The gut microbiota, as the “second genome” of the human body, plays a central regulatory role in maintaining host physiological homeostasis; conversely, its dysbiosis can impair male reproductive function via the “gut-testis axis”, leading to a series of pathological manifestations such as abnormal semen quality, sexual dysfunction, and reproductive organ damage. Gut microbiota exerts multidirectional effects on host metabolism, immunity, endocrinology, and the neural system, collectively forming a complex regulatory network for male reproduction. Among these, microbiota-derived metabolites such as short-chain fatty acids (SCFAs), serotonin (5-HT), and secondary bile acids, function as systemic signaling molecules that exert direct and indirect effects on the testis through blood circulation and modulation of gut barrier integrity, regulation of systemic inflammation, epigenetic reprogramming, respectively. The potential and limitations of microbiota-targeted intervention strategies, including probiotics, prebiotics, synbiotics, traditional natural herbal extracts, and fecal microbiota transplantation (FMT), are also discussed. Finally, we propose that future interventions should be tailored to individual gut microbiota profiles to achieve precise regulation of male reproductive function. This review aims to provide a new systems biology perspective for understanding the complex etiology of male infertility and to lay a theoretical foundation for the development of innovative microbiome-based diagnostic tools and therapeutic strategies.

The corpus luteum (CL) arises from the luteinization of granulosa cells (GCs) and theca cells, marked by rapid progesterone elevation and angiogenesis. Intriguingly, angiogenesis lags behind progesterone elevation, creating an avascular phase during which luteal cells must fuel intensive steroidogenesis without perfusion. How the avascular CL meets this energetic demand remains a mystery. Here, we reveal a cellular adaptive mechanism-granulosa cell energy storage (GCES)-that resolves this enigma. We demonstrate that upon luteinization initiation, GCs enter a metabolically quiescent state yet enhance glucose uptake, converting the glucose into glycogen. Catabolism of this glycogen reserve supplies the energy required for the avascular CL, ensuring normal luteogenesis. Disruption of GCES induces luteal insufficiency, whereas timely glucose administration enhances GCES, improving luteal function and optimizing reproductive outcome in both mouse and ovine models. In human study, oral intake of glucose post-hCG significantly augments GCES and enhances progesterone production. These results advance our understanding of luteinization.

Yaks are a critical livestock species for plateau regions; however, their poor reproductive efficiency—characterized by low estrus and pregnancy rates—significantly hampers genetic improvement and industry sustainability. Although synchronization protocols are widely utilized in cows to enhance reproductive performance, research on yaks is limited. Our study systematically evaluated 3 estrus synchronization protocols (CIDR-GPPG, CIDR-GPG, GPPG) on 99 female yaks. The GPPG treatment resulted in the lowest estrus detection (40.63%) and pregnancy rate (15.38%), while the CIDR-GPG protocol significantly improved these rates, achieving an estrus rate of 65.71% and a pregnancy rate of 52.17%. Notably, serum estradiol concentrations were significantly elevated in the CIDR-GPG group compared to the GPPG group. Given the importance of body condition for synchronization outcomes, we analyzed correlation between body measurements and reproductive performance. Among 10 body measurements, chest girth demonstrated the strongest positive correlation with both estrus (R = 0.75) and pregnancy success (R = 0.72). Further logistic regression modeling based on chest girth generated robust predictors for estrus (AUC = 0.934) and pregnancy (AUC = 0.923). Importantly, CIDR-GPG protocol effectively lowered the body condition threshold required for successful reproductive outcomes, indicating that yaks with smaller chest girths can still achieve comparable probabilities of estrus and pregnancy when employing this protocol. Overall, our findings supported the CIDR-GPG protocol as an effective strategy for optimizing yak reproduction and highlighted chest girth as a valuable indicator for predicting reproductive outcomes, providing a practical approach to enhance reproductive management, even in animals with suboptimal body conditions.

