Secreted Frizzled-Related Protein 1 (SFRP1) expression is decreased during breast cancer progression and inversely associated with breast tissue age-related lobular involution in women. In this study, we have investigated the potential causal role of the decrease in SFRP1 expression in early breast carcinogenesis. In order to do so, we characterized mammary epithelial cells from both nulliparous and multiparous mice in organoid culture ex vivo, and modulated SFRP1 expression in breast non-tumoral and tumoral cell lines from multiple molecular subtypes. Our results support the hypothesis that a lack of SFRP1 could have a causal role in early breast carcinogenesis.Secreted Frizzled-Related Protein 1 (SFRP1) as decreased during breast cancer progression. In addition, SFRP1 was inversely associated with breast tissue age-related lobular involution, and differentially regulated in women with regard to their parity status and the presence of microcalcifications. The causal role of SFRP1 in breast carcinogenesis remains, nevertheless, not well understood. In this study, we characterized mammary epithelial cells from both nulliparous and multiparous mice in organoid culture ex vivo, in the presence of estradiol (E2) and/or hydroxyapatite microcalcifications (HA). Furthermore, we have modulated SFRP1 expression in breast cancer cell lines, including the MCF10A series, and investigated their tumoral properties. We observed that organoids obtained from multiparous mice were resistant to E2 treatment, while organoids obtained from nulliparous mice developed the luminal phenotype associated with a lower ratio between Sfrp1 and Esr1 expression. The decrease in SFRP1 expression in MCF10A and MCF10AT1 cell lines increased their tumorigenic properties in vitro. On the other hand, the overexpression of SFRP1 in MCF10DCIS, MCF10CA1a, and MCF7 reduced their aggressiveness. Our results support the hypothesis that a lack of SFRP1 could have a causal role in early breast carcinogenesis.A human transcriptome array on ERα-positive breast cancer continuum of risk identified  ESR1, GATA3, and XBP1), luminal B (high expression of ESR1 and HER2), HER2-enriched (high expression of HER2 and EGFR), basal-like  and normal-like (high expression of CD36 and GPX3) [Recent decades have seen the advent of systematic mammographic screening in women older than 50 years old in developed countries, as well as the development of targeted therapies such as tamoxifen, a selective estrogen receptor modulator antagonizing the estrogen receptor-alpha (ERα) in the breast. To personalize breast cancer care, tumors can nowadays be classified by clinical subtypes, assessed by immunohistochemistry and described by the St. Gallen expert consensus, i.e., luminal breast cancer expressing ERα and/or the progesterone receptor (PR), overexpressing —or not —the human epidermal growth factor receptor 2 (HER2) and/or having —or not —a high proliferative index (Ki-67) ,2,3. In nd GPX3) ,5.If ERα is a crucial marker of breast cancer diagnosis and a well-known therapeutic target against the disease, it is important to keep in mind that, during a woman’s lifetime, ERα expression in breast tissue does not remain constant, even in a physiological context. Indeed, ERα is more expressed during breast morphogenesis periods, including embryogenesis and puberty ,7,8. It Secreted Frizzled-Related Protein 1 (SFRP1) was identified [Sfrp1 demonstrated that it negatively regulates mammary branching and the expression of ERα, raising the question of the causal role the lack of SFRP1 plays in luminal breast cancer development in women [In order to better understand the early ERα-positive carcinogenesis, our group previously performed a human transcriptome array on normal tissue, atypical ductal hyperplasia (ADH), ERα-positive ductal carcinoma in situ (DCIS) and ERα-positive invasive ductal carcinoma (IDC) . Among tentified . At the entified ,21. In ain women .In another study, our group demonstrated that SFRP1 was largely expressed in non-involuted breast epithelial tissue, while it dramatically decreased after perimenopausal lobular involution completion in nulliparous women. Furthermore, we observed that SFRP1 expression was also associated with the presence of microcalcifications in breast tissue, once more in nulliparous women specifically . HoweverSFRP1 in non-tumoral breast tissue results in mammary gland branching in nulliparous mammalians, and in the acquisition of a malignant phenotype, increasing ESR1 expression and, consequently, sensitizing breast cells to estradiol (E2) proliferative and migratory effects. We also anticipate that an increase in SFRP1 expression in breast cancer cell lines should decrease cell viability and reduce ESR1 expression, at least in luminal breast cancer cell lines.Hence, we hypothesize that the lack of SFRP1 expression and promoter methylation among breast non-tumoral and tumoral tissues. Thereafter, we compared the correlation between SFRP1 and biomarkers of breast cancer molecular subtyping, such as ESR1, PGR, and HER2 in both breast non-tumoral and tumoral tissues. We then compared the expression of Sfrp1 in nulliparous and multiparous mice mammary glands cultured ex vivo in the presence, or not, of E2 and/or HA crystals. We also reported on the phenotypic characteristics of the organoids depending on parity, the presence of E2 and/or HA, and SFRP1 expression. In addition, we analyzed the causal role of SFRP1 modulations of expression by lentiviral transduction on early breast carcinogenesis and breast cancer aggressiveness in vitro. We also investigated the existence of an E2-dependent modulation of SFRP1 expression, as well as a potential SFRP1-dependent response to E2 in breast cell lines. Finally, we examined the association between SFRP1 expression in tumoral tissue and patients’ outcomes after stratification for ERα status or breast cancer molecular subtypes.In the present study, we first analyzed public datasets to compare SFRP1 expression across breast non-tumoral, tumoral, and metastatic tissues was obtained by using the TNMplot public dataset  [SFRP1 expression analyses regarding breast cancer degree of differentiation, molecular subtypes (assessed by immunohistochemistry), and grade were obtained from the GENT2 public dataset  [https://xenabrowser.net/, accessed on 18 September 2021) on the TCGA Breast Cancer (BRCA) tumoral dataset (n = 1247) and on normal breast tissue . Breast cancer molecular subtypes in the TCGA BRCA tumoral dataset were assessed by the PAM50 diagnostic method. Survival curves were drawn using the KM-plot online tool (accessed on 15 April 2021) [SFRP1 expression was used to segregate high SFRP1 expression from low SFRP1 expression. Each molecular subtype pathological response to chemotherapy in association with SFRP1 expression was obtained from the ROCplot.org database . The same probe set (202037_s_at), targeting SFRP1, was used for all previously cited analyses. PREdiction of Clinical Outcomes from Genomic Profiles  [SFRP1 expression and breast cancer overall survival  [il 2021) . SFRP1 eil 2021) . Co-expril 2021) . Overallil 2021) . Follow-il 2021)  was usedil 2021) .g for 5 min. Supernatant was removed and 2 mL of warm dispase  and 0.1–1 mg/mL of DNAse I  were added. After homogenization by pipetting, 10 mL of cold 1X complete HBSS solution was added, and the suspension was filtered through a 40 μm cell strainer  before centrifugation at 350× g for 5 min. Cells were then counted, and a purification process was performed using the EasySep Mouse Epithelial Cell Enrichment kit II (StemCell) according to the manufacturer’s protocol. Thereafter, cells were plated for 1 h for differential plating (to avoid overgrowth of fibroblasts). Purified mammary epithelial cells were centrifuged at 350× g for 5 min and resuspended in complete EpiCult-B mouse medium (StemCell) + 5% FBS and 75% growth-factor-reduced Matrigel  + 100 ng hydroxyapatite crystals (Sigma), or not, for a total of 30,000 cells by 40 μL droplet. Droplets were plated in 24-well plates with cold tips and then incubated upside down for 15 min at 37 °C. Subsequently, 3 mL of warm complete EpiCult-B mouse medium (StemCell) + 5% FBS at 37 °C with 5% CO2 was added. Medium was replaced 24 h later by serum-free complete EpiCult-B mouse medium with 10 nM EtOH or E2, and changed every 3 days. Pictures were taken every 3 days with the EVOS M5000 Imaging System  and analyzed using ImageJ software.Primary culture of mouse organoids was performed as previously detailed by Lacouture et al. . Protoco® CRL-10317™) breast cancer risk continuum obtained from a Caucasian 36-year-old woman was used to investigate the role of SFRP1 on early breast carcinogenesis, and was obtained from the Cell Resources Facility of the Barbara Ann Karmanos Cancer Institute, Wayne State University. The MCF10AT1 cell line, representing ADH, was derived from a stable transfection of MCF10A with mutant Ha-ras while MCF10DCIS.com, representing DCIS, was obtained from a xenograft of MCF10AT1 on an immunodeficient mouse. The MCF10CA1a and MCF7 (ATCC® HTB-22™) cell lines were used to explore the impact of SFRP1 modulation of expression on IDC aggressiveness in vitro. For all cell lines, culture media were changed every 48 to 72 h after washing with 1% PBS. Cells were split by adding 1 mL trypsin once they reached 60 to 70% confluency. Cells were maintained at 37 °C and 5% CO2. The hydrocortisone, insulin, and EGF were purchased from Sigma-Aldrich . E2 was obtained from Steraloids . All other cell culture reagents were purchased from Wisent Inc. . All cell lines were shown to be mycoplasma free.The embryonic kidney HEK293T cell line was used for transfection and lentivirus production, and was kindly provided by Dr. Stéphane Gobeil (CHU de Québec Research Centre). The HEK293T cell line was grown in DMEM medium supplemented with 10% fetal bovine serum on previously coated (5 mg/mL poly-L-lysine) petri dishes. All breast cell line characteristics and culture media used in the present study are summarized in SFRP1, i.e., TRCN0000062169, TRCN0000062170, and negative control shScramble in pLKO.1-puro vector were used to knock down SFRP1. These shRNAs were graciously provided by Dr. Stéphane Gobeil from Laval University, Quebec City, Canada and are respectively referred to as sh62169, sh62170, and shScramble throughout the manuscript. One lentivirus containing an open reading frame (ORF) plasmid construct for the SFRP1 gene (BRDN0000460838) in pLX_TRC304 vector was used to overexpress SFRP1 in breast cell lines. This ORF, graciously provided by Dr. Mathieu Laplante from Laval University, Quebec City, Canada, is referred to as OxSFRP1 throughout the manuscript. Empty pLX_TRC304 vector was used as negative control. The transfection was done in HEK293T cells plated in 60 