated with a 1:1 ratio of 0.05% trypsin and 2 mg.ml−1 soybean trypsin inhibitor . To end the digest, an equal volume of 2 mg/ml soybean trypsin inhibitor was added to the well and incubated for 10 min at room temperature. Dishes were scraped, and the cells were collected and spun at 2,000 ×g for 5 min at 4°C. The supernatant was removed, and the pellet was washed with 500 µl 2 mg/ml soybean trypsin inhibitor before repeating the spin and removing the supernatant. The cell pellet was lysed in 85 µl RIPA buffer containing proteinase inhibitor cocktail  for 30 min on rotator at 4°C. Lysates were spun at 13,000 ×g/4°C for 10 min, and the supernatant was collected for SDS-PAGE as below.Confluent RPE1 cells were treated with growth media + 50 µg/ml ascorbic acid for 1.5 h at 37°C/5% CO2 independent media + 50 µg/ml ascorbic acid overnight at either 20°C or 37°C/5% CO2. Cells were then fixed and stained or used in a secretion assay as above—including the control that lacked cycloheximide treatment.Confluent WT and giantin KO RPE1 cells stably expressing pro-SBP-GFP-COL1A1 were incubated with 50 µg/ml L-ascorbic acid-2-phosphate  ± 100 µg/ml cycloheximide for 16 h to promote secretion of existing procollagen and prevent synthesis of new procollagen, respectively, to remove preexisting protein. As a control to test the efficiency of the cycloheximide treatment, one cycloheximide-treated sample was collected after this incubation. Remaining cells were rinsed 3× with PBS and once with growth media before incubating with COSecretion assays, IP, and lysate samples were separated by SDS-PAGE on precast 3–8% tris-acetate and 4–12% bis-tris acrylamide gels (Invitrogen) followed by transfer to 0.2 µm nitrocellulose membranes (Amersham). Membranes were blocked in 5% milk 0.05% Tween (Sigma-Aldrich) in Tris-buffered saline for at least 1 h. Primary and HRP-conjugated secondary antibodies were diluted in block and sequentially incubated with the membrane for 2–16 h each. HRP was detected by enhanced chemiluminescence .n numbers, and sample sizes are indicated in the figure legends; P values where significant (P < 0.05) are shown on the figures. All experiments were analyzed with nonparametric tests as data were assumed not to be normally distributed. Sample sizes were chosen based on previous similar experimental outcomes. No samples were excluded.Measurements of images and immunoblots were performed using ImageJ. Statistical analyses were performed using GraphPad Prism 7.00. The tests used, golgb1 HET and HOM zebrafish. Review HistoryClick here for additional data file.


Chronic lymphocytic leukemia (CLL) is considered a clonal B cell malignancy. Sporadically, CLL cases with multiple productive heavy and light-chain rearrangements were detected, thus leading to a bi- or oligoclonal CLL disease with leukemic cells originating either from different B cells or otherwise descending from secondary immunoglobulin rearrangement events. This suggests a potential role of clonal hematopoiesis or germline predisposition in these cases. During the screening of 75 CLL cases for kappa and lambda light-chain rearrangements, we could detect a single case with CLL cells expressing two distinct kappa and lambda light chains paired with two separate immunoglobulin heavy-chain variable regions. Furthermore, this patient also developed a prostate carcinoma. Targeted genome sequencing of highly purified light-chain specific CLL clones from this patient and from the prostate carcinoma revealed the presence of a rare germline polymorphism in the POLE gene. Hence, our data suggest that the detected SNP may predispose for cancer, particularly for CLL. Chronic lymphocytic leukemia (CLL) is a very frequent B cell malignancy of the elderly characterized by the accumulation of mature CD5/CD19 double-positive B cells in peripheral blood and lymphoid organs. Although novel therapies have dramatically improved the treatment outcome of patients, CLL is still considered incurable . TypicalFrom the initially screened cohort of 75 CLL patients , we deteOur analysis revealed that the kappa and lambda clones in this sample had different IGHV gene usage, with the kappa clone being IGHV 3-7*03 and the lambda clone being IGHV 3-74*03 and distinct IGHD, IGHJ, and CDR3 