Understanding the physiology and molecular mechanisms of lactogenesis is crucial for enhancing mammalian milk production. Yes-associated protein 1 (YAP1) regulated mammary epithelial cell survival during pregnancy, but its role in lactation maintenance remains unclear. We found that YAP1 was highly expressed in mammary gland across specie, with elevated expression levels during murine gestation and lactation, particularly localized in alveoli epithelial cells. In vivo administration of a YAP1 inhibitor impaired murine milk yield, mammary gland weight, alveolar structure, and mammary epithelial cell dynamics. In vitro, YAP1 positively affected mammary epithelial cell growth and the synthesis of triglyceride and α-casein. Notably, the primary lactogenesis hormone Prolactin induced cell growth and triglyceride secretion while enhancing YAP1 expression and activity. In contrast, Melatonin inhibited cell growth and triglyceride synthesis, decreasing YAP1 expression and activity. YAP1 knockdown compromised prolactin induced effects, whereas YAP1 overexpression partially rescued cell functions inhibited by melatonin. Finally, Bioinformatics analyses revealed that YAP1 regulated multiple biological processes related to lactogenesis, including cell cycle, Apoptosis, endoplasmic reticulum, amino acid transport and biosynthesis, etc. These finding indicated that YAP1 is essential for mammary epithelial cells growth and secretion and played an essential role in the lactating endocrine network by mediating key hormone functions.

The most notable progress in renal clear cell carcinoma (ccRCC) in the past decades is the introduction of drugs targeting the VHL-HIF signaling pathway-associated angiogenesis. However, mechanisms underlying the development of VHL mutation-independent ccRCC are unclear. Here we provide evidence that the disrupted Hippo-YAP signaling contributes to the development of ccRCC independent of VHL alteration. We found that YAP1 and its primary target genes are frequently upregulated in ccRCC and the upregulation of these genes is associated with unfavorable patient outcomes. Research results derived from our in vitro and in vivo experimental models demonstrated that, under normoxic conditions, hyperactivated YAP1 drives the expression of FGFs to stimulate the proliferation of tumor and tumor-associated endothelial cells in an autocrine/paracrine manner. When rapidly growing cancer cells create a hypoxic environment, hyperactivated YAP1 in cancer cells induces the production of VEGF, which promotes the angiogenesis of tumor-associated endothelial cells, leading to improved tumor microenvironment and continuous tumor growth. Our study indicates that hyperactivated YAP1 is essential for maintaining ccRCC progression, and targeting the dual role of hyperactivated YAP1 represents a novel strategy to improve renal carcinoma therapy.

Introduction:Bactrian camels have diverse agricultural roles, yet their nutritional needs, particularly for trace minerals like zinc, are not well understood. Methods:This study investigated zinc supplementation's effects on milk production, hump morphology, and hair development in lactating camels, and its impact on growth and hair development in calves. Seventy-nine lactating camels and their calves were assigned to four groups: a control group without zinc and three treatment groups receiving differential zinc sulfate concentrations. Results:Results indicated that zinc supplementation had no significant effect on milk yield or composition compared to the control. Furthermore, zinc did not improve the proportion of camels with tilted humps across groups. However, Dose 3 (2.00 g/camel/day) significantly stimulated hair growth compared to the control, whereas Dose 1 (1.00 g/camel/day) and Dose 2 (1.50 g/camel/day) showed no significant differences. In calves, zinc demonstrated more pronounced effects: both Dose 1 (0.50 g/camel/day) and Dose 2 (0.75 g/camel/day) doses markedly enhanced body weight gain and hair development compared to the control. Dose 3 did not benefit calves. Discussion:Collectively, in the present study, zinc at 2.00 g/camel/day improved hair growth in lactating camels without affecting milk production, while 0.50-0.75 g/camel/day enhances growth and hair development in calves, establishing a basis for zinc supplementation in camel husbandry.

Differential N-glycosylations of follicle-stimulating hormone (FSH) influence follicular development dynamics, significantly affecting assisted reproductive technology outcomes. However, the ovarian microenvironment induced by different FSH glycoforms remains incompletely understood. In this study, in vivo analyses using RT-qPCR, single-cell RNA sequencing, and bulk RNA sequencing demonstrated that FSH administration rapidly activated responses in both granulosa cells (GCs) and theca cells (TCs) within 2 h. Hypo-glycosylated FSH (hgFSH) initiated GCs and TCs differentiation more effectively than fully glycosylated FSH (fgFSH), and genes with higher expression in hgFSH were predominantly located in these cell types. HgFSH significantly upregulated 20.2