to 70% confluency in 10 cm petri dishes coated with 5 mg/mL poly-L-lysine for 30 min at 37 °C. Lentiviruses were assembled by co-transfecting HEK293T with 12 μg shRNAs or ORF, 10.8 μg pMD2.G (VSVG coding plasmid), and 1.2 μg psPAX2 (lentivirus packaging plasmid) in 350 μL LBS buffer (pH = 4). After 48 h incubation at 37 °C and 5% CO2, the media containing lentiviruses were collected and filtered using 0.45 μm pore size filters . The lentiviruses were aliquoted in 1.5 mL tubes and stored at −80 °C until further use. Subsequently, 1.5 × 106 cells were plated in 10 cm petri dishes, and each infected with 2 mL of either shRNAs, shScramble, or OxSFRP1 viruses. One mL of medium and 3 µL polybrene (10 µg/mL) were also added to each petri dish, and cells were incubated overnight at 37 °C and 5% CO2. After 12 to 16 h of incubation, the media were changed after washing 3 times with 1X PBS. After 24 h, cells were split and fresh selective media, containing 2.5 to 22 µg/mL of blasticidin  for OxSFRP1, and 1 µg/mL of puromycin  for shRNAs and shScramble, were added. Three independent infections were done on 3 different cryovials for each cell line and each condition  in technical triplicate.Three lentiviruses, each containing a different shRNA targeting ® . The plates were then incubated at 37 °C with 5% CO2 until the first appearance of pink coloration. The absorbance was measured at 570 nm and 585 nm wavelengths with the TECAN Infinite M1000 . The experiments were performed in triplicate and repeated three times independently.Cell lines were automatically counted with the BIORAD TC20 automated cell counter  and 10,000 cells were plated in 200 µL of fresh media in 96-well plates. After 24 h incubation, media were replaced by fresh media containing 10 nM E2 or ethanol (EtOH). After 72 h incubation at 37 °C and 5% CO2, the media were changed with fresh medium containing 10% alamarBlueCell lines were automatically counted with the BIORAD TC20 automated cell counter  and 10,000 cells were plated in 200 µL of fresh media containing 10 nM E2 or EtOH in Ultra-Low Attachment (ULA) 96-well plates . Pictures were taken with the EVOS™ M5000 imaging system  every 24 h for 5 days. The spheroid size was measured with ImageJ, and the average size relative to the respective negative control at day 1 was calculated. The experiments were performed in triplicate and repeated three times independently.Cell lines were automatically counted with the BIORAD TC20 automated cell counter  and 25,000 cells were plated in 100 µL of fresh media in 2 chamber wells . Cells were then incubated at 37 °C with 5% CO2. After 24 h, the chambers were removed, and the culture media were replaced with fresh media containing 10 µM mitomycin C to stop cell proliferation and 10 nM E2 or EtOH. Pictures were taken with the EVOS™ M5000 imaging system  immediately (0 h) and every 6 to 12 h until the gap between the cells was filled. The wound area was measured with ImageJ and the percentage of wound area was calculated relative to the wound area at 0 h of migration. The experiments were performed in triplicate and repeated three times independently.Total RNA from organoids and cell lines was isolated with the Qiagen RNeasy mini kit  following the manufacturer’s instructions, using 30 µL of warmed (37 °C) RNAse-free water for elution. Thereafter, 2 to 3 µg of total RNA were diluted in RNAse-free water for a final volume of 10 µL. Thereafter, 1 µL of dNTP (10 mM), 1 µL oligo dT (50 µM), and 1 µL random hexamers (50 µM) were added to the mixture before incubation at 65 °C in a dry bath for 5 min, followed by 1 min on ice. Subsequently, 1 µL of SuperScript IV Reverse Transcriptase, 4 µL of 5X Buffer, 1 µL RNAse out and 1 µL DTT (100 mM) were added to the mixtures and incubated for 10 min at room temperature followed by 15 min at 50 °C and 10 min at 80 °C in a dry bath. All reagents were purchased from Thermo Fisher Scientific . Purification was performed with the QIAquick PCR Purification Kit  following the manufacturer’s instructions, using 20 to 30 µL of warmed (37 °C) RNAse-free water for elution.® Green PCR Master Mix , 20 ng of cDNA, 2 µL of oligo mix (5 µM each primer), and DNAse-free water. Thereafter, the qPCRs were performed in the StepOne Plus Real-Time PCR System . The runs were started by 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C followed by 1 min at 60 °C. Melt curves were performed in order to confirm the melting temperatures of the amplicons. Data calculation and normalization were performed using the second-derivative and double-correction methods. Three housekeeping genes, i.e., Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hypoxantine-guanine phosphoribosyltransferase (HPRT1), and Mitochondrial membrane ATP synthase (ATP5O) for human cell lines, and 2 housekeeping genes, i.e., Pumilio homolog 1 (Pum1) and TATA binding protein 1 (Tbp1) for mouse epithelial cells, were quantified. DNAse-free water was used for no-template control for each oligo primer pair in order to detect potential contamination.Oligo primers  were deshttp://www.rstudio.com/ (accessed on 1 November 2021)). Comparisons between the average mouse weight and average weight of the mammary glands (n = 57) were performed using the unpaired two-sided Student’s t-test and using the t test R function from the stats package, version 4.1.2. For ex vivo and in vitro experiments (n = 3), comparisons of means between the two groups were performed by one-sided Mann–Whitney testing with the R function wilcox.test from the stats package, version 4.1.2. In both figures and the main text, ex vivo and in vitro results are presented as mean ± standard deviation. All experiments were performed in technical triplicate and performed three times independently. p-value lower than 0.05 was considered significant.All analyses described were performed with RStudio v1.2.5033 (RStudio Team (2019), RStudio: Integrated Development for R, RStudio, Inc., Boston, MA, USA, URL SFRP1 expression decreases with the progression of breast lesions [SFRP1 expression was lower in tumoral breast tissue (fold change (FC) = 0.20) compared to normal breast tissue, and lower in metastatic (FC = 0.77) compared to breast tumors  and well-differentiated breast tumors  and grade 1  tumors  and HER2  breast tumors and in basal compared to luminal  and HER2  breast tumors  tumors . SFRP1 et tumors B.SFRP1 decreased expression in tumor tissue compared to normal breast tissue seemed to be mediated by its promoter, methylation , PGR , and HER2  expression, while it was negatively correlated with ESR2 expression . Corroborating our previous study, which demonstrated that SFRP1 expression in non-tumoral breast tissue was inversely associated with age-lobular involution, we saw that SFRP1 expression in non-tumoral tissue from the Benz 2020 dataset was positively correlated with the number of terminal ductal lobular units , PGR , and HER2  expression, while it was positively correlated with ESR2 . The average age was also significantly different between both groups, i.e., 235.1 ± 56.9 days in the nulliparous group compared to 319.4 ± 48.8 days in the multiparous group . The average mammary gland weight was higher in multiparous (0.4 ± 0.2 g) than in nulliparous  meaning that mice mammary gland weight is associated with the parity status, independently from age.As we previously observed a difference in SFRP1 expression between nulliparous and parous women after perimenopausal lobular involution , and in To attest that mice ex vivo models could be a good model for a lobular involution study, we first performed hematoxylin-eosin (H&E) staining on formalin-fixed paraffin-embedded mice mammary gland tissues –F. A mamWe previously observed that SFRP1 expression was different among nulliparous and parous women, in premenopausal compared to postmenopausal women, as well as in the presence compared to in the absence of breast microcalcifications. Along the same lines, we decided to mimic these conditions ex vivo by cultivating organoids obtained from nulliparous and multiparous mice, in the presence or in the absence of E2 and/or HA crystals .Sfrp1 expression was similar between organoids obtained from nulliparous and multiparous mice and grown in EtOH condition , and the organoids from nulliparous mice grown in the E2 + HA condition . Such a difference was not observed in organoids obtained from multiparous mice, corroborating the hypothesis that nulliparous and multiparous mammary glands do not respond to E2 stimulation in the same way . Similar results were obtained in E2 + HA condition compared to EtOH condition, without any difference between E2 and E2 + HA conditions. No difference between EtOH + HA and EtOH conditions was observed in organoids obtained from nulliparous mice. These results suggest that E2 promotes luminal organization of the mammary epithelial cells obtained from nulliparous mice.Moreover, the difference in E2 response between nulliparous and multiparous groups was confirmed by the organoid phenotype analyses I–Q. Aftep-value < 0.05; p-value < 0.05, p-value = ns; Sfrp1 in murine organoids does not seem to be impacted by parity status, while it was decreased by E2 treatment. Altogether, these results confirm what was described in women in Sfrp1 expression is positively correlated with Esr1 and Pgr expression in non-tumoral mammary epithelial cells and is associated with the breast lobulo-alveolar phenotype.On the other hand, E2 treatment had no statistically significant impact on the phenotype of organoids from multiparous mice. Indeed, after 15 days of growth in EtOH condition, 32.0 ± 9.4% of total organoids were luminal, 60.9 ± 8.6% were opaque, and 8.6 ± 3.4% were lobular, while in E2 condition, 46.8 ± 13.4% of total organoids were luminal, 49.5 ± 13.4% were opaque, and 3.7 ± 1.6% were lobular. No difference was observed between organoids obtained from multiparous mice grown in the presence of HA + EtOH compared to EtOH alone. However, after 15 days of growth, 56.9 ± 5.7% of total organoids obtained from multiparous mice cultivated in E2 + HA condition were luminal, 38.8 ± 4.0% were opaque, and 4.3 ± 1.7% were lobular, while in EtOH condition, only 30 ± 1.2% were luminal (SFRP1 expression pattern across breast cell lines was similar to what we observed in breast cancer tissues from different molecular subtypes (https://xenabrowser.net/ (accessed on 18 September 2021)). We saw that, indeed, breast cancer cell lines from luminal subtypes express less SFRP1 than those from basal subtypes, and that normal breast cell lines express a higher SFRP1 level than invasive breast cancer cell lines . qPCR analyses confirmed the efficiency of the two shRNAs compared to shScramble condition. In the MCF10A cell line, sh62169 induced a 57.3 ± 26.5% decrease in SFRP1 expression, while sh62170 