sequences. The IGHV chains had a sequence homology to the germline IGHV sequences of 95% and 92%, respectively, and therefore, they are classified as mutated IGHV C. The seAs coverage for these two positions was very low in the germline sample (135 reads mean coverage versus 22 or 40 reads coverage for POLE or ATXN1) and as both deletions were annotated as polymorphisms in the SNP database , we perfIn this study, we present an in-depth molecular analysis of a unique case with two independent CLL clones and a prostate carcinoma. Although previous investigations already suggested an origin from two independent B cells, thorough genetic evidence for this assumption was missing. We found that all three cancers and particularly the two CLL clones had no shared mutations, which confirms that the two CLL clones likely originated from two different B cells and not from a common B cell precursor with a distinct profile of somatic mutations. In contrast, we describe the presence of a hemizygous POLE deletion already present in the germline DNA of the patient, which impedes correct splicing and thus likely results in translation of a dysfunctional polymerase epsilon protein.Although we cannot exclude the presence of mutations not covered by our 6110 gene exome panel or of additional unfavorable SNPs that remained undetected in our study, our data suggest that POLE SNP rs761516512 predisposes to a higher incidence for cancer development. Although POLE mutations are not recognized as known driver genes in CLL, the gene is listed as a cancer driver in the cosmic database ,18. In cWe initially monitored a cohort of 75 CLL patients  during ow/o pheno red; Life Technologies, Carlsbad, CA, USA; cat 32404-014) supplemented with 2% fetal calf serum, they were analyzed on a Cytomix FC500 flow cytometer (Beckman Coulter) or sorted on a FACS Aria III (BD Biosciences) for kappa or lambda-positive CLL cell populations . Measurements and sorts were evaluated with Kaluza Analysis Software 2.1. (Beckman Coulter) or FACS Diva Software 8.0.1 (BD Biosciences).To determine the amount of IGL-k and IGL-l positive cells in CLL samples, we performed a flow cytometric staining specific for these two clones in combination with CLL and T cell detection of thawed biobanked and density gradient purified PBMCs. After thawing and washing of a PBMC sample, about 20 million cells were resuspended in 1 mL of PBS supplemented with 0.1% BSA. We added 20 µL anti-lambda-FITC , 20 µL anti-kappa-PE , 15 µL anti-CD5-PC5 , and 15 µL anti CD19-PC7  antibodies. The cells were incubated with the antibodies for 25 min at 4 °C. After washing, filtering, and resuspending of the cells in RPMI 1640  according to the manufacturer’s recommendation with minor changes. In short, we extracted DNA from the sorted populations according to Qiagen’s protocol of the DNeasy Blood & Tissue Kit . We prediluted the extracted DNA to about 10 ng/µL and used 2.5 µL for further processing. We added 22.5 µL IGHV leader master mix (Invivoscribe) and 0.1 µL Eagle Taq DNA polymerase  to a final volume of 25.1 µL. We used the PCR program according to the LymphoTrack® Dx protocol and a Biometra T3000 thermocycler . Further PCR product cleanup was performed starting with 25 µL of AMPure XP reagent  per cleanup and further steps according to the LymphoTrack® Dx protocol. We eluted each sample with 15 µL of 10 mM Tris solution with pH 8.0. After PCR product check with the 4200 TapeStation analyzer and D1000 screen tapes , the concentration was determined with Qubit and a dsDNA HS Assay Kit . Further processing strictly followed the LymphoTrack Dx protocol. Then, 15 pM final NGS library were sequenced with the MiSeq reagent kit v3 and a 2x 301 bp read length on a MiSeq Sequencer . Analysis of the sequencing runs was performed using LymphoTrack Software (Invivoscribe). The obtained sequences were further subjected to IMGT/V-QEST [The sorted kappa and lambda-positive CLL populations were further processed for NGS-based IGHV mutation analysis by LymphoTrackT/V-QEST  for detaTo analyze 6110 disease-associated genes in the kappa or lambda-sorted CLL samples, the prostate cancer FFPE sample from radical prostatectomy, and the buccal swab sample for germline analysis, we used the Agilent SureSelect