Follicle-stimulating hormone is the primary gonadotropin regulating ovarian follicular development and is widely utilized in assisted reproduction. Our previous research indicated that different glycoforms of the follicle-stimulating hormone Beta-subunit influence follicular development in vivo, but their specific roles across various folliculogenesis stages remained unclear. In this study, we compared two commercial follicle-stimulating hormone preparations: pituitary-derived follicle-stimulating hormone, which contains both fully and hypo- glycosylated Beta-subunit forms, and recombination follicle-stimulating hormone, predominantly consisting of fully glycosylated forms. Using an in vitro follicular culture model, we observed that pituitary-derived follicle-stimulating hormone was more effective than recombination follicle-stimulating hormone in stimulating follicular growth and promoting antrum formation in preantral follicles. Pituitary-derived follicle-stimulating hormone also rescued insufficient preantral follicle growth and antrum formation caused by recombination follicle-stimulating hormone. Besides, pituitary-derived follicle-stimulating hormone more efficiently drove small antral follicle growth than recombination follicle-stimulating hormone. Additionally, pituitary-derived follicle-stimulating hormone significantly increased the granulosa cells proliferation and effectively inhibited apoptosis, thereby protecting growing follicles from atresia. Pituitary-derived follicle-stimulating hormone also induced higher steroidogenesis than recombination follicle-stimulating hormone. Mechanistically, pituitary-derived follicle-stimulating hormone activated key downstream signaling including protein kinase A/cAMP-response element binding protein, mitogen-activated protein kinase/extracellular signal-regulated kinase, and phosphatidylinositol 3-kinase/AKT more efficiently than recombination follicle-stimulating hormone. Importantly, pre-treating antral follicles with pituitary-derived follicle-stimulating hormone significantly improved ovulation rate following human chorionic gonadotropin administration. This was linked to a rapid upregulation of luteinizing hormone/choriogonadotropin receptor expression and enhanced mitogen-activated protein kinase/extracellular signal-regulated kinase signaling activity. Collectively, our data support that pituitary-derived follicle-stimulating hormone, with its mixture of fully and hypo-glycosylated forms, has greater bioactivity in promoting preantral-antral transition, antral follicle growth, and ovulation compared to recombination follicle-stimulating hormone, which mainly consists of fully glycosylated follicle-stimulating hormone. Application of mixed glycoforms or hypo-glycosylated follicle-stimulating hormone may improve the outcomes in assisted reproduction.

Several microRNAs (miRNAs) have been identified as potential biomarkers widely dispersed in animals since 1993, and they have become a significant molecular biology research area. Because of their ability to activate extracellular molecules, stabilise bodily tissues, control cell-to-cell signals, and be easily extracted, miRNAs are outstandingly nominated as biomarkers. However, there is growing interest in targeting miRNAs to monitor physiological reproductive performance, including reproductive system development, embryo development, fertilisation, endocrinology, and animal welfare in stressful conditions. Moreover, miRNAs play significant roles in gene expression regulation; single miRNAs may have overlapping roles, and on a broader scale, multiple mRNAs govern a single function. Also, miRNAs serve as an intermediary messenger between the environment and reproductive performance, making them a vital component of miRNAs as performance biomarkers under environmental conditions like heat stress. This makes describing a unique miRNA's consequences and functions exceptionally challenging, which may confound many researchers. Also, enhancing our comprehension of miRNAs in response to testicular heat stress could potentially aid in preventing and treating spermatogenesis disorders. Therefore, the present review highlights miRNA's regulatory mechanisms on reproductive performance under heat stress to employ these findings in improving reproduction physiology research.

Inflammation disrupts the normal function of granulosa cells (GCs), which leads to ovarian dysfunction and fertility decline. Inflammatory conditions such as polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI), endometriosis, and age-related ovarian decline are often associated with chronic low-grade inflammation. Nicotinamide mononucleotide (NMN) is an important precursor of NAD+ and has gained attention for its potential to modulate cellular metabolism, redox homeostasis, and mitigate inflammation. This study investigated the protective roles of NMN against lipopolysaccharide LPS-mediated inflammation in GCs. The results of this experiment demonstrated that LPS had negative effects on GCs in term of reduced viability and proliferation rates and upregulated the production of pro-inflammatory cytokines, including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), cyclooxygenase-2 (Cox-2), and tumor necrosis factor-alpha (TNF-α). Notably, the levels of NAD+ and NAD+/NADH ratio in GCs were reduced in response to inflammation. On the other hand, NMN supplementation restored the NAD+ levels and the NAD+/NADH ratio in GCs and significantly reduced the expression of pro-inflammatory markers at both mRNA and protein levels. It also enhanced cell viability and proliferation rates of GCs. Furthermore, NMN also reduced apoptosis rates in GCs by downregulating pro-apoptotic markers, including Caspase-3, Caspase-9, and Bax while upregulating anti-apoptotic marker Bcl-2. NMN supplementation significantly reduced reactive oxygen species ROS and improved steroidogenesis activity by restoring the estradiol (E2) and progesterone (P4) levels in LPS-treated GCs. Mechanistically, this study found that NMN suppressed the activation of the TLR4/NF-κB/MAPK signaling pathways in GCs, which regulates inflammatory processes. In conclusion, the findings of this study revealed that NMN has the potential to reduce LPS-mediated inflammatory changes in GCs by modulating NAD+ metabolism and inflammatory signaling pathways. NMN supplementation can be used as a potential therapeutic agent for ovarian inflammation and related fertility disorders.