induced a 45.5 ± 26.6% decrease in SFRP1 expression compared to shScramble in EtOH condition  increase in MCF10A sh62169 + EtOH cell viability and a 26.9 ± 21.1%  increase in MCF10A sh62170 + EtOH viability compared to MCF10A shScramble + EtOH after 72 h of growth. Interestingly, treatment with E2 induced a 47.7 ± 9.0% increase in MCF10A shScramble cell viability, a 62.0 ± 29.0% increase in MCF10A sh62169 cell viability, and an 84.0 ± 21.0% increase in MCF10A sh62170 cell viability compared to MCF10A shScramble + EtOH , suggesting an additive effect of SFRP1 knockdown and E2 treatment on promoting non-tumoral cell line MCF10A viability.To further assess if the decrease in SFRP1 expression increases cell viability and proliferation , while spheroids obtained from MCF10A shScramble did not grow  while spheroids obtained from MCF10A shScramble did not grow  and an 18.6 ± 14.8% decrease in MCF10A sh62169 spheroid growth compared to MCF10A sh62169 EtOH . Combined with results presented in Spheroid assays confirmed that the decrease in feration H. Spheronot grow H. Similanot grow H. SurpriSFRP1 was associated with mammary branching , confirming the additive effects of both SFRP1 knockdown and E2 treatment on the non-tumoral MCF10A breast cell line. MCF10A sh62170 + EtOH demonstrated a 29 ± 8.3% increase in cell migration after 24h compared to MCF10A shScramble + EtOH , compared to shScramble, after 12 h of migration , and a 1.6 ± 1.1-fold increase in ESR2 in MCF10AT1 sh62170 + EtOH compared to shScramble + EtOH  and a 2.0 ± 0.9-fold  decrease, respectively, in c-MYC expression, compared to MCF10AT1 scramble + EtOH . Similarly, MCF10AT1 sh62170 + E2 showed a 17.3 ± 7.4%, a 44.0 ± 2.1%, and a 25.8 ± 11.7% increase in wound healing after 12 h, 24 h, and 30 h of migration, respectively, compared to MCF10AT1 shScramble + E2  and pre-tumoral (MCF10AT1) breast lesions was inversely correlated with ESR1 expression . However, this difference disappeared after 3 days of growth. On the other hand, SFRP1 overexpression was responsible for a decrease in MCF10DCIS cell line migratory abilities in EtOH condition . Inversely, in E2 condition, the overexpression of SFRP1 induced a 12.9 ± 8.4% increase in wound healing after 36 h of migration, compared to MCF10DCIS empty pLX_TRC304 . Altogether, these results suggest that SFRP1 reduced the migratory abilities of the MCF10DCIS cell line in the absence of E2 only. On the other hand, these results also raise the question of the potential oncogenic role of SFRP1 in pre-invasive lesions in the presence of E2.In order to assess the potential of iability G nor sphiability H, regardondition I, but noondition J. More pSFRP1 on breast cancer progression, we then overexpressed it in the basal-like invasive ductal carcinoma MCF10CA1a cell line. Like in MCF10DCIS, we detected very low SFRP1 expression in MCF10CA1a. Furthermore, as we previously observed that SFRP1 overexpression in MCF10DCIS induced an increase in ESR1 expression  and MCF10CA1a OxSFRP1 + EtOH . After 5 days of growth, MCF10CA1a pLX_TRC304 + E2 demonstrated a 12.3 ± 13.2% decrease in spheroid growth, while MCF10CA1a OxSFRP1 + EtOH demonstrated a 23.3 ± 0.5% decrease in spheroid growth and MCF10CA1a OxSFRP1 + E2 demonstrated a 26.9 ± 11.6% decrease in spheroid growth compared to MCF10CA1a pLX_TRC304 + EtOH . In addition, MCF10CA1a OxSFRP1 + EtOH showed an 8.2 ± 10.3% decrease in spheroid growth, while MCF10CA1a OxSFRP1 + E2 showed a 12.1 ± 4.4% decrease in spheroid growth compared to MCF10CA1a pLX_TRC304 + E2 . In addition to the reduction in MCF10CA1a viability, SFRP1 overexpression also reduced cell migratory abilities, in both EtOH . In E2 condition, SFRP1 overexpression induced a 17.0 ± 2.6% decrease and an 18.8 ± 8.1% decrease in MCF10CA1a wound healing after 24 h and 36 h of migration, respectively, compared to MCF10CA1a empty pLX_TRC304 . Taken together, these results confirm the potential of SFRP1 to act as a tumor suppressor in basal-like triple-negative breast cancer, notably by reducing both canonical  had a lower SFRP1 expression than patients who did not relapse . After SFRP1 expression was observed in the continuum of breast cancer risk and in breast cancer, compared to non-tumoral tissue, by analyzing public datasets [SFRP1 expression among tumoral tissue compared to non-tumoral tissue could be mediated by its promoter, hypermethylation, in luminal and HER2 invasive carcinomas [In the present manuscript, we first corroborated previous studies from our group and others, in which a decrease in datasets ,46,47,48rcinomas ,42,46.SFRP1 expression, as well as a different mammary gland phenotype, according to parity and microcalcification status [Sfrp1 and Esr1 expression in both groups. We also observed that the decrease in both Sfrp1 and Esr1 expression following E2 treatment was associated with a lobulo-alveolar phenotype in organoids obtained from nulliparous mice only. Interestingly, nulliparous women have a majority of type 1 lobules (>12 acini), which are more positive to ERα and PR than type 3 lobules (>80 acini), while parous women have more type 3 lobules, expressing less ERα and PR than type 1 lobules, which is concordant with the fact that organoids obtained from nulliparous mice expressed more Esr1 than those obtained from multiparous mice [Sfrp1 developed branched mammary glands, similar to pregnant mice in vivo, corroborating that the decrease in Sfrp1 expression by E2 treatment in organoids obtained from nulliparous mice increased organoid luminal organization ex vivo [Our results also supported those from another study performed by our group, in which we observed a differential regulation of n status . Indeed,lobules > acini, wous mice ,15,49,50 ex vivo .Sfrp1 expression. Interestingly, a study by dos Santos et al. reported the existence of an epigenetic memory of pregnancy in mice mammary glands [Sfrp1 expression by E2 treatment would not modify mammary gland phenotype, as we observed ex vivo in organoids obtained from multiparous mice. Moreover, Kanadys et al. observed that the duration of oral contraceptive use in women was associated with breast cancer risk, when the calculation of period of consumption was limited to the period preceding the first full-term pregnancy [SFRP1 was decreased in nulliparous compared to parous women following age-related lobular involution [Sfrp1 expression between both groups.In the present study, we also observed that organoids obtained from nulliparous mice developed luminal organization upon E2 treatment, while organoids obtained from multiparous mice seemed resistant to E2 treatment and became progressively opaque, while both demonstrated a decrease in y glands . Feigmany glands . If mammregnancy , corroboregnancy . Few sturegnancy ,60,61,62volution . Here, nSFRP1 expression in both non-tumoral MCF10A and hyperplasic MCF10AT1 cell lines induced an increase in the tumorigenic properties of the cells in vitro. In addition, a decrease in SFRP1 expression in non-tumoral and pre-tumoral cell lines also increased their sensitivity to E2 and the expression of ESR1. Interestingly, the public dataset analyses we performed also reported that SFRP1 expression was positively correlated with tumoral biomarkers such as ESR1, PGR, and HER2 in non-tumoral tissue, while it became negatively correlated with such genes in breast tumoral tissues in women, raising the potential causal role of SFRP1 dysregulation in breast carcinogenesis initiation. However, as a limit of these analyses, it is important to keep in mind that these correlations were not adjusted for age or for parity status. That being said, combined with the results we obtained in vitro on both MCF10A and MCF10AT1 cell lines, it is tempting to speculate that the “switch” in SFRP1 expression positive correlation with ESR1 expression is already observable in MCF10A and MCF10AT1 cell lines, corroborating the hypothesis that SFRP1 could be involved in early ERα-positive breast carcinogenesis. These results are also in concordance with the study of Gregory et al., in which the authors reported that breast hyperplasia samples expressed less SFRP1 than paired benign tissue, as well as that treatment of human breast explant cultures with E2 + recombinant SFRP1 decreased the response to E2 compared to treatment with E2 alone [SFRP1 expression.We also demonstrated that a decrease in E2 alone . HoweverESR1 expression, and a decrease in cell viability and migratory abilities in MCF7 overexpressing SFRP1 compared to the negative control. This was also in concordance with public dataset analyses, which demonstrated that in patients treated with tamoxifen adjuvant therapy, the 5-year relapse rate was lowest in patients expressing more SFRP1 than the median of expression, compared to patients who expressed lower SFRP1 than the median. Once more, these analyses were not adjusted for age and parity status, mainly because this last variable remains rarely available in public datasets. Therefore, the knowledge regarding the molecular impact of parity on breast cell phenotype is not well understood. Nevertheless, a few studies showed that high parity (>3 children) has been associated with an increased breast cancer mortality compared to nulliparous women [Subsequently, we confirmed the potential of SFRP1 to reduce luminal A aggressiveness by increasing its expression in the MCF7 cell line. Indeed, we observed a reduction in us women , and havus women . ConsideSFRP1 overexpression in MCF10DCIS pre-invasive and MCF10CA1a invasive triple-negative breast cancer cell lines reduced their aggressiveness in vitro. However, we observed that SFRP1 overexpression in MCF10DCIS increased ESR1 expression, and then increased cell migration abilities in E2 conditions, while it decreased this expression in EtOH condition, suggesting that the role of SFRP1 in ERα-negative ductal carcinoma in situ could be E2-dependent. On the other hand, SFRP1 overexpression in the MCF10CA1a cell line did not modify ESR1 expression in EtOH condition. However, in E2 condition, SFRP1 expression was further increased, and ESR1 expression further decreased, resulting in an exacerbation of the reduction in cell viability compared to SFRP1 overexpression alone. In accordance with a previous work from Bernemann et al., we found that, in the public dataset, TNBC patients who had a complete pathological response to chemotherapy had a higher SFRP1 expression than non-responder, suggesting that SFRP1 could be a potential tumor suppressor in TNBC, as well as a good prognostic marker [Finally, we showed that c marker . NeverthSFRP1 expression is positively correlated with ESR1 in non-tumoral breast tissue, while it is negatively correlated with ESR1 in invasive breast cancer. In addition, Sfrp1 expression is reduced upon E2 treatment in murine mammary organoids, independently from the mouse’s parity status. They also confirmed the existence of the memory of pregnancy, responsible for a decrease in organoid sensitivity to E2. In addition, the decrease in SFRP1 expression in non-tumoral cell lines induced an increase in mammary branching in vitro and an increase in tumorigenic properties, such as viability and migration, suggesting a potential causal role of the decrease in SFRP1 expression in early breast carcinogenesis. On the other hand, the overexpression of SFRP1 in pre-invasive and invasive breast cancer cell lines induced a reduction in cell viability and migratory abilities, suggesting that SFRP1 could be a potential tool against breast cancer progression. Further analysis to better understand the functional crosstalk between SFRP1 and ERα should be performed in order to assess the causal role of SFRP1 in luminal carcinogenesis.To conclude, the presented results showed that