QXT focused exome reagent kit and capture library . DNA extraction was performed as described above. Samples were prepared according to Agilent’s SureSelectQXT Target Enrichment protocol for the Illumina paired-end multiplexed sequencing library using the 200 ng DNA input option or the FFPE-optimized option for the prostate specimen, respectively. The prepared library was analyzed on a NextSeq 550 platform with 2x 100 bp read length and a 300 cycle kit v2. As seeding concentration, 1.6 pM was used.p-value 0.05, somaticFilter. Bam-readcount (v0.8.0) [Raw sequencing basecount files were demultiplexed using Illumina bcl2fastq software (v2.19.1.403), and the demultiplexed fastq files were trimmed using Trimmomatic  . Trimmed(v0.8.0)  was used(v0.8.0)  and the (v0.8.0) .g and analyzed on a 3500 Series 8-capillary Genetic Analyzer (ThermoFisher) with POP-7TM polymer separation matrix (ThermoFisher). Sequences were evaluated using Chromas Lite Software 2.01 .For confirmation of POLE and ATX1 aberrations, we used DNA obtained as described above from the sorted kappa and lambda clone CLL cells, the prostate cancer FFPE sample from radical prostatectomy, and the buccal swab sample. Primer sequences for POLE were TAAACACAGCCCAAATCTGTAAGGAACC (forward), GGAGCAGAAGTCTACGTCCATCAGCAC (reverse), and for ATXN1, they were GGAAATGTGGACGTACTGGTTCTGCTG (forward), CTATTCCACTCTGCTGGCCAACATGG (reverse). For PCR amplification, we prepared PCR reactions consisting of 10 µL of 2x Phusion Master Mix with HF buffer , 1 µL of each forward and reverse primer diluted to 10 pmol/µL, 20 ng DNA, and added nuclease-free water to a total reaction volume of 20 µL. For POLE, we used the following temperature gradient: (1) 98 °C 30 s, (2) 98 °C 10 s, (3) 63 °C 5 s, (4) 72 °C 20 s, go to step 2 30 times, (5) 72 °C 10 min, (6) 4 °C hold. For ATXN1, we used the following conditions: (1) 98 °C 30 s, (2) 98 °C 10 s, (3) 72 °C 20 s, go to step 2 30 times, (4) 72 °C 10 min, 4 °C hold. All reactions were performed on a Biometra Trio . After PCR amplification, 5 µL of all PCR products were cleaned up with 2 µL ExoSAP-ITTM solution  at 37 °C for 15 min, 80 °C for 15 min, 4 °C hold and prepared for Sanger sequencing by adding 2 µL cleaned up PCR product to 2 µL 5x Cycle Sequencing buffer, 1 µL BigDyeTM Terminator v3.1, and 0.5 µL forward or reverse primer. The reactions were filled to a final volume of 10 µL with nuclease-free water. The cycle sequencing program was as follows: (1) 96 °C 1 min, (2) 96 °C 10 s, (3) 50 °C 10 s, (4) 60 °C 3 min, go to step 2 31 times, 4 °C hold. Before subjecting the samples to Sanger sequencing, a XTerminatorTM purification  was performed. To each 10 µL sample, 40 µL SAMTM solution and 6 µL XTerminatorTM solution were added and shook for 30 min at 1800 rpm on a ThermoMixer , centrifuged 2 min at 1000×


Neurotrauma is a worldwide public health problem which can be divided into primary and secondary damge. The primary damge is caused by external forces and triggers the overproduction of peroxides and superoxides, leading to long-lasting secondary damage including oxidative stress, wound infection and immunological reactions. The emerging catalysts have shown great potential in the treatment of brain injury and neurogenic inflammation, but are limited to biosafety issues and delivery efficiency.2O2 of the ultrasmall gold clusters is 10 times that of glassy carbon (GC) electrodes, indicating excellent catalytic activity. The gold clusters can relieve the oxidative stress and decrease the excessive O2·− and H2O2 both in vitro and in vivo. Besides, gold clusters can accelerate the wound healing of brain trauma and alleviate inflammation via inhibiting the activation of astrocytes and microglia through noninvasive adminstration. decrease the peroxide and superoxide of brain tissue.Herein, we proposed the noninvasive delivery route to brain trauma by employing highly active gold clusters with enzyme-like activity to achieve the early intervention. The decomposition rate to HPresent work shows noninvasive treatment is a promising route for early intervention of brain trauma.The online version contains supplementary material available at 10.1186/s12951-021-01071-4. Copper nitrate (Cu(NO3)2), Cadmium nitrate (Cd(NO3)2), 3-Mercaptopropionic