Accurate identification of cows' likelihood of conception during the period from recent calving to the first artificial insemination (AI) will provide assistance in managing the fertility of dairy cows and contribute to the economic prosperity and sustainability of farms. The purpose of this study was to use Fourier-transform infrared (FTIR) spectroscopy data collected between recent calving and the first AIto predict the likelihood of a cow conceiving after the first AI and the first or second AI. This study specifically focused on the role of FTIR spectral and farm data collected during different time windows in improving the accuracy of models for predicting a cow's likelihood of conceiving after the first AI and the first or second AI. From 2019 to 2023, fertility information of 10,873 Holstein dairy cows in China were collected, coupled with 21,928 spectral data. First, cows were classified as having a good or poor likelihood of conception. In strategy 1, cows conceiving after the first AI were classified as having a good likelihood of conception and as others as having a poor likelihood of conception. In strategy 2, cows conceiving after the first or second AI were classified as having a good likelihood of conception and others as having a poor likelihood of conception. Second, partial least squares discriminant analysis was used to develop models for predicting the likelihood of conception after the first AI and the first or second AI. The model was assessed using a cross-validation set and herd-independent external validation set. The study also focused on examining the potential correlation between the accuracy of prediction and the period of spectral and farm data collection by analyzing the diagnostic performance of the model in 8 different time windows: from 0 to 7 d postpartum (dpp), 8 to 14 dpp, 15 to 21 dpp, 22 to 30 dpp, 31 to 45 dpp, 46 to 60 dpp, >= 61 dpp, and 0 to 7 d before the first AI. The results showed that the model based on strategy 1 performed better when in proximity to the first AI, with AUC for the cross-validation and herd-independent external validation sets of 0.621 and 0.633, respectively. The model based on strategy 2 exhibited superior performance throughout the late phase of uterine involution. The optimal model was developed by using spectral data collected from 22 to 30 dpp. The AUC for the cross-validation and herd-independent external validation sets were 0.644 and 0.660, respectively, which were higher than those of strategy 1. This study demonstrates the potential of using FTIR spectral data to predict a cow's ability to conceive. The model developed from data collected within a certain time window exhibited better prediction accuracy, particularly from 22 to 30 dpp and 0 to 7 d before the first AI. This study offers novel perspectives on alternate approaches for assessing the fertility of cows, which will contribute to the regularization and sustainability of farms, as well as to the precision management of agriculture.

Large-scale cancer genetic/genomic studies demonstrated that papillary renal cell carcinoma (pRCC) is featured with a frequent shallow deletion of the upstream tumor suppressors of the Hippo/YAP signaling pathway, suggesting that this signaling pathway may play a role in pRCC development. Here we develop a transgenic mouse model with a renal epithelial cell-specific hyperactivation of YAP1 and find that hyperactivation of YAP1 can induce dedifferentiation and transformation of renal tubular epithelial cells leading to the development of pRCC. We analyze at the single-cell resolution the cellular landscape alterations during cancer initiation and progression. Our data indicate that the hyperactivated YAP1, via manipulating multiple signaling pathways, induces epithelial cell transformation, MDSC (Myeloid-derived suppressor cells) accumulation, and pRCC development. Interestingly, we find that depletion of MDSC blocks YAP1-induced kidney overgrowth and tumorigenesis. Inhibiting YAP1 activity with MGH-CP1, a recently developed TEAD inhibitor, impedes MDSC accumulation and suppresses tumor development. Our results identify the disrupted Hippo/YAP signaling as a major contributor to pRCC and suggest that targeting the disrupted Hippo pathway represents a plausible strategy to prevent and treat pRCC.