Neural stem cells (NSCs) proliferation and differentiation rely on proper expression and posttranslational modification of transcription factors involved in the determination of cell fate. Further characterization is needed to connect modifying enzymes with their transcription factor substrates in the regulation of these processes. Here, we demonstrated that the inhibition of KAT2A, a histone acetyltransferase, leads to a phenotype of small eyes in the developing embryo of zebrafish, which is associated with enhanced proliferation and apoptosis of NSCs in zebrafish eyes. We confirmed that this phenotype is mediated by the elevated level of PAX6 protein. We further verified that KAT2A negatively regulates PAX6 at the protein level in cultured neural stem cells of rat cerebral cortex. We revealed that PAX6 is a novel acetylation substrate of KAT2A and the acetylation of PAX6 promotes its ubiquitination mediated by the E3 ligase RNF8 that facilitated PAX6 degradation. Our study proposes that KAT2A inhibition results in accelerated proliferation, delayed differentiation, or apoptosis, depending on the context of PAX6 dosage. Thus, the KAT2A/PAX6 axis plays an essential role to keep a balance between the self-renewal and differentiation of NSCs. The developmental stages of the neural lineage cells are usually defined by the stage-specific markers including transcription factors. These transcription factors are responsible for the self-renewal, proliferation, neurogenesis, and astrogliogenesis of different neural stem cells (NSCs) or progenitors , 2. For Kat2a gene, is the first histone acetyltransferase that links histone acetylation to chromatin remodeling, which results in transcriptional activation of the target gene , Pax6-null progenitor cells proliferate more slowly than wildtype controls, which indicates that the effects of Pax6 on the proliferation of cortical progenitors are context dependent. Mi et al.  as the E3 ligase that is responsible for the PAX6 ubiquitination. Our findings provide insight into the mechanism of the proliferation and differentiation of NSCs through posttranslational modifications on PAX6.In this study, we found that KAT2A inhibition gives rise to a phenotype of small eyes accompanied by an increased PAX6 level during zebrafish development. This result linked a HAT to the PAX6 level that was also verified in et al.  . The developing eyes are composed of many NSCs at 24 hpf, and the developing retinae and lens could be observed (kat2a MO (D and E), indicating the phenotype might be due to exhausted NSCs in the developing zebrafish. We further speculated whether kat2a knockdown gives rise to more cell death. We utilized terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to detect apoptosis in the developing eyes and observed an increase in the number of apoptotic cells in developing eyes with reduced kat2a expression (F and G). These data showed that both proliferation and apoptosis were augmented in kat2a morphants, which proposed that early exhaustion of NSCs might occur in developing eyes. Then, the phenotype of small eyes might be the result of overproliferation and subsequent apoptosis of NSCs during development.The roles of Kat2a during development have been defined through genetic studies in mice and other vertebrates , 23. Loset al.  reporteddes (MO) . Along wcontrols , A–C. Thobserved , 26 at tkat2a MO , D and Epression , F and Gkat2a morphants caused a phenotype of small eyes. Owing to the similarity of the phenotype of kat2a knockout/knockdown and that of pax6 mutations .utations , 29, 30,affected , A–D.Figpax6a in zebrafish embryos. The results showed that upregulation of pax6a was sufficient to cause a reduction in eye size that was similar to the phenotype of Pax6 MO (E and F), and the higher dosage of Pax6 led to a severer abnormality in the eyes. We thus detected cell proliferation and apoptosis in developing eyes. The forced expression of PAX6 at doses from 50 pg to 500 pg were verified by Western blotting, and PAX6 overexpression caused accelerated proliferation and increased apoptosis (G–L). Moreover, we downregulated pax6a using MO for comparison purposes, and the individuals with downregulation of pax6a exhibited small eyes too (A–D). This suggests that Pax6 is essential for the expansion of NSCs in developing eyes. Similar to those with kat2a knockdown, embryos with excessive Pax6 had more apoptotic cells.To find out if an increase in Pax6 level was part of the reason for reduced eye size, we overexpressed  Pax6 MO , E and Fpoptosis , G–L. Moeyes too , A–D. Thkat2a knockdown by downregulating Pax6 at the same time. We then performed a double knockdown of kat2a and pax6a in zebrafish embryos using morpholino oligonucleotides  and observed the morphology of the eyes. The results showed that kat2a MO increased protein level of PAX6, which could be rescued by pax6a MO (M and N). Although solely downregulating kat2a using 0.3 mmol MO results in eye malformation, 0.01 mmol pax6a MO rescued the eye phenotype in kat2a morphants (O and P). These results strongly implicated that the small eye phenotype of kat2a MO was due to the accumulation of Pax6 protein in the developing eyes of zebrafish.Kat2a harbors more function than just acetylating Pax6, but if the regulation of NSC differentiation by Kat2a is mainly mediated by the reduction of Pax6, we could rescue the effect of pax6a MO , M and Norphants , O and PIn vitro differentiation of NSCs was induced using fibronectin and removal of FGF and EGF in the medium. PAX6 and SOX2 are transcription factors tightly related to neurogenesis. The RT-qPCR assay showed that mRNA of Pax6 did not vary significantly; however, protein PAX6 levels reduced along with differentiation that was verified by Western blotting and immunostaining assay (A–D). Meanwhile, another transcription factor, SOX2, also showed decreased levels (E–H). In contrast, the KAT2A level increased gradually during differentiation (I–L). These data were consistent with the negative correlation between the levels of KAT2A and PAX6 in the developing eyes.To confirm the relationship between KAT2A and PAX6 during neural development, we investigated the expression pattern of KAT2A and PAX6 in primarily cultured and differentiated NSCs from E14 rat embryos. ng assay , A–D. Med levels , E–H. Inntiation , I–L. ThA–D). The proliferation of NSCs increased upon treatment, while the apoptosis did not vary significantly (E–H) at the dosages tested, which might be due to the sufficient supply of nutrients and space in the culture system. Then, we investigated whether the MB-3 treatment could affect the level of PAX6. As expected, in comparison with the control where PAX6 gradually declined along with differentiation, the expression of PAX6 remained steady throughout the 48 h of induction of differentiation (I and J). In contrast, the SOX2 level declined during NSCs differentiation with statistical significance at 48 h and 72 h after induction of differentiation, and the treatment of MB-3 did not hamper the decline of SOX2 level (I and K). Hence, we propose that the reduction of PAX6 level during NSC differentiation is specially regulated by KAT2A.As PAX6 and KAT2A are negatively related in their protein levels during NSC differentiation, there could be a direct or indirect connection between the decrease of PAX6 and the activity of KAT2A in differentiating NSCs. First, we examined if the change in the level of PAX6 is in response to KAT2A. We applied KAT2A specific inhibitor MB-3  to confificantly , E–H at ntiation , I and JX2 level , I and KKat2a to downregulate the expression of KAT2A by electroporation and observed the NSCs after induction of differentiation for morphology of the cells, proliferation, and expression of PAX6. After 24 h of induction, the cells showed shorter processes, suggesting differentiation was inhibited after depletion of KAT2A (A and B). We used EdU to label proliferating cells and found more proliferating cells after the knockdown of Kat2a (C and D). Western blotting of PAX6 showed an elevated level of PAX6 after knockdown of Kat2a (E–G). These results suggest that KAT2A downregulates the level of PAX6 and promotes differentiation in NSCs.Also, we transfected NSCs with siRNA targeting of KAT2A , A and Bof Kat2a , C and Dof Kat2a , E–G. ThA), with Pearson’s R values of 0.68 and 0.41, respectively. Meanwhile, the separate signals were also observed in NSCs. We believed that the KAT2A protein is multifunctional, and then both colocalized and separate signals with PAX6 protein could be present in cells.KAT2A negatively regulates PAX6 level in NSCs; we then asked what the underlying mechanism is. The first question we addressed is whether the two molecules colocalize or interact in NSCs. We performed immunofluorescence staining on cultured differentiation NSCs to detect any interaction between KAT2A and PAX6. We detected colocalization of KAT2A and PAX6 both in undifferentiated and differentiated NSCs A, with PB). Meanwhile, the interaction between KAT2A and SOX2 was not detected (C) in NSCs. We also detected the expression of other HAT during NSCs differentiation. The results showed that CREB-binding protein (CBP) maintained a stable expression, and P300/CBP-associated factor (PCAF) displayed low expression level in NSCs, which was different from the pattern of KAT2A (A–D).Next, we performed an immunoprecipitation assay to find evidence for any interaction between KAT2A and PAX6. Using an anti-PAX6 antibody for immunoprecipitation, both Pax6 and KAT2A could be detected in the precipitate by Western blot B. Meanwhdetected C in NSCsof KAT2A , A–D.D–F). Then, we dissected the N-terminal region into smaller truncated proteins and performed GST-pulldown for each of them and did not observe an interaction between these truncates and KAT2A (A–E). These results suggested that the N-terminal