acid (MPA), Sodium borohydride (NaBH4), Sodium hydroxide (NaOH), were purchased from Aladdin. Ultrapure water (18.2 MΩ*cm) was used for all the experiments. Kits and fluorescent probes were purchased from commercial sources and used as per the instructions.All chemicals and reagents were purchased from commercial sources and used without further purification. Gold chloride  and MPA  were added to water (2.35 mL) and stirred for 5 min at room temperature. Then, NaOH solution  was added to the reaction mixture, followed by the addition of 0.1 mL of NaBH4 (43 mg of NaBH4 powder in 10 mL of 0.2 M NaOH solution). Au25MPA18 was collected after the final reaction mixture stirred at room temperature for 3 h in the dark and aged at 4 °C for 12 h. The Au24Cu1 and Au24Cd1 were synthesized based on the same method, except the Au atoms in HAuCl4  were replaced by various nitrate metal ions  at a 4% molar ratio. Ultrafiltration tubes of 3 and 10 K at 3500 rpm/min were used for ultrafiltration to remove smaller organic ligands and larger-sized clusters, and lyophilization was used to collect the purified product for further test and investigation.The MPA-protected Au reports , 59, 60.A JEM-2100 F electron microscope  was employed to acquire transmission electron microscopic (TEM) images. A Malvern Zetasizer Nano ZS90 (UK) was employed to measure dynamic light scattering (DLS) to test the hydrodynamic size and determine the zeta potential of clusters.The XPS spectrum of the metal elements was performed using ESCALAB Xi+ spectrometer, with a monochromatic Al Kα X-ray source (Thermo Fisher Scientific). All XPS spectra are processed with XPSPEAK41 software and corrected with the peak of C1s (standard: 428.8 eV). ICP-MS  was used to determine the content of each metal element in the clusters.25, Au24Cu1 and Au24Cd1 clusters  were deposited onto the surface of GC electrodes and dried naturally, then Nafion solution (3 µL) was dropped onto the surface of GC electrodes and dried, the modified electrodes were used for catalytic activity tests.The clusters were deposited onto the surface of glassy carbon (GC) electrodes for electrochemical assay , 40. Bri−1; OER, 100 mV s−1) and Linear Sweep Voltammetry  measurements were taken electrochemical analyzer, CHI760E, Shanghai) to evaluate the performance of the clusters modified electrodes for HER and OER. HER was carried out in 0.5 M H2SO4, and a graphite rod and a saturated calomel electrode were used as the counter electrode and reference electrode, respectively. OER was carried out in 1 M KOH, and a platinum wire and an Ag/AgCl electrode were used as the counter electrode and reference electrode, respectively.Cyclic Voltammetry  and LSV (scan rate: 5 mV s−1) measurements were taken  to evaluate the catalytic activities for O2 and H2O2 reduction. A three-electrode cell was adopted for both O2 and H2O2 reduction, a platinum wire and a saturated calomel electrode were used as the counter electrode and reference electrode, respectively. Oxygen reduction reaction (ORR) was carried out in O2-saturated in 0.01 M PBS (pH 7.4), and the H2O2 reduction was performed in the presence of 9.8 mM H2O2 in N2-saturated 0.01 M PBS (pH 7.4).CV (scan rate: 50 mV s3), mouse microglia (BV2) (4 × 103), and mouse astrocytes (MA-c) (4 × 103) were seeded in 96-well plates filled in 100 µL medium with 0.01 M PBS (Gibco) at the border overnight. The culture medium was replaced by different doses of Au25, Au24Cu1, or Au24Cd1 dissolved in the DMEM, and then cells were incubated for another 24 h. Wells were washed with 0.01 M PBS once, and the medium were replaced by fresh culture medium with serum-free DMEM. Cell survival was analyzed using MTT -2,5-diphenyl-2 H tetrazolium bromide, Beyotime) of 5 mg/mL for 2.5 h and detected at optical density (OD) 490 nm.Cytotoxicity assay. mouse hippocampal neuronal cell line (HT22)  were seeded in 6-well plates for 12 h and stimulated with 100 µM H2O2 for 6 h before being treated with the clusters (12 ng/µL). Cell survival was determined at the MTT concentration of 5 mg/mL for 2.5 h and detected at optical density (OD) 490 nm. Fluorescent staining was carried out to evaluate the intracellular oxidative stress levels with different probes such as DHE for O2·− and DCFH-DA