Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) Proportion of PB integration sites across the genome: promoters (15.13%), untranslated region (UTR) (1.04%), exons (2.09%), introns (32.12%), and intergenic regions (49.64%). ( B ) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the top 200 genes with the most frequent insertions from the S-iTSCs. VEGF, vascular endothelial growth factor; EGFR, epidermal growth factor receptor. ( C ) The top 10 candidate genes among the inserted genes from the S-iTSCs. ( D ) Sense (red) and antisense (green) insertions of PB in Catip and Dyrk1a gene bodies from the S-iTSCs. The rectangles indicate the exons. ( E ) Schematic diagram of the strategy to Catip -KO and Dyrk1a -KO in WT-ESCs by using CRISPR-Cas9 system. ( F ) Sequences of genotypes in the Catip -KO and Dyrk1a -KO ESCs.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: CRISPR

Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) The live cell numbers of the Catip -KO ESCs, Dyrk1a -KO ESCs, and WT-ESCs during daily culture. Catip -KO and Dyrk1a -KO did not affect the proliferation of ESCs. n.s., not significant. ( B ) Chromosome spread analysis of Catip -KO ESCs, Dyrk1a -KO ESCs, and WT-ESCs (diploid). Scale bars, 10 μm. ( C ) Statistical analysis of chromosome numbers in the Catip -KO ESCs, Dyrk1a -KO ESCs, and WT-ESCs. ( D ) Teratomas formed from the Catip -KO ESCs, Dyrk1a -KO ESCs, and WT-ESCs were identified by hematoxylin and eosin staining. Scale bars, 100 μm. The tissues shown were glands (endoderm), blood (mesoderm), and neural tissue (ectoderm). ( E ) GO analysis of DEGs between Catip -KO ESCs and WT-ESCs. ( F ) GO analysis of DEGs between Dyrk1a -KO ESCs and WT-ESCs. Pol II, polymerase II.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: Staining

Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) Phase-contrast images of established Catip -KO iTSCs and Dyrk1a -KO iTSCs. Scale bars, 100 μm. ( B ) Immunofluorescence of TSC-specific markers CDX2 (green) and EOMES (green) in the Catip -KO iTSCs and Dyrk1a -KO iTSCs. DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm. ( C ) Immunofluorescence of the trophoblast lineage–specific markers PROLIFERIN (green), TPBPA (green), and SDC1 (green) in differentiated cells derived from the Catip -KO iTSCs and Dyrk1a -KO iTSCs. DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm. ( D ) PCA of RNA-seq datasets from the WT-ESCs, Catip -KO ESCs, Dyrk1a -KO ESCs, Cdx2 -OE ESCs, WT-TSCs, Catip -KO iTSCs, Dyrk1a -KO iTSCs, and Cdx2 -OE iTSCs. ( E ) Genome browser tracks showing CpG methylation status in Pou5f1 and Elf5 loci of WT-ESCs, Catip -KO ESCs, Dyrk1a -KO ESCs, WT-TSCs, Catip -KO iTSCs, and Dyrk1a -KO iTSCs. ( F ) Bubble plot representing the expression of Catip , Dyrk1a , Cdx2 , Zscan4b , Dux , and Oct4 in early embryonic developing stages and ESCs. ( G ) Schematic diagram of the DYRK1A inhibitor–harmine. ( H ) Phase-contrast images of established harmine + iTSCs and WT-TSCs. Scale bars, 100 μm.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: Immunofluorescence, Staining, Derivative Assay, RNA Sequencing, CpG Methylation Assay, Expressing

Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) Heatmap showing some DEGs between Dyrk1a -KO ESCs and WT-ESCs mapping to Notch pathway genes, Hippo/Yap pathway genes, and totipotent genes. ( B ) PCA results of RNA-seq datasets from the WT-ESCs, Dyrk1a -KO ESCs, TBLCs, TLSCs, and ciTotiSCs. ( C ) GSEA of DEGs associated with totipotent genes in Dyrk1a -KO ESCs. NES, normalized enrichment score; FDR, false discovery rate. ( D ) The Venn diagram of the up-regulated DEGs between Dyrk1a -KO ESCs and TBLCs, versus WT-ESCs. ( E ) Top 10 genes in the co–up-regulated DEGs between Dyrk1a -KO ESCs and TBLCs, versus WT-ESCs. ( F ) GO analysis of co–up-regulated DEGs between Dyrk1a -KO ESCs and TBLCs, versus WT-ESCs.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: RNA Sequencing

Journal: Science Advances

Article Title: Haploid-genetic screening of trophectoderm specification identifies Dyrk1a as a repressor of totipotent-like status

doi: 10.1126/sciadv.adi5683

Figure Lengend Snippet: ( A ) Immunofluorescence of CDX2 (red) in chimeric blastocyst from GFP-labeled Dyrk1a -KO ESCs and WT-ESCs. Donor cells were GFP + (green), and DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm. ( B ) Images of E12.5 chimeras (including fetus, placenta, and yolk sac) derived from GFP-labeled Dyrk1a -KO ESCs, with uninjected embryo as a control. Scale bars, 5 mm. ( C ) FACS analysis of GFP + cells in the fetus, placenta, and yolk sac in E12.5 chimeras derived from the GFP-labeled Dyrk1a -KO ESCs. The percentages of GFP + cells in the fetus, placenta, and yolk sac of Dyrk1a -KO chimera were 43.6, 14.9, and 54.4%, respectively; uninjected ones (fetus, placenta, and yolk sac) at E12.5 were as a control. ( D ) Immunofluorescence staining of placental-specific markers (TPBPA, PROLIFERIN, TFAP2C, and GCM1, each in red) in the GFP-labeled Dyrk1a -KO ESC–derived E12.5 placentas. The white arrows indicated that GFP-positive cells could contribute to the tissues expressing specific markers. Scale bars, 75 μm. ( E ) Phase-contrast images of blastoid derived from the Dyrk1a -KO ESCs. WT-blastocyst was used as a control. Scale bars, 10 μm. ( F ) Immunofluorescence of CDX2 (green) and OCT4 (red) in blastoid derived from the Dyrk1a -KO ESCs. WT-blastocyst was used as a control. DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm. ( G ) Immunofluorescence of CDX2 (green) and GATA6 (red) in blastoid derived from the Dyrk1a -KO ESCs. WT-blastocyst was used as a control. DNA was stained with Hoechst 33342 (blue). Scale bars, 50 μm.

Article Snippet: For inhibition of DYRK1A, different concentrations of harmine (MCE, HY-N0737A) were used as indicated.

Techniques: Immunofluorescence, Labeling, Staining, Derivative Assay, Control, Expressing

Journal: Global challenges (Hoboken, NJ)

Article Title: Cancer-Derived Extracellular Vesicle ITGB2 Promotes the Progression of Triple-Negative Breast Cancer via the Activation of Cancer-Associated Fibroblasts.

doi: 10.1002/gch2.202400235

Figure Lengend Snippet: Figure 5. ITGB2 in TNBC EVs promotes fibroblast-to-CAF transition and activation. A) Western blot analysis of ITGB2 expression in wild-type (Control), ITGB2-overexpressing (ITGB2-OE), and ITGB2-knockdown (ITGB2-KD) MDA-MB-231 cells. B) ITGB2 levels in EVs derived from wild-type, ITGB2-OE, and ITGB2-KD MDA-MB-231 cells, measured by western blot. C) Confocal fluorescence microscopy showing the uptake of ITGB2-labeled EVs by fibroblasts after 24 h of incubation. D) Migration and invasion assays of fibroblasts treated with EVs from ITGB2-OE, ITGB2-KD, or control cells. E–G) Western blot analysis of 𝛼-SMA, FAP, and FSP levels in fibroblasts treated with ITGB2-expressing or ITGB2-knockdown EVs. Data are expressed as mean ± SD, *P < 0.05 versus control.