region from the PD domain to the HD domain is required for the interaction between PAX6 and KAT2A.To investigate whether KAT2A and PAX6 could interact directly, we conducted a GST-pulldown assay. The results showed that the GST-tagged KAT2A could successfully pulldown the His-tagged PAX6. We further generated several PAX6 truncated proteins and performed a GST-pulldown assay to locate the specific domain of PAX6 responsible to interact with KAT2A. We revealed that the N-terminal region containing the PD and HD domains is essential for the interaction , D–F. Thnd KAT2A , A–E. ThA). Also, we purified the His-tagged KAT2A and PAX6 protein expressed in Escherichia coli and conducted in vitro acetylation assay. The results clearly showed that acetylated PAX6 was observed in the presence of KAT2A and acetyl-CoA (B and C).KAT2A is known for its acetyltransferase activity. To verify whether PAX6 is a substrate of KAT2A, we observed acetylated lysine in the precipitate obtained using PAX6 antibody from NSCs, suggesting lysine acetylation indeed occurred in NSCs A. Also, etyl-CoA , B and CD and E). Then, we immunoprecipitated PAX6 in NSCs in the presence of MG-132 and detected the ubiquitinated PAX6 with an antibody against ubiquitinated lysine residues. The results showed that the ubiquitinated Pax6 could be detected both in undifferentiated and differentiated NSCs (F). Then, we questioned whether the ubiquitination of PAX6 was regulated by the acetylation of PAX6 by KAT2A. We thus inhibited the activity of KAT2A in NSCs by MB-3 and found the ubiquitination level of PAX6 decreased (G and H). These results support that, along with NSC differentiation, PAX6 is ubiquitinated, which is promoted by its acetylation catalyzed by KAT2A.Since there was a decline in the PAX6 level, which might be mediated by ubiquitination, we examined situations of PAX6 ubiquitination in differentiated NSCs and investigated whether the inhibition of KAT2A could affect PAX6 ubiquitination. For the reason that ubiquitinated PAX6 will experience degradation by proteasome, we applied proteasome inhibitor MG-132 , D and Eted NSCs F. Then, ecreased , G and Hvia site-directed mutagenesis to mimic the lysine acetylation were transfected into COS7 cells. Compared with WT PAX6, K75Q and K264Q showed markedly reduced PAX6 protein (A and B). Meanwhile, the protein level of the K270Q mutant did not change significantly. We then confirmed the phenotype of K75Q and K264Q mutant in NSCs. In this assay, the double-mutant K75Q/K264Q was also tested, and the control mutants K75R and K264R were introduced as well. The results showed that the mutants K75Q, K264Q, and K75Q/K264Q led to a reduced level of PAX6, and the wildtype and K75R and K264R mutants did not show a similar effect (C–E). Therefore, our data proposed that the acetylation of K75 and K264 contributed to PAX6 degradation.To determine the site of acetylation of PAX6, we conducted mass spectrometry analysis of PAX6 from the immunoprecipitates from NSCs, and the results proposed three potential lysine acetylation sites, K264, K75, and K270 of PAX6 . To veri protein , A and Br effect , C–E. Thhttp://bdmpub.biocuckoo.org/index.php), which indicates K246 as a potential candidate with the highest score. We designed the mutant K246Q to test the effect on the PAX6 level. The results showed that the mutation led to attenuation of Pax6 degradation, which proposed that K246 was the site of ubiquitination. Moreover, K264Q mutation resulted in augmented degradation of PAX6, while the double mutation K246Q/K264Q significantly rescued the effect of K264Q (F and G). These results strongly indicated that K246 ubiquitination was a site of ubiquitination that might be regulated by acetylation modification of PAX6.Then, we further investigated the ubiquitination sites of PAX6 during NSCs differentiation. We did not obtain the information on ubiquitinated PAX6 in the data of mass spectrometry, which might be due to the instability of ubiquitinated proteins. We thus predicted the sites of ubiquitination for PAX6 using the online software BDM-PUB . Moreover, we performed the coimmunoprecipitation assay in HEK293T cells that were transfected with the tagged RNF8 and PAX6. The results clearly confirmed the interaction of RNF8 and PAX6 (D). In addition, the endogenous RNF8 could be coimmunoprecipitated with PAX6 in cultured NSCs (I). Inhibition of RNF8 expression protected PAX6 protein from degradation (E and F). Actually, another E3 ligase, Tripartite Motif Containing 11 (TRIM11) had been reported to play a role in the degradation of insoluble PAX6. Tuoc et al. , suggesting that RNF8 was the involved E3 ubiquitin ligase. The results above suggest that KAT2A promotes ubiquitination of PAX6 by RNF8 during the differentiation of NSCs.To search for E3 ubiquitin ligases involved in the PAX6 degradation pathway, we retrieved the mass spectrometry results of the immunoprecipitation reaction of PAX6 from NSCs and found an E3 ligase RNF8, which is involved in DNA damage response in previous reports , 34. Theand PAX6 D. In addred NSCs I. Inhibiradation , E and Fc et al.  detectedt Trim11 , G and HA–C). In developing zebrafish embryos, the downregulation of rnf8 reduced eye sizes, promoted the accumulation of Pax6, and increased both proliferation and apoptosis (D–K), which showed similar phenotype as the embryos with downregulation of kat2a. These data also support the idea that RNF8 participates in the PAX6 degradation that is facilitated by the KAT2A-mediated acetylation.To confirm that RNF8 is an essential player in the regulation, we observed the effect of RNF8 inhibition in differentiating NSCs and developing zebrafish embryos. Effective downregulation of RNF8 appeared to cause a mimic phenotype of downregulation or inhibition of KAT2A in NSCs, in terms of promoting proliferation and reducing the length of neurites in neuron-like cells , A–C. Inpoptosis , D–K, whIn summary, we revealed that KAT2A binds to and acetylates PAX6 to promote its ubiquitination mediated by RNF8, which in turn intensifies the downregulation of PAX6 to inhibit the proliferation and drive the differentiation of NSCs. Upon knockdown or inhibition of KAT2A, a decrease in KAT2A weakens the KAT2A-dependent degradation of PAX6, and the accumulated PAX6 leads to overproliferation and apoptosis . This stTranscription factors modification is essential for their transcriptional activity, distribution, stability, and degradation. In this study, we revealed a KAT2A/PAX6/RNF8 axis that controls the level of PAX6 during NSCs differentiation. We found that KAT2A-mediated acetylation of PAX6 leads to enhanced degradation of PAX6, which modulated the proliferation, differentiation, and apoptosis of NSCs.Drosophila germline stem cells  were provided by the Zebrafish Facility at Nantong University under standard laboratory conditions of 14 h light/10 h darkness at 28.5 °C in E3 buffer, which contains 6.4 mM KCl, 0.22 mM NaCl, 0.33 mM CaClEach embryo received about 1 nl of the Morpholino Oligonucleotide solution  by the Animal Care and Use Committee of Nantong University.For immunofluorescence, 15 zebrafish embryos were pooled in a 1.5-ml tube and soaked using 1 ml 4% paraformaldehyde (PFA) (in PBS) at room temperature for 5 min and repeated with fresh 4% PFA for another 5 min. Then the sample was fixed in another 1 ml of 4% PFA for 30 min. The embryos were then washed with 1× PBST (contains 0.1% Tween-20) three times, 10 min each. Then the embryos were placed in 1× PBST (1% TritonX-100) at room temperature for 30 min. Next, the embryos were placed in 500 μl of blocking reagent  for 2 h at room temperature. The embryos were incubated with 500 μl of 1:400 primary antibody solution at 4 °C for 4 days. The embryos were washed with 1× PBST (containing 0.1% Tween-20) four times, 10 min each. The embryos were incubated with 300 μl of secondary antibody solution (1:500) at 4 °C for 2 days. Again, the embryos were washed with 1× PBST (containing 0.1% Tween-20) four times, 10 min each. The embryos were stained with 500 μl of DAPI (10 μg/ml) for 1 h and finally washed with 1× PBST (containing 0.1% Tween-20) three times, 10 min each. Antibodies used were Rabbit-anti-PAX6  and Cy3 AffiniPure Goat Anti-Rabbit IgG (H + L) .For BrdU staining, the embryos were first placed on ice in E3 buffer for 15 min, and then changed with 1 ml 10 mM BrdU/15% dimethyl sulfoxide (DMSO) and kept on ice for 30 min. Then they were transferred into E3 buffer at 28.5 °C and kept at 28.5 °C for 5 min. Then they were fixed with PFA and transferred into methanol and kept at −20 °C overnight. The embryos were rehydrated in methanol: PBST (3:1), methanol: PBST (1:1), and methanol: PBST (1:3) in tandem, 5 min each, and washed with PBST twice, 5 min each. Then the embryos were digested with Proteinase K (10 μg/ml) for 10 min, washed with PBST for 10 min, and postfixed using PFA for 20 min. The embryos were washed with water quickly three times, in 2 mol/l HCl two times, and incubated in 2 mol/l HCl for 1 h, and washed in PBST three times, 5 min each. The samples were then soaked in blocking solution  for 30 min and then incubated in 1:100 BrdU (Bu20a) Mouse mAb anti-BrdU antibody (CST #5292) at 4 °C for 5 days and washed five times in PBST, 10 min each. Next, the samples were incubated in 1:500 Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H + L)  at 4 °C for 2 days and washed in PBS five times, 10 min each. The embryos were finally stained with DAPI (10 μg/ml) for 1 h and washed with 1× PBS five times, 5 min each.For the TUNEL experiments, the manufacturer’s instructions were followed .NSCs were isolated and induced for differentiation as reported , 42. BriCOS7 cells were maintained and cultured in a DMEM medium  plus 10% FBS . Cells were transfected using Lipofectamine 3000  according to the supplier’s guidelines.For MB-3 and MG132 treatments, the doses used were 50 μM for MB-3 (Sigma M2449) and 20 μM for MG132 (Selleck S2619).The cultured cells were fixed in 4% paraformaldehyde for 20 min and were incubated with Rabbit anti-KAT2AL2 C26A10 (KAT2A) antibody , Mouse anti-KAT2A/KAT2A Monoclonal Antibody , Rabbit anti-PAX6 antibody , monoclonal Mouse anti-PAX6 antibody , Rabbit anti-RNF8 antibody (1:400 Abcam ab105362), Rabbit anti-SOX2 antibody (1:400 Abcam ab97959), Rabbit anti-TUJ1 (1:400 Abcam ab18207), respectively. Fluorescence-labeled secondary antibodies (Alexa Fluor 488 affinipure Goat anti-rabbit IgG (H + L) , Cy3 affinipure Goat anti-rabbit IgG (H + L) , and Alexa Fluor 488 AffiniPure Goat Anti-Mouse IgG (H + L) ) were added and further incubated, followed by Hoechst 33342 staining. Visualization was performed on a Leica TCS SP5 confocal microscope.EdU Cell Proliferation Detection Kit (RiboBio) was used for cell proliferation detection. Briefly, EdU (Reagent