for ROS, and cell images were captured by a fluorescence microscope.Cell viability and Immunofluorescence. HT22 was used in all the cellular experiments. Cells were seeded into the 96-well plates and grew in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C with 5% CO25, TBI+Au24Cu1, and TBI+ Au24Cd1 group (n = 15) randomly. All mice were anesthetized with 10 % chloral hydrate (10 mg/kg) and the scalp was cut before placed on the stereotaxic frame. The craniotomy was carried out by drilling the skull in a circle of 2 mm in diameter. The scalp was sewn together carefully, and the clusters were added to the wound of TBI mice at a concentration of 50 mg/kg. The healing process was recorded photographically and the wound remaining was calculated after treatment.TBI models: C57BL/6 mice at 21–23 g were employed to establish TBI models using an electromagnetically CCI injury device , with an impactor of 5 m/s velocity, 0.61 mm depth, 150 ms duration, and 20º angle of dura mater on the vertical axis. The mice were divided into control, TBI, TBI+Au2O2 using commercially available kits. All testing methods are carried out as per the instructions (Beyotime).Oxidative stress level: Brain tissues were taken out on days 7 and 14 post-treatment, then homogenized in 0.9% physiological saline and analyzed for SOD, GSH/GSSG, MDA, and HMorris water maze tests: Morris water maze (MWM) was conducted on days 13–17 and 26–30 post-treatment as described in the previously reported literature , 61, 62.Western Blotting: The total protein in the brain tissue was extracted and the content of IL-1β (1:1000), IL-6 (1:1000) and TNF-α (1:1000) was analyzed. SDS-PAGE electrophoresis was performed before transferring to the membrane. Immune responses of the specific antibodies were carried out and the images were captured with autoradiography. All antibodies were purchased from Abcam.ELISA analysis: Inflammatory cytokines including IL-6, IL-1β and TNF-α were determined by ELISA kits , and the assays were performed as per the instructions provided by the manufacture.Tissue staining: Brain tissues were taken out at 30 days post-injury and fixed in 4% paraformaldehyde and embedded in paraffin. Immunofluorescent staining was performed with primary antibodies including anti-GFAP, IL-6, IL-1β antibodies as per the instructions of (Abcam). Then the slices were incubated with Alexa Fluor 488/594-conjugated goat secondary antibody for 1-1.5 h at room temperature under dark and counterstained with DAPI. Immunohistochemical staining for IL-6 and mice brain tissue was performed according to the instructions (Proteintech).Toxicological studies: The biosafety of clusters was measured on male C57BL/6J mice at 7-8 weeks (21-23 g). Mice were treated with 200 µL of clusters at a dose of 50 mg/kg every other day. Hematology and blood biochemical indicators were tested on day 30. The blood sample was obtained from the retroorbital sinus and stored in a test tube containing K2EDTA for testing. The blood sample used for biochemical analysis was allowed to stand for 30 min, and then centrifuged twice at 3500 rpm for 15 min each time, the supernatant was collected and tested. The main organs of the mice were collected and fixed in 4 % paraformaldehyde for 24 h, embedded in paraffin, and mounted on a glass slide . The slices were stained with hematoxylin and eosin to observe the toxicity of clusters in major organs, including the heart, liver, spleen, lung, kidney and testis.Data are presented as mean ± standard deviation (SD) or standard error of the mean (SEM). For multiple comparisons, one-way analysis of variance (ANOVA) was performed using the SPSS 22 software to assess difference in means among groups and compared with the Con and TBI groups, analyzed by ANOVA.Additional file 1: Figure S1. The content of metal elements in the clusters were quantified by ICP-MS. Figure S2. X-ray photoelectron spectroscopy of clusters. Figure S3. Cell survival of (a) HT22, (b) BV2 and (c) MA-c cells in the presence of various concentrations of clusters. Figure S4. Hematology of mice treated with or without gold clusters on day 30. Figure S5. Blood biochemistry analysis of mice treated with or without gold clusters on day 30. Figure S6. Histology of major organs in mice.