The PPARGC1A gene plays a fundamental role in regulating cellular energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, adipogenesis, gluconeogenesis, and glucose/fatty acid metabolism. In a previous study, our group investigated seven SNPs in Mediterranean buffalo associated with milk production traits, and the current study builds on this research by exploring the regulatory influences of the PPARGC1A gene in buffalo mammary epithelial cells (BuMECs). Our findings revealed that knockdown of PPARGC1A gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis. Additionally, we observed downregulated triglyceride secretion after PPARGC1A knockdown. Furthermore, the critical genes related to milk production, including the STATS, BAD, P53, SREBF1, and XDH genes were upregulated after RNAi, while the FABP3 gene, was downregulated. Moreover, Silencing the PPARGC1A gene led to a significant downregulation of β-casein synthesis in BuMECs. Our study provides evidence of the importance of the PPARGC1A gene in regulating cell growth, lipid, and protein metabolism in the buffalo mammary gland. In light of our previous research, the current study underscores the potential of this gene for improving milk production efficiency and overall dairy productivity in buffalo populations.

Codon-based analyses of the PPARGC1A gene across 38 vertebrate species were deployed to elucidate patterns of evolutionary change. Employing maximum likelihood assessments through MEGA, we scrutinized 447 codon positions addressing the entire coding region, excluding positions mired by gaps or missing data. Distinct codons manifested variance in selection pressures, particularly codons 4, 11, 66, and 123, which exhibited positive dN-dS values suggestive of positive selection. Codon 137 displayed the most pronounced dN-dS value, signifying intensified selective advantage. Meanwhile, codons 30 and 90 portrayed near-neutral scores, indicative of purifying selection. Complementary computational methods (IFEL, REL, FUBAR, and SLAC) confirmed positive selection at specific codon sites, with varying degrees of corroboration. The integration of mixed-effect modeling (MEME) identified episodic diversifying selection, pinpointing codons that underwent selection episodes in certain lineages. Refined codon model selection lent insight into substitution rates, revealing nuanced degrees of evolutionary conservation among different codons. Supporting these genetic insights, the phylogenetic analysis highlighted relationships among the PPARGC1A sequences and domain analysis confirmed conserved features across species, while protein-protein interaction networks suggested a complex web of functional interdependencies. These findings advance our understanding of the PPARGC1A gene's evolutionary trajectory and underscore the gene's potential adaptive significance within diverse vertebrate lineages.

High grade serous ovarian carcinoma (HGSOC) is the most common and aggressive ovarian malignancy. Accumulating evidence indicates that HGSOC may originate from human fallopian tube epithelial cells (FTECs), although the exact pathogen(s) and/or molecular mechanism underlying the malignant transformation of FTECs is unclear. Here we show that human papillomavirus (HPV), which could reach FTECs via retrograde menstruation or sperm-carrying, interacts with the yes-associated protein 1 (YAP1) to drive the malignant transformation of FTECs. HPV prevents FTECs from natural replicative and YAP1-induced senescence, thereby promoting YAP1-induced malignant transformation of FTECs. HPV also stimulates proliferation and drives metastasis of YAP1-transformed FTECs. YAP1, in turn, stimulates the expression of the putative HPV receptors and suppresses the innate immune system to facilitate HPV acquisition. These findings provide critical clues for developing new strategies to prevent and treat HGSOC.

Female subfertility is an increasing reproductive issue worldwide, which is partially related to abnormal ovarian follicular development. Granulosa cells (GCs), by providing the necessary physical support and microenvironment for follicular development, play critical roles in maintaining female fertility. We previously showed that ectopic expression of four and a half LIM domains 2 (FHL2) promoted ovarian granulosa cell tumor progression. However, its function in follicular development and fertility remains unknown. Here, we confirmed that FHL2 is highly expressed in human and mouse ovaries. FHL2 immunosignals were predominantly expressed in ovarian GCs. A Fhl2 knockout (KO) mouse model was generated to examine its roles in follicular development and fertility. Compared with wildtype, knockout of Fhl2 significantly decreased female litter size and offspring number. Furthermore, Fhl2 deficiency reduced ovarian size and impaired follicular development. RNA-sequencing analysis of GCs isolated from either KO or WT mice revealed that, Fhl2 deletion impaired multiple biological functions and signaling pathways, such as Ovarian Putative Early Atresia Granulosa Cell , ErbB , Hippo/YAP , etc. In vitro studies confirmed that FHL2 silencing suppressed GCs growth and EGF-induced GCs proliferation, while its overexpression promoted GC proliferation and decreased apoptosis. Mechanistic studies indicated that FHL2, via forming complexes with transcriptional factors AP-1 or NF-κB, regulated Egf and Egfr expression, respectively. Besides, FHL2 depletion decreased YAP1 expression, especially the active form of YAP1 (nuclear YAP1) in GCs of growing follicles. EGF, serving as an autocrine/paracrine factor, not only induced FHL2 expression and nuclear accumulation, but also stimulated YAP1 expression and activation. Collectively, our study suggests that FHL2 interacts with EGFR and Hippo/YAP signaling to regulate follicular development and maintain fertility. This study illuminates a novel mechanism for follicular development and a potential therapeutic target to address subfertility.