A) was diluted into culture media at 1:1000 to a final volume of 50 mM, and cells were treated for 2 h before harvesting. Cells were then fixed with 4% PFA for 30 min and washed with 2 mg/ml glycine in PBS once and with PBS twice. Cells were then stained with 1× Apollo staining solution for 30 min and washed with PBS with 0.5% TritonX-100 for 10 min twice. Cells were stained with 1 μg/ml Hoechst 33342 for 15 min and washed with PBS for 5 min three times before Imaging.Western blot analysis was performed as described . BrieflyAntibodies used were anti-PAX6 antibody , anti-KAT2A antibody , Mouse anti-β-Tubulin III (Tuj1) antibody , Mouse anti-GAPDH antibody , Mouse anti-beta Actin antibody , Rabbit anti-SOX2 antibody , Rabbit anti-CBP antibody , Mouse anti-PCAF antibody , Mouse anti-Ubiquitin antibody , Rabbit anti-acetylated-lysine antibody , HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) , and HRP-conjugated Goat anti-rabbit IgG (Santa Cruz sc-2054).Expression plasmids were transformed into BL21 bacteria (TransGen). Expression was induced with 0.4 mM IPTG at 16 °C shaking at 120 rpm overnight. Proteins were quantified using the BCA method and purified with His or GST beads.GST-pulldown was performed using Pierce GST Protein Interaction Pull-Down Kit (Pierce 21516) following the manufacturer’s instructions. Briefly, 50 μl GST beads were mixed with 125 μl TBS. 1 ml GST-bait protein was mixed with the beads and spun at 175 rpm for 30 min. The samples were placed on a magnetic stand for 10 s. The supernatant was collected and mixed with 6× loading buffer and boiled to form the postbinding control. The beads were washed with 250 μl TBS, mixed with a prey protein solution, and spun at room temperature at 175 rpm for 30 min. The beads were placed on the magnetic stand for 10 s. The supernatant was collected, mixed with 6× loading buffer, and boiled to form the post-IP control. The beads were washed with 250 μl TBS and spun at 175 rpm for 1 min and placed on the magnetic stand for 10 s to remove supernatant and washed repeatedly three times. A volume of 125 μl Elution Buffer was added to the beads for elution. A volume of 25 μl loading buffer was added to the elution before boiling for 10 min.g for 20 min. After measurement of protein concentration, part of the solution was mixed with 6× SDS loading buffer and boiled for 5 min and diluted to 2 μg/μl for the input control. The rest of the solution was diluted with PBS to 1.5 μg/μl, and 30 μl S-protein Agarose was added per 500 μl supernatant. The antibodies were then added to the samples and mixed by slow spinning at 4 °C overnight. The precipitate was collected by centrifuge at 14,000g for 15 min and washed with cool IP lysis buffer twice and resuspended with 60 μl 2× loading buffer. The samples were boiled for 8 min, briefly spun, and the supernatant was used for Western blotting.Coimmunoprecipitation was performed using Pierce Co-Immunoprecipitation (Co-IP) Kit (Pierce 26149) following the manufacturer’s instructions. Briefly, 30 μl of S-protein Agarose was used for each reaction. Cells were treated with lysis buffer at 4 °C and centrifuged at 14,000A 50-μl reaction was prepared consisting of 5 μg PAX6, 1 μg KAT2A, 2 mM Ac-CoA, 50 mM Tris-HCl (pH 8.0), 100 mM EDTA, 1 mM PMSF, 5 mM DTT, 2.5 mM NAM, 1 μM TSA, and 10% glycerol. The reaction was placed at 37 °C for 2 h and stopped by adding 10 μl 6× loading buffer and incubating in boiling water for 5 min before detection with immunoblotting.Full-length Pax6 cDNA was cloned by RT-PCR from a rat and inserted into pEasy-T3 (Transgen) and transcribed using mMessage mMachine Kit (Thermo Fisher Scientific).Reverse transcription was performed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher K1622), and PCR reactions were prepared using AceQ qPCR SYBR Green Master Mix . qRT-PCR reactions were performed on a StepOne plus Real-Time PCR System. Primers are listed in Cultured cells were observed using a Leica DMi8 inverted fluorescent microscope. Bright-field images of zebrafish embryos were taken using an Olympus SZ61 stereomicroscope. Fluorescent images were taken using a Leica TCS SP5 confocal microscope.Plasmids used in this study (and their origins): pmCherry-N1-KAT2A, pEGFP-N1-PAX6, pEGFP-N1-RNF8, pEGFP-N1-Trim11, pEGFP-N1-PAX6-K75R, pEGFP-N1-PAX6-K75Q, pEGFP-N1-PAX6-K264R, pEGFP-N1-PAX6-K264Q, pEGFP-N1-PAX6-K246Q, pEGFP-N1-PAX6-K75Q+264Q, pEGFP-N1-PAX6-K246Q+264Q, pET-22b(+)-PAX6, pET-22b(+)-KAT2A His-FLAG-RNF8, and His-FLAG-TRIM11. All plasmids were purchased from Anhui General Biology.For acetylation site analysis, pcDNA3.1(+)-stag-PAX6 was transfected into HEK293T cells and S-Tag-PAX6 was harvested by immunoprecipitation in 48 h using Anti-S-tag antibody  and sent to Shanghai Applied Protein Technology Co, Ltd for mass spectrometry analysis on acetylated lysine.m/z scan range was 350 to 1800 for full scans, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between 1 MS scan and 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control was set at 5E4.Gel pieces were destained in 50 mM NH4HCO3 in 50% acetonitrile (v/v). Gel pieces were dehydrated with 100 μl of 100% acetonitrile for 5 min and rehydrated in 10 mM dithiothreitol and incubated at 56 °C for 60 min. Gel pieces were once again dehydrated in 100% acetonitrile and rehydrated with 55 mM iodoacetamide. Samples were incubated at room temperature in the dark for 45 min. Samples were washed with 50 mM NH4HCO3 and dehydrated with 100% acetonitrile. Samples were rehydrated with 10 ng/μl trypsin and resuspended in 50 mM NH4HCO3 on ice for 1 h. Samples were digested with trypsin at 37 °C for 20 h. Peptides were extracted with 50% acetonitrile/5% formic acid and then 100% acetonitrile. Peptides were lyophilized to completion and resuspended in 2% acetonitrile/0.1% formic acid. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a home-made reversed-phase analytical column . The gradient comprised an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23% to 35% in 8 min, and up to 80% in 3 min, then was kept at 80% for the last 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The Based on the raw data, Mascot 2.2 was used to search the rat protein database uniprot_Rattus_norvegicus_36135_20200217/zjk_3_20200721 established by Shanghai Applied Protein Technology Co, Ltd with these parameters: Enzyme, Trypsin; Fixed modifications, Carbamidomethyl (C); Variable modifications, Oxidation (M)/Acetyl(K)/GlyGly(K)/Phospho(STY); Missed Cleavage, 2, Peptide Mass Tolerance, 20 ppm; Fragment Mass Tolerance, 0.1 Da; Filter by score ≥20.https://imagej.net/software/fiji) per imaged area. Bright field and fluorescence images were processed using Fiji , Adobe Photoshop (image rotation), and Adobe Illustrator . Representative images were taken from measurements of at least three repeats. Proliferating or apoptotic cells in each imaged eye were counted using Fiji on z-stack images after maximum-projection, and data are presented as a total number of proliferating or apoptotic cells. In the cultured NSCs, the proliferating and apoptotic cells were counted and quantified in five areas in each sample, and the ratios were calculated. Blinding was performed by hiding grouping information when conducting measurement. Axon lengths were determined by measuring the distance from the cell body to the growth cone of the longest process.The number of cells were counted using Fiji (Relative mRNA expression levels were normalized to endogenous GAPDH and calculated using the ΔΔCt method. Differences were determined comparing with control samples, which were treated with control siRNA or control Morpholino Oligonucleotides.For Western blotting, protein expression was normalized to the levels of endogenous GAPDH or β-Actin. Negative controls used for Western blotting were control Morpholino Oligonucleotides for gene specific Morpholino Oligonucleotides, mCherry mRNA for PAX6 mRNA injections, DMSO for MB-3 treatment. Relative ubiquitinated PAX6 level was normalized by dividing the amount of ubiquitinated protein by the amount of PAX6.t test, one-way ANOVA, and two-way ANOVA were used for comparisons. Following one-way ANOVA, Tukey’s multiple comparisons or Dunnett’s multiple comparisons were performed for comparison between every two groups or between one control group and all other groups, respectively. Following two-way ANOVA, Fisher’s LSD was performed for multiple comparisons. One-tailed p values are provided in statistical diagrams.All statistical analysis was performed in GraphPad Prism 8. Unless stated otherwise, all measurements are shown as mean ± SEM. Student’s All data generated in this study are included in this article and the supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Sodium-Glucose Cotransporters (SGLT) mediate the uphill uptake of extracellular sugars and play fundamental roles in sugar metabolism. Although their structures in inward-open and outward-open conformations are emerging from structural studies, the trajectory of how SGLTs transit from the outward-facing to the inward-facing conformation remains unknown. Here, we present the cryo-EM structures of human SGLT1 and SGLT2 in the substrate-bound state. Both structures show an occluded conformation, with not only the extracellular gate but also the intracellular gate tightly sealed. The sugar substrate are caged inside a cavity surrounded by TM1, TM2, TM3, TM6, TM7, and TM10. Further structural analysis reveals the conformational changes associated with the binding and release of substrates. These structures fill a gap in our understanding of the structural mechanisms of SGLT transporters. SGLT plays key roles in sugar uptake and reabsorption. Here the authors provided the cryo-EM structures of human SGLT1 and SGLT2 in the substrate-bound occluded state, uncovering the conformational changes of SGLT during sugar transport. These transporters utilize the electrochemical gradient to drive the uphill transport of glucose and play essential roles in human health2. SGLT1 (SLC5A1) and SGLT2 (SLC5A2) are two important subtypes of this family, and their physiological functions are well-documented. The loss-of-function mutations of genes encoding SGLT1 and SGLT2 could lead to intestinal glucose-galactose malabsorption and familial renal glucosuria in humans, respectively1. SGLT2-specific inhibitors are widely prescribed for the treatment of diabetes3. Moreover, SGLT inhibitors also show promise in the treatment of cardiovascular diseases, constipation, and certain types of cancer7. Pioneering structural work on the bacterial homolog vSGLT from Vibrio parahaemolyticus revealed that SGLT proteins have 14 transmembrane helices, which assemble into a LeuT-like fold8. In the presence of substrate and sodium, vSGLT resembles an inward-facing conformation8. The recent development