Decidualization is a crucial step for human reproduction, which is a prerequisite for embryo implantation, placentation and pregnancy maintenance. Despite rapid advances over recent years, the molecular mechanism underlying decidualization remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of a mouse uterus during decidualization. By analyzing the undecidualized inter-implantation site of the uterus as a control, we were able to identify global gene expression changes associated with decidualization in each cell type. Additionally, we identified intercellular crosstalk between decidual cells and niche cells, including immune cells, endothelial cells and trophoblast cells. Our data provide a valuable resource for deciphering the molecular mechanism underlying decidualization. Decidualization is a crucial step for human reproduction. It is a prerequisite for embryo implantation, placentation and pregnancy maintenance . DeciduaDue to ethical restrictions, studies on human decidualization are limited to the in vitro level. The in vivo investigation of decidualization heavily relies on mice. Slightly different from humans, the decidual reaction in mice is an embryo-dependent process. Prolactin family 8 subfamily a member 2 (Prl8a2), a paralog of human dPRL, is a well-established marker for mouse decidualization . By usinWith advances in the single-cell RNA-seq techniques, it is now possible to analyze the global gene expression profile within highly heterogeneous tissues at a single-cell level . In the To create a cell-type resolved map of a mouse uterus upon decidualization, we performed single-cell RNA-seq analysis A. The imUnsupervised clustering analysis revealed 21 distinct cell clusters for all the cells from the IIS and the IS combined A. The maHormone-responsive cells included epithelial cells expressing Epcam and Krt19  Figure C, stromaEndothelial cells had four clusters: VEC and its proliferating subset VECp were vascular endothelial cells expressing Sox17 , while L+Nkg7+Cd3e−) [+Nkg7−Cd3e+Mki67+), T cells  [+Cd79a+Ms4a1+) [+Adgre1+) [+Itgax+) [+Itgax+Mki67+) and plasmacytoid dendritic cells  [There were eight immune cell clusters I,J. Incl7+Cd3e−) , prolife7−Cd3e+) , B cells+Ms4a1+) , macrophAdgre1+) , dendrit+Itgax+) , prolifeiglech+) .Finally, we aimed to discover novel markers for each cell type. We selected the genes that were expressed significantly higher in the cell type of interest than the other cell types using the Wilcoxon rank sum test. A complete list of marker genes is presented in p = 1.2 × 10−10), RNA metabolism (p = 4.4 × 10−9), protein metabolism (p = 1.1 × 10−5), cell cycle and proliferation (p = 0.00017), cell adhesion (p = 0.014) and cell death (p = 0.038). GeneSet#2, with 475 genes, was S1-specific. These genes were enriched in cell adhesion (p = 1.6 × 10−11), developmental processes (p = 4.4 × 10−10), stress response (p = 9.0 × 10−7), cell organization and biogenesis (p = 0.00019), cell death (p = 0.00095) and protein metabolism (p = 0.0070). Geneset#3 of 562 genes gradually decreased during the decidualization process. Based on GO, the enriched terms were cell cycle and proliferation (p = 2.1 × 10−11), protein metabolism (p = 5.9 × 10−9), cell organization and biogenesis (p = 1.2 × 10−8), DNA metabolism (p = 2.3 × 10−6) and transport (p = 0.0021). GeneSet#4 of 717 genes gradually increased during the decidualization process. The enriched GO terms were transport (p = 8.7 × 10−9), protein metabolism (p = 0.016) and cell cycle and proliferation (p = 0.038). Hsd11b2, C3, Mki67 and Hand2 were representative genes for gene sets 1–4, respectively , S1p , S2 , S3p  and S3 . We selected signature genes for each cell cluster by using the Wilcoxon rank sum test. After the removal of redundancy, we identified a total of 2113 signature genes . Throughectively B.To further reveal the relationship between these five stromal cell clusters, pseudotime trajectory analysis was conducted by using the Monocle2 software . The celWe are particularly interested in GeneSet#4, the S3-enriched genes. On GD8, the entire IS, from the anti-mesometrial region to the mesometrial region, is fully decidualized. Meanwhile, the IIS shows no sign of decidualization. Thus, the S3-enriched genes could be validated using qTR-PCR with bulk tissues. The top 12 S3-enriched genes  were selected . Althoug2 test was employed to assess the significance of difference between the two groups. By using the criteria of p < 0.05 and fold change > 2, the proportions of all the cell types are unchanged, except DC and B cells , the PI3K-Akt signaling pathway (FDR = 1.00 × 10−34), the Cytokine–cytokine receptor interaction (FDR = 1.00 × 10−16), the regulation of actin cytoskeleton (FDR = 1.00 × 10−14), the MAPK signaling pathway (FDR = 5.01 × 10−8), the Rap1 signaling pathway (FDR = 1.26 × 10−7), endocytosis (FDR = 1.58 × 10−6), the natural killer cell-mediated cytotoxicity (FDR = 3.98 × 10−6), phagosome (FDR = 6.31 × 10−6), the