Optimized reproduction management enhances fertility of dairy cows, and thus improves their milk production efficiency. Comparing different synchronization protocols under variable ambient conditions would be conducive to protocol selection and production efficiency improvement. Here, 9538 primiparous Holstein lactating cows were enrolled to either Double-Ovsynch (DO) or Presynch-Ovsynch (PO) to determine the outcomes under different ambiences. We found that averaged THI of 21-days before the first service (THI-b) was the best indicators in a total of 12 environmental indexes to explain changes in conception rate. And the conception rate decreased linearly in DO treated cows when THI-b was over 73, whereas the threshold was 64 in cows subjected to PO. Compared with PO treated cows, DO increased conception rate by 6%, 13% and 19%, when THI-b was lower than 64, from 64 to 73, and over 73, respectively. Furthermore, employing treatment of PO would lead greater risk for cows staying open compared with DO when THI-b below 64 (hazard ratio, 1.3) and over 73 (hazard ratio, 1.4). Most importantly, calving intervals were 15 days shorter in DO treated cows compared PO when THI-b over 73, while no difference was detected when THI-b below 64. In conclusion, our results supported that, fertility of primiparous Holstein cows could be improved by employing DO, especially in hot weather (THI-b ≥ 73), and the benefits of DO protocol were abated under cool conditions (THI-b < 64). Considering the impacts of environmental heat load is necessary to determine reproductive protocols for commercial dairy farm.

As an important source of genomic variation, copy number variation (CNV) contributes to environmental adaptation in worldwide buffaloes. Despite this importance, CNV divergence between swamp buffaloes and river buffaloes has not been studied previously. Here, we report 21 152 CNV regions (CNVRs) in 141 buffaloes of 20 breeds detected through multiple CNV calling strategies. Only 248 CNVRs were shared between river buffalo and swamp buffalo, reflecting great variation of CNVRs between the two subspecies. Population structure analysis based on CNVs successfully separated the two buffalo subspecies. We further assessed CNV divergence by calculating FST for genome-wide CNVs. Totally, we identified 110 significantly divergent CNV segments and 44 putatively selected genes between river buffaloes and swamp buffaloes. In particular, LALBA, a key gene controlling milk production in cattle, presented a highly differentiated CNV in the promoter region, which makes it a strong functional candidate gene for differences between swamp buffaloes and river buffaloes in traits related to milk production. Our study provides useful information of CNVs in buffaloes, which may help explain the genetic differences between the two subspecies.

Supplementary Figure S1. G-1 suppresses breast cancer cell proliferation regardless of expression status of GPER and classic estrogen receptors. Supplementary Figure S2. G-1 inhibits proliferation of triple negative breast cancer (TNBC) cells in a GPER independent manner. Supplementary Figure S3. G-1 Induced Caspase activation and Apoptosis of breast cancer cells. Supplementary Figure S4. Effects of G-1 treatment on the cell cycle progression of breast cancer cells. Supplementary Figure S5. G-1 arrests cell cycle in the prophase of mitosis in breast cancer cells regardless of GPER expression. Supplementary Figure S6. Effects of G-1 on the cell cycle progression in three breast cancer cell lines examined by fluorescent immunocytochmemistry. Supplementary Figure S7. Comparison of the effects of G-1 and known microtubule targeting agents on the morphology and cell cycle progression of breast cancer cells. Supplementary Figure S8. G-1 disrupts cellular microtubule network in a concentration-dependent manner.