of cryo-EM facilitates the structure determination of human SGLT (hSGLT). A consensus-mutated hSGLT1 in the apo state shows an inward-open structure (hSGLT1inward-open)9. The SGLT2-specific inhibitor empagliflozin and the non-specific inhibitor LX2761 occupy not only the sugar substrate-binding site but also the extracellular vestibule to lock hSGLT2 and hSGLT1 in the outward-open conformation11. Despite this progress, the structure of SGLT in the occluded conformation is yet unknown, representing a key missing state during the transporting cycle of SGLT, which precludes a comprehensive mechanistic understanding of how SGLT functions.Sugars, such as glucose, are one of the most important nutrients across all kingdoms of life. The uptake of glucose against its concentration gradient is mediated by the sodium-dependent glucose cotransporters (SGLT)In this work, we present the cryo-EM structures of hSGLT1 and hSGLT2 in the substrate-bound occluded conformation. We also show the structure of hSGLT2 in the apo state in an inward-open conformation. Structural comparisons of SGLT between states reveal how the essential structural elements concertedly move during the transport cycle.GFP-MAP17nb protein for structural studies12. It was reported that replacing the equatorial hydroxyl group at the 4-position of glucose with a fluorine atom yielded a glucose analogue, 4-deoxy-4-fluoro-d-glucose (4D4FDG), with an enhanced affinity for SGLT1 (K0.5 is 0.07 mM) compared with glucose (K0.5 is 0.5 mM)  into the hSGLT2GFP-MAP17nb protein in nanodisc , viewed from the extracellular side . Since the structure of hSGLT in the outward-open state without any ligand bound is not available, we use the LX2761-bound hSGLT1 structureing Fig. . In the ing Fig. . In addiide Fig. . These cide Fig.  and sugaide Fig. , while tide Fig. .Fig. 3St9 10 when further sharpening was applied  and SGLT2 has one sodium-binding site (Na2)14. However, we found that in hSGLT1 or hSGLT2, the conformation of the moving region  changes largely, but the structural changes of the less-mobile region  are less pronounced  moves relative to the “hash motif”  in a rigid body fashion during conformational transition11, hSGLT2 absolutely requires the auxiliary subunit MAP17 for uptake activity, but hSGLT1 does not15. Our structural analysis reveals that MAP17 might play a structural role in the transporter complex because its structure and its interaction with hSGLT stay largely unchanged during the transport cycle. The surface labeling experiment using the antibody against the extracellular side of hSGLT2 showed that robust surface expression of hSGLT2 requires MAP17. This is in contrast to the previous study showing that co-expression of MAP17 did not enhance the surface expression of hSGLT215. However, that study was based on confocal imaging, which might be limited in spatial resolution and less quantitative than our surface labeling experiment. Our structural and functional data favor a model in which hSGLT2 alone might be subjected to intracellular retention and MAP17 is essential for the transportation of hSGLT2 to the plasma membrane to perform the uptake function. In contrast, hSGLT1 alone could traffic to the cell surface to perform sugar uptake.Although hSGLT2 and hSGLT1 share high structural and sequence similarities, and both of them could interact with MAP17Taken together, the structures of human SGLTs described here provide a glimpse of the key intermediate occluded state during sugar transportation and provide a solid foundation to further study the mechanisms and dynamics of these transporters.2 and 70% humidity. AD293 adherent cells (Agilent Technologies) were cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% FBS at 37 °C with 5% CO2. The cell lines were routinely checked to be negative for mycoplasma contamination but have not been authenticated.Sf9 insect cells  were cultured in Sf-900 III serum-free medium (Thermo Fisher Scientific) or SIM SF serum-free medium  at 27 °C. HEK293F suspension cells (Thermo Fisher Scientific) were cultured in FreeStyle 293 medium (Thermo Fisher Scientific) or SMM 293-TI medium  supplemented with 1% fetal bovine serum (FBS) at 37 °C with 6% CO10. AD293 cells cultured in the 12-well plates were transfected with wild-type hSGLT1 and hSGLT2 with or without wild-type MAP17. One day post-transfection, cells were seeded into a 96-well plate coated with poly-D-lysine. After attachment, cells were washed with 200 μl per well of PBS  twice, followed by incubation at 37 °C with 5% CO2 for 1 h in uptake buffer  supplemented with 600 μM 1-NBD-glucose and 0.3% bovine serum albumin (BSA). Subsequently, cells were washed three times with 200 μl per well of PBS to stop the uptake. Then the cells were lysed with 150 μl per well of TBS buffer containing 1% SDS for 30 min at 25 °C. The lysates were transferred to a clear-bottom black 96-well plate for 1-NBG-glucose detection with excitation at 445 nm, and emission at 525 nm on an Infinite 200Pro imager (Tecan Life Sciences). Protein concentration in 96-well plate was determined using a BCA protein assay kit (CWBIO). The 1-NBD-glucose fluorescence signals in each well were normalized to the protein concentration. The specific 1-NBD-glucose uptake of each well was calculated by subtracting NBD fluorescence signals in wells transfected with empty vector. The IC50 of 4D4FDG was calculated by a three parameters Dose-response equation in GraphPad Prism.1-NBD-glucose uptake assay was used to measure the sugar transport activity of SGLT21. AD293 cells were plated onto a poly-D-lysine-treated 24-well plate and transfected with wild-type hSGLT2 with or without MAP17 and incubated for 40–48 h. The cells were washed with 500 μl PBS per well twice, fixed using 300 μl 4% formaldehyde in PBS for 30 min, and washed with 500 μl PBS per well twice again. For surface expression, the cells were blocked with 300 μl 3% goat serum in PBS per well for 30 min and labeled with 150 μl primary antibody mAb90 (diluted 3350 times in blocking buffer) per well for 1 h. After washing with PBS three times, cells were incubated with horseradish-peroxidase (HRP) labeled goat anti-mouse IgG secondary antibody  for 30 min. After extensive washing, the cells were incubated with High-Sig ECL Western Blotting Substrate (Tanon) for 2 min, and chemiluminescence signals were measured with an Infinite M Plex plate reader (Tecan).Monoclonal antibody mAb90 was generated by injecting purified hSGLT2-MAP17 protein into mice, followed by hybridoma screening. The work was carried out by Proteintech Group. The surfacing labeling was carried out as described previouslyGFP-MAP17nb complex and the hSGLT2GFP-MAP17nb complex were carried out as described previously with minor modifications11. BacMam virus was generated using sf9 cells. For protein expression, HEK293F cells cultured in SMM 293-TI medium at a density of 2.5g  for 10 min at 4 °C and washed with TBS buffer  containing 2 μg/ml aprotinin, 2 μg/ml pepstatin and 2 μg/ml leupeptin. The cells were then flash-frozen and stored at −80 °C.The expression and purification of the hSGLT1GFP-MAP17nb, the membrane pellets corresponding to 1 liter of culture were resuspended and homogenized in 15 ml TBS buffer containing protease inhibitors ), 10 mM MgCl2, 0.7 μg/ml benzonase, and 5 mM 4D4FDG . The crude membrane solution was incubated at 37 °C for 1 h to facilitate 4D4FDG binding. Digitonin (BioSynth) was added to a final concentration of 1% (W/V) after cooling the membrane to 4 °C, and the mixture was stirred for 1 h to solubilize membrane proteins. The insoluble debris was removed by centrifugation at 193,400 × g  for 30 min. Subsequently, the supernatant was loaded onto a 5 ml Streptactin Beads 4FF (Smart Lifesciences) column and washed with 50 ml wash buffer 1 , 5 mM 4D4FDG, 10 mM MgCl2 and 1 mM adenosine triphosphate (ATP)) to remove the contamination of heat shock proteins. Then, the column was extensively washed with 50 ml wash buffer 2 (TBS buffer supplemented with 40 μM GDN and 5 mM 4D4FDG). The target protein was eluted with elution buffer containing 50 mM Tris pH 8.0 at 25 °C, 150 mM NaCl, 40 μM GDN, 5 mM 4D4FDG, and 10 mM d-desthiobiotin (IBA). The eluate was loaded onto a HiTrap Q HP column  and the hSGLT1-MAP17 complex was separated from aggregates with a linear gradient from 0 mM NaCl to 1000 mM NaCl in buffer containing 20 mM Tris pH 8.0 at 4 °C, 40 μM GDN, and 5 mM 4D4FDG. The fractions containing the hSGLT1-MAP17 complex were collected for nanodisc reconstitution. MSP2X which contains two copies of MSP1E3D1 was purified according to the previously described method22. The hSGLT1-MAP17 complex was mixed with MSP2X and soybean polar lipids extract  GDN, Avanti) at a molar ratio of protein: MSP2X: SPLE = 1: 4: 400. The mixture was allowed to equilibrate for 1 h at 4 °C, and then Bio-beads SM2 (Bio-Rad) were added to initiate the reconstitution with constant rotation at 4 °C. Bio-beads SM2 were added to the mixture four times within 24 h to gradually remove detergents from the system. Afterward, the proteins that were not reconstituted in lipid nanodisc were removed by centrifugation at 86,600 × g for 30 min in TLA 100.3 rotor (Beckman Coulter). The supernatant containing the hSGLT1-MAP17 complex reconstituted in lipid nanodisc was loaded onto the 1 ml Streptactin Beads 4FF column to remove empty nanodisc. The elution from the Streptactin Beads 4FF column was concentrated and subjected to a Superose 6 increase 10/300 GL column  in the buffer that contained 20 mM HEPES pH 7.5, 150 mM NaCl and 5 mM 4D4FDG. The peak fractions corresponding to the hSGLT1-MAP17 complex in lipid nanodiscs were collected and diluted to a concentration of approximately 0.1 mg/ml for cryo-EM sample preparation.For purification of the hSGLT1GFP-MAP17nb complex, the membrane pellets were resuspended and homogenized in TBS buffer containing protease inhibitors, 10 mM MgCl2 and 0.7 μg/ml benzonase. The crude membrane solution was incubated at 37 °C for 1 h and then cooled to 4 °C. The hSGLT2-MAP17 complex was solubilized by 1% (W/V) GDN for 1 h with constant rotation. The insolubilized materials were removed by centrifugation at 193,400 × g for 30 min in Ti50.2 rotor (Beckman Coulter). The supernatant was loaded onto a Streptactin Beads 4FF (Smart Lifesciences) column. The column was washed sequentially with buffer W1  and buffer W2 (TBS buffer plus 40 μM GDN). The hSGLT2-MAP17 complex was eluted with buffer E ). The hSGLT2-MAP17 complex in elution was further purified by a HiTrap Q HP  column to remove D-desthiobiotin. The fractions from the HiTrap Q HP column, which contained the hSGLT2-MAP17 complex, were collected for nanodisc reconstitution. The hSGLT2-MAP17 complex was mixed with SPLE (solubilized in 1% GDN) and NW9 at a molar ratio of protein: NW9: SPLE = 