TGF-beta signaling pathway (FDR = 1.58 × 10−5), the Hippo signaling pathway (FDR = 2.51 × 10−4), the NOD-like receptor signaling pathway (FDR = 3.98 × 10−4), the Toll-like receptor signaling pathway (FDR = 3.98 × 10−4), the mTOR signaling pathway (FDR = 2.00 × 10−3) and the Ras signaling pathway (FDR = 2.00 × 10−4) (We then used the CellChat software  to predi × 10−4) B.p < 0.05 and fold change > 2, we found that the proportion of LEC was unchanged, whereas the proportions of VEC were significantly increased , the ECM-receptor interaction (FDR = 1.00 × 10−46), the Rap1 signaling pathway (FDR = 1.00 × 10−20), the Cytokine-cytokine receptor interaction (FDR = 1.00 × 10−20), the Ras signaling pathway (FDR = 1.00 × 10−17), the regulation of actin cytoskeleton (FDR = 1.00 × 10−15), the HIF-1 signaling pathway (FDR = 1.26 × 10−9), the MAPK signaling pathway (FDR = 1.58 × 10−9), endocytosis (FDR = 6.31 × 10−8), the Phospholipase D signaling pathway (FDR = 3.16 × 10−6), the Hippo signaling pathway (FDR = 3.98 × 10−6), the TGF-beta signaling pathway (FDR = 2.00 × 10−5), the mTOR signaling pathway (FDR = 5.01 × 10−4), phagosome (FDR = 1.26 × 10−3), the NOD-like receptor signaling pathway (FDR = 6.31 × 10−3) and the Toll-like receptor signaling pathway (FDR = 6.31 × 10−3)  B.Trophoblast cells may interact with decidual cells and pay an important role during the decidualization process. However, due to the relatively small number of embryonic cells at the implantation site, we did not find any fetal cell clusters in our single-cell RNA-seq data. Alternatively, we re-analyzed a published single-cell RNA-seq dataset on mouse E7.5 embryos (GSM3457437) . E7.5 is−37), the PI3K-Akt signaling pathway (FDR = 1.00 × 10−29), the Cytokine-cytokine receptor interaction (FDR = 1.00 × 10−11), the regulation of actin cytoskeleton (FDR = 1.00 × 10−9), the Rap1 signaling pathway (FDR = 2.00 × 10−8), the TGF-beta signaling pathway (FDR = 3.98 × 10−6), the Ras signaling pathway (FDR = 7.94 × 10−7), the Phospholipase D signaling pathway (FDR = 7.94 × 10−5), the Hippo signaling pathway (FDR = 1.26 × 10−3) and the MAPK signaling pathway (FDR = 1.26 × 10−3)  E.We have recently established a single-cell atlas of mouse uterus during embryo implantation using the 10× Genomics approach . In thisWe used 2 mg/mL Collagenase II and 10 mg/mL Dispase II for a single-cell suspension preparation. Collagenase II is a crude collagenase preparation with weak trypsin-like activity and, thus, trypsin was not used in our method. It turned out that the cell viability was >80% and the percentage of cell clumps was <10%, indicative of the high efficiency of our method. Nevertheless, we noticed that the percentage of decidualized stromal cells was much lower than expected: S3  was 4.4% and S3p  was 1.7%. As we observed lower cell viability at the implantation site compared to the inter-implantation site, we suspected that the collagenase treatment might selectively destroy the decidualized cells. This was in line with the finding that the highly expressed decidual-specific marker gene Prl8a2 contaminated all the other cells, indicative of the rupture of the decidual cells during the single-cell suspension preparation. Moreover, we would like to note that the collagenase treatment might cause transcriptional disturbances, leading to artifacts . We compIn this study, the embryo at the IS was kept. We estimated that there were around 2000 cells per embryo. We usually obtained two million cells per sample in the single-cell dissociation procedure. Approximately 5000 cells were sequenced per sample by the 10× platform. By calculating the probability, we found that only ~5 cell per embryo could be captured in our single-cell RNA-seq data. This was in line with our findings that there were no embryo-derived cell clusters in our single-cell RNA-seq data. The embryo-derived cells, if they existed at all, might be scattered among uterine cells in our single-cell RNA-seq data. Due to their small number, these cells might not have an obvious effect on the assigning cell type label for each cell cluster. Moreover, these cells might not interfere with the process for identifying differentially expressed genes either, because we set min.pct to 0.20, which means that we required the differentially expressed genes to be expressed in a minimum of 20% of cells within the cluster of interest.In mice, embryo implantation initiates at midnight on GD4. Upon embryo implantation, the superficial stromal cells proximally surrounding the implantation chamber exhibit proliferation and spreading. These cells differentiate and form the primary decidual zone (PDZ) on the afternoon of gestational day five. The PDZ subsequently undergoes apoptosis and disappears by GD8. Meanwhile, the stromal cells next to the PDZ continue to proliferate and differentiate into decidual cells, forming the secondary decidual zone (SDZ). The SDZ is full developed on GD8. Here, we found five stromal cell clusters