Article Snippet: Western blot analysis was performed to determine the levels of α-SMA, FAP, and FSP using antibodies against α-SMA (bs-10196R; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), FAP, and FSP (BA1471-2; Wuhan Boster Biological Technology Co., Ltd., Wuhan, China).

Techniques: Activation Assay, Western Blot, Expressing, Control, Knockdown, Derivative Assay, Microscopy, Labeling, Incubation, Migration

Journal: Global challenges (Hoboken, NJ)

Article Title: Cancer-Derived Extracellular Vesicle ITGB2 Promotes the Progression of Triple-Negative Breast Cancer via the Activation of Cancer-Associated Fibroblasts.

doi: 10.1002/gch2.202400235

Figure Lengend Snippet: Figure 6. ITGB2 promotes TNBC tumorigenesis and CAF activation in vivo. A) Tumor volumes in nude mice inoculated with wild-type, ITGB2- overexpressing, or ITGB2-knockdown MDA-MB-231 cells, measured on day 50 post-inoculation (n = 6). B) Representative images of tumors excised from nude mice in each group. C) Western blot analysis of CAF activation markers (𝛼-SMA, FAP, FSP) in tumor tissues from each group. Data are expressed as mean ± SD, *P < 0.05 versus control, #P < 0.01 versus knockdown group.

Article Snippet: Western blot analysis was performed to determine the levels of α-SMA, FAP, and FSP using antibodies against α-SMA (bs-10196R; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), FAP, and FSP (BA1471-2; Wuhan Boster Biological Technology Co., Ltd., Wuhan, China).

Techniques: Activation Assay, In Vivo, Knockdown, Western Blot, Control

Journal: Global challenges (Hoboken, NJ)

Article Title: Cancer-Derived Extracellular Vesicle ITGB2 Promotes the Progression of Triple-Negative Breast Cancer via the Activation of Cancer-Associated Fibroblasts.

doi: 10.1002/gch2.202400235

Figure Lengend Snippet: Figure 7. Graphical summary of the study: ITGB2 in TNBC progression and tumor microenvironment remodeling. Tumor-derived EVs enriched in ITGB2 are secreted by TNBC cells. ITGB2 facilitates the uptake of EVs by fibroblasts, promoting their transition into CAFs through the upregulation of activation markers (𝛼-SMA, FAP, and FSP). Activated CAFs, in turn, enhance tumor-stroma interactions, fostering tumor growth, migration, and invasion. Additionally, ITGB2 overexpression correlates with poorer patient prognosis and higher tumor aggressiveness. These findings highlight ITGB2 as a key mediator of TNBC progression and a potential target for therapeutic intervention.

Article Snippet: Western blot analysis was performed to determine the levels of α-SMA, FAP, and FSP using antibodies against α-SMA (bs-10196R; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), FAP, and FSP (BA1471-2; Wuhan Boster Biological Technology Co., Ltd., Wuhan, China).

Techniques: Derivative Assay, Activation Assay, Migration, Over Expression

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Inhibition of micro RNA miR-122-5p on lipopolysaccharide-induced myocardial injury. Wistar rats were intravenously injected with miR-122-5p antagomir, followed by lipopolysaccharide (LPS) stimulation (n = 6 rats per group). (a-b) After LPS treatment for 12 h, heart tissues were harvested for the relative expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) using real-time quantitative PCR (RT-qPCR) or western blot assay. (c) The ratio of heart weight/body weight (HW/BW) was calculated. (d) Hematein and eosin (H&E) staining revealed the effect of miR-122-5p on LPS-induced histopathological changes in heart. (e) Levels of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were examined by enzyme-linked immunosorbent assay (ELISA). *** p < 0.001 versus control; ## p < 0.01, ### p < 0.001 versus LPS + NC antagomir