1: 2: 100. The mixture was incubated for 1 h at 4 °C, followed by adding Bio-beads SM2 (Bio-Rad) to initiate the reconstitution. A total of four batches of Bio-beads SM2 were added during the reconstitution process. After reconstitution, the hSGLT2-MAP17 complex in lipid nanodisc was clarified by centrifugation at 86,600 × g for 30 min in TLA 100.3 rotor (Beckman Coulter). The empty nanodisc was removed by a Streptactin Beads 4FF column according to the previously described method in buffer without detergent. The hSGLT2-MAP17 complex reconstituted in lipid nanodisc was further purified by a Superose 6 increase 10/300 GL column  in TBS buffer. The peak fractions were collected. For the hSGLT2GFP-MAP17nb complex in the apo state, the protein was diluted to a concentration of approximately 0.1 mg/ml for cryo-EM sample preparation. For the hSGLT2GFP-MAP17nb complex in the AMG binding state, the protein was incubated with 100 mM AMG for 1 h at 37 °C for substrate binding before cryo-EM sample preparation.For purification of the hSGLT223. Aliquots of 2.5 µl of the hSGLT1-MAP17 complex or the hSGLT2-MAP17 complex in presence of 0.5 mM fluorinated octyl-maltoside  were applied to the grids. After incubation at 4 °C under 100% humidity for 60 s, the grids were blotted for 4 s using a blot force of 4, and then plunge-frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). The grids were transferred to a Titan Krios electron microscope (Thermo Fisher) operating at 300 kV. SerialEM-3.6.11 was used for automated data collection. Movies from the dataset of the hSGLT1-MAP17 were recorded on a K3 Summit direct detector (Gatan) mounted post a quantum energy filter (slit width 20 eV) in super-resolution mode with a defocus range of −1.4 to −1.8 μm and a magnification of 105,000×, resulting in a calibrated pixel size of 0.417 Å. Movies from datasets of the hSGLT2-MAP17 in the apo state and the AMG binding state were collected on a K2 Summit direct detector (Gatan) at a magnification of 165,000× with a calibrated pixel size of 0.4105 Å. Each stack of 32 frames was exposed for 8 s, with an exposing time of 0.25 s per frame at a dose rate of 4.7 electrons per Å−2 per second.The surface of Quantifoil Au 300 mesh R 0.6/1.0 grids were coated with graphene oxide24 and 2× binned to a pixel size of 0.834 Å. The contrast transfer function (CTF) parameters of dose-weighted micrographs were estimated by Gctf25. Micrographs were manually screened, and a total of 3746 micrographs were used for the following process. A total of 2,794,295 particles were auto-picked using Gautomatch-0.56 (developed by K. Zhang). All particles were extracted with a box size of 120 and 2× binned (pixel size 1.668 Å) in Relion-3.126. These particles were subjected to 2D classification with cryoSPARC-3.1.027. After two rounds of 2D classification, 537,777 particles from classes exhibiting recognizable hSGLT1 features were selected for further processing. A batch size of 50,000 particles was used to generate an initial 3D reference model. The initial model was low-pass filtered to resolutions of 8 Å, 15 Å, 25 Å and 35 Å. The low-pass filtered maps were used as references for a heterogeneous refinement without symmetry imposed. Subsequently, 246,748 particles from the best classes were re-extracted using a box size of 240 at 0.834 Å per pixel and re-centered in Relion-3.1 and then refined with NU-refinement and local refinement subsequently in cryoSPARC-3.1.028, resulting in a reconstruction with an overall resolution of 3.15 Å. Further, seed-facilitated 3D classification29 was performed in cryoSPARC-3.1.0 and a set of 788,321 particles was enriched. NU-refinement and local refinement of these particles yielded a reconstruction at 2.52 Å. This model was low-pass filtered to resolutions of 4 Å, 8 Å, 12 Å, 16 Å and 20 Å. The maps were used as references for a multi-reference 3D classification with an adapted mask. The classes of the last 12 iterations with the best features of hSGLT1 and MAP17 in 3D classification were combined, and the duplicated particles were removed. The final 318,616 particles were re-extracted using a box size of 240 and a pixel size of 0.834 Å in Relion-3.1. These particles were subjected to NU-refinement and local refinement in cryoSPARC-3.1.0, yielding a reconstruction at 3.26 Å. The resolution was estimated using the Fourier shell correlation (FSC) = 0.143 criterion. Local resolution was calculated using cryoSPARC-3.1.0. ThreeDFSC curves were calculated using the 3DFSC server30 .For the hSGLT1-MAP17 in complex with 4D4FDG, a total of 4600 micrographs were collected. Beam-induced drift was corrected using MotionCor224 and binned to a pixel size of 0.821 Å. Dose-weighted micrographs were used for CTF estimation by Gctf25. A total of 3,313,984 particles (the apo state) and 3,310,006 particles (the AMG binding state) were auto-picked using Gautomatch-0.56 (developed by K. Zhang). Two rounds of 2D classification in CryoSPARC-3.1.027 were performed to remove ice spots, contaminants and aggregates, yielding 139,915 and 301,506 particles for the apo state and the AMG binding state, respectively. After ab-initio reconstruction and heterogeneous refinement using CryoSPARC-3.1.027, 25,599 particles (the apo state) and 124,469 particles (the AMG binding state) were selected and used as seeds for seed-facilitated classification. After that, 107,441 particles (the apo state) and 647,871 particles (the AMG binding state) were retained and yielded reconstructions at 3.61 Å and 3.61 Å, respectively. To further improve the map quality of the hSGLT2-MAP17 complex, multi-reference 3D classification without alignment was performed with an adapted mask, resulting in a reconstruction at 3.48 Å from 39,476 particles for the apo state and 3.79 Å from 31,089 particles for the AMG binding state. After that, the particles were re-extracted from micrographs of AMG binding state, and the map of 3.79 Å was input as a seed. In total, 71,638 particles were extracted and performed a 2D classification, leaving 44,393 particles. After NU refinement and local refinement, a map at 3.33 Å was obtained. Resolutions were estimated using the gold-standard Fourier shell correlation criterion of 0.143. Local resolution was calculated using CryoSPARC-3.1.027.For the hSGLT2-MAP17 complex, a total of 9841 and 7393 micrographs of the hSGLT2-MAP17 complex in the apo state and the AMG binding state were collected respectively, and the beam-induced drift was corrected using MotionCor231 and manually adjusted using Coot-0.8.9.332. The substrates were manually fitted into the electron densities. The models were further refined against the maps using Phenix-1.1933. The RMSD between different states were calculated with Pymol-1.7.0.5.The homology model of the hSGLT1-MAP17 complex in 4D4FDG binding state was from the hSGLT1-MAP17 complex with LX2761 (PDB ID: 7WMV). The models of the hSGLT2-MAP17 complex in the apo state and the AMG binding state were from the model of the hSGLT2-MAP17 complex with empagliflozin (PDB ID: 7VSI). The models were fitted into the cryo-EM maps using UCSF Chimera34. The local resolution was estimated using Relion-3.126. The number of independent reactions (N) and the relevant statistical parameters for each experiment (such as mean or standard deviation) are described in the figure legends. No statistical methods were used to pre-determine sample sizes.Global resolution estimations of cryo-EM density maps are based on the 0.143 Fourier Shell Correlation criterionFurther information on research design is available in the Supplementary informationPeer Review FileReporting Summary


It is worth noting that some IDHs exhibit dual coenzyme specificity characteristics. Herein, a dual coenzyme-dependent IDH from Umbonibacter Marinipuiceus (UmIDH) was expressed, purified, and identified in detail for the first time. SDS-PAGE and Gel filtration chromatography analyses showed that UmIDH is an 84.7 kDa homodimer in solution. The Km values for NAD+ and NADP+ are 1800.0 ± 64.4 μM and 1167.7 ± 113.0 μM in the presence of Mn2+, respectively. Meanwhile, the catalytic efficiency (kcat/Km) of UmIDH is only 2.3-fold greater for NADP+ than NAD+. The maximal activity for UmIDH occurred at pH 8.5 (with Mn2+) or pH 8.7 (with Mg2+) and at 35 °C (with Mn2+ or Mg2+). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn2+ or Mg2+. Moreover, three putative core coenzyme binding residues  of UmIDH were evaluated by site-directed mutagenesis. This recent work identified a unique dual coenzyme-dependent IDH and achieved the groundbreaking bidirectional modification of this specific IDH’s coenzyme dependence for the first time. This provides not only a reference for the study of dual coenzyme-dependent IDH, but also a basis for the investigation of the coenzyme-specific evolutionary mechanisms of IDH.Isocitrate dehydrogenase (IDH) can be divided into NAD Three scans were performed for each sample, and the average value was recorded. The protein secondary structure was estimated following the method described by Raussens et al. [Circular dichroism (CD) spectra were acquired using a Jasco model J-810 spectropolarimeter. UmIDH and mutants concentrations were diluted to 0.2 mg/mL using a CD buffer containing 20 mM NaHs et al. .2+, Mg2+, Ca2+, Co2+, Cu2+, Ni2+, K+, Na+, and Li+) on MmIDH were assessed using the standard assay method. All data for enzyme activity were tested in at least three independent experiments.The pH-dependent activity of the recombinant UmIDH was investigated in 50 mM Tris-HCl buffer ranging from pH 7.7 to 9.5 at 25 °C. The temperature dependence of the recombinant UmIDH activity was assessed across the temperature range of 25–60 °C at pH 8.5. The thermostability of the recombinant UmIDH was evaluated by subjecting enzyme aliquots to heat inactivation at temperatures ranging from 25 °C to 65 °C at pH 8.5 for 20 min. Following incubation, the aliquots were rapidly cooled on ice, and the residual enzyme activity was determined using the standard enzyme assay. The effects of various metal ions + at a temperature of 25 °C. The increase in NAD(P)H absorbance was monitored at 340 nm using a Cary 300 UV-Vis spectrophotometer  with an extinction coefficient (ε340) of 6.22 mM−1·cm−1. One unit (U) of enzyme activity was defined as the generation of 1 μmol of NADPH per minute. Protein concentrations were determined using a Bio-Rad protein assay kit . The enzyme’s kinetic parameters were determined by measuring its activity at various concentrations of NADP+ or NAD+. The apparent kinetic parameters were calculated using nonlinear regression analysis with Prism 5.0 software . All experiments were conducted at least three times to ensure reproducibility.Enzyme assays were performed in a 1 mL reaction volume consisting of 50 mM Tris-HCl (pH 8.5), 2 mM MnCl