for the IS  and three stromal cell clusters for the IIS . S2 was superficial stromal cells and S1/S1p was deep stromal cells, which is in line with our previous study . S3 and We are particularly interested in GeneSet#4, which may represent the key molecular mechanism of decidualization. The best strategy to validate our findings is to obtain a pure cell population by using fluorescence activated cell sorting (FACS). Previously, CD13-specific antibodies were used to sort stromal cells by FACS from human endometrial biopsies ,36. Howep < 0.05 and fold change > 2, the proportions of all the cell types are unchanged, except for DC and B cells. As B cells were a minor cell type (< 5%) in the uterus, we thus focused on DC cells. It has been demonstrated that the depletion of DC cells resulted in a severe impairment of the decidualization process, leading to embryo resorption [Immune cells play an important role in embryo implantation and decidualization in mice . We invesorption . We founEndothelial cells are in the niche of decidual cells. We hypothesized that endothelial cells might contribute to decidualization. We found that VEC cells were significantly increased, while LEC cells were slightly decreased upon decidualization. This was in line with the fact that the decidualization process is accompanied by angiogenesis . Though Previously, E7.5 embryos obtained from the GD8 uterus have been subjected to single-cell RNA-seq . ThroughIn summary, we revealed intercellular crosstalk between decidual cells and niche cells, including immune cells, endothelial cells and trophoblast cells. This cell atlas of mouse uterus on GD8 provides an essential resource for understanding the molecular mechanism underlying decidualization.Adult CD-1 mice of the SPF grade were used in this study. All mice were caged under light-controlled conditions (14 h/10 h light/dark cycles) with free access to regular food and water. Female mice were mated with fertile males and mating was confirmed the next morning by the presence of a vaginal plug. The day of the vaginal plug was denoted as gestation day 1 (GD1). On GD8, the implantation sites  and non-implantation sites  were collected separately.The total RNA from uterine tissues were extracted with the TRIzol reagent (Invitrogen). RNA-seq libraries were generated by using the TruSeq RNA sample preparation kit  and sequenced on an Illumina HiSeq 2500 system with the paired-end 150-bp protocol. Raw data were analyzed using TopHat v2.0.4  and Cuffg at 4 °C for 4 min and the pelleted cells were washed using centrifugation. In order to measure cell viability, cells were strained with AO/PI solution  and counted using a Cellometer Auto 2000 instrument . The single-cell suspension was carried forward to single-cell RNA-seq only if the cell viability was >80% and the percentage of cell clumps was <10%.Uterine samples from 3 mice for each group were pooled and minced with a blade. Samples were then incubated in dissociation buffer containing 2 mg/mL Collagenase II , 10 mg/mL Dispase II  and 50,000 U/mL DNase I  for up to 30 min at 37 °C in a shaking incubator. The digestion progress was checked every 5 min with a microscope until a single cell suspension was achieved. The single-cell suspension was then passed through a 40-micrometer cell strainer to remove undigested tissues. Cells were spun down at 250× TM Single Cell B Chip , aiming at recovering 8000–10,000 cells. The Chromium Single Cell 3′ Library and Gel Bead Kit v3  was used for single-cell bar-coding, cDNA synthesis and library preparation, following the manufacturer’s instructions. Library sequencing was performed on an Illumina NovaSeq 6000 system configured with the paired-end 150-bp protocol at a sequencing depth of approximately 400 million reads.The final concentration of single-cell suspension was adjusted to 1000 cells/μL and a volume of 15 µL was loaded into one channel of the ChromiumRaw data (bcl files) from the Illumina NovaSeq 6000 platform were converted to fastq files using the bcl2fastq tool . These fastq files were aligned to the UCSC mm10 mouse reference genome by using the CellRanger software . The resulting gene counts matrix was analyzed with the R package Seurat (v3.1.3) . Cell wiThe Monocle2 package v2.18.0 was used for pseudotime analysis . The coup < 0.05 was used as the significance threshold to identify enriched GO terms.Gene ontology (GO) analysis was performed as described previously . GO termPathway enrichment analysis was conducted by using the Metascape v7.4 online tools . The sigp < 0.05 was considered significant.The CellChat v1.1.0 R package  was usedThe TRIzol reagent  was used to extract total RNAs. The cDNAs were synthesized with the PrimeScript reverse transcriptase reagent kit . A quantitative RT-PCR was performed on Applied Biosystems 7500  with the THUNDERBIRD SYBR qPCR Mix . The Rpl7 gene was used as the reference gene for data normalization. A complete list of primer sequences is provided in