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Inhibition, Injection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: In vitro analysis for beneficial role of inhibiting micro RNA miR-122-5p in lipopolysaccharide (LPS)-induced apoptosis. (a-b) Rat H9c2 cells were treated with LPS for 12 h or 24 h, and the expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were assessed by real-time quantitative PCR (RT-qPCR) or western blot analysis. (c-d) H9c2 cells were transfected with NC inhibitor or miR-122-5p inhibitor for 24 h, followed by LPS treatment for another 24 h under proper culture conditions. After that, miR-122-5p and GIT1 expression levels were measured. (e) The contents of cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) were detected by appropriate kits. (f) Flow cytometry showed the apoptosis of LPS-stimulated myocardial cells. (g) Western blot analysis illustrated the changes of caspase-3 expression. ** p < 0.01, *** p < 0.001 versus control; ++ p < 0.01, +++ p < 0.001 versus LPS + NC inhibitor

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Flow Cytometry, Control

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: Potential downstream target gene of micro RNA miR-122-5p. H9c2 cells were transfected with NC mimics, miR-122-5p mimics, NC inhibitor and miR-122-5p inhibitor for 48 h. (a-b) The expression levels of miR-122-5p and G-protein-coupled receptor kinase interacting protein-1 (GIT1) were verified by real-time quantitative PCR (RT-qPCR) assay. (c) The predicted binding sites of miR-122-5p in the 3ʹ-UTR of GIT1, and the sequence information of miR-122-5p and GIT1 (wild- or mutant- type) was displayed. (d) Luciferase assay verified the correlation between miR-122-5p and GIT1. aa p < 0.01, aaa p < 0.001 versus NC mimics; bbb p < 0.001 versus NC inhibitor; dd p < 0.01 versus NC mimics + GIT1 3ʹUTR (WT)

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Binding Assay, Sequencing, Mutagenesis, Luciferase

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency attenuates the effects of micro RNA miR-122-5p loss on myocardial injury. (a) H9c2 cells were transfected with GIT1 siRNA to downregulate GIT1 expression. (b) The cells were transfected with GIT1 siRNA and/or miR-122-5p inhibitor, and then induced by lipopolysaccharide (LPS). GIT1 expression at mRNA and protein levels was then measured using real-time quantitative PCR (RT-qPCR) or western blot. (c) Apoptosis of myocardial cells was analyzed by flow cytometry. (d) Reactive oxygen species (ROS) production was examined using flow cytometry. (e-g) The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and tumor necrosis factor alpha (TNF-α) were assessed by the enzyme-linked immunosorbent assay (ELISA) kits. XXX p < 0.001 versus NC siRNA; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001 versus LPS + miR-122-5p inhibitor + NC siRNA

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Bioengineered

Article Title: Inhibition of micro RNA miR-122-5p prevents lipopolysaccharide-induced myocardial injury by inhibiting oxidative stress, inflammation and apoptosis via targeting GIT1

doi: 10.1080/21655979.2021.1926201

Figure Lengend Snippet: G-protein-coupled receptor kinase interacting protein-1 (GIT1) deficiency inhibits nuclear factor erythroid 2-related factor 2 (Nrf-2) activation. (a) Real-time quantitative PCR (RT-qPCR) assay was used to measure the heme oxygenase-1 (HO-1) and NAD(p)H: quinone oxidoreductase 1 (NQO-1) expression. (b) Nuclear Nrf-2 level was revealed using western blot analysis. ^^ p < 0.01 versus LPS + miR-122-5p inhibitor + NC siRNA

Article Snippet: After blocking with 5% nonfat milk for 1 h, the proteins were incubated overnight at 4°C with corresponding primary antibodies against GIT1 (1: 500; Boster, USA), caspase 3 (1: 1000; CST, USA), nuclear factor erythroid 2-related factor 2 (Nrf-2; 1: 500; Affinity, China) and GAPDH (1: 10000; Proteintech, China).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Western Blot