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: Cell Death & Disease

Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

doi: 10.1038/s41419-024-06828-x

Figure Lengend Snippet: A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.

Article Snippet: F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis.

Techniques: Flow Cytometry, Expressing, RNA Sequencing, ChIP-qPCR, Binding Assay, Negative Control, Activity Assay

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Neural plate stage Xenopus laevis embryos were processed for co-immunoprecipitation (IP) assays. Example of Western blot assay from immunoprecipitates with FOLR1 or GFP (control) antibodies and probed for CD2AP or FOLR1. Similar results were observed in N = 3 independent experiments. b Neural plate stage Xenopus laevis embryos were fixed and processed for immunostaining. Images are transverse single z-sections of immunostained neural plate showing apical colocalization of phospho-CD2AP (p-CD2AP) and p-c-Cbl with C-cadherin. Scale bar, 10 μm. Similar results were observed in N = 3 independent experiments. c Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 9.9 pmol of Control-morpholino (Control, Control-MO), 2.6 pmol CD2AP-MO1 (CD2AP KD1) or 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD2/KD) per blastomere until neural tube closed in control embryos, when they were fixed and photomicrographed. Examples of whole embryos in each group. Arrowheads indicate open neural tube (neural tube defect, NTD). Numbers are embryos presenting closed (green) or open (purple, NTD) neural tube. Bar graph represents proportion of phenotypes in each group. d Two-cell stage Xenopus laevis embryos were unilaterally microinjected with 9.9 pmol Control-MO (Control) and 7.4–9.9 pmol CD2AP-MO2 (CD2AP KD) along with GFP and mCherry mRNA, respectively, and allowed to develop until they reached mid-neural plate stages, when they were fixed and processed for immunostaining. Image is a transverse section of the neural plate, immunostained for GFP (Control), mCherry (CD2AP KD) and C-cadherin. Double arrows indicate apical surface length of medial superficial Control (white) and CD2AP KD (magenta) neural plate cells. Scale bar, 20 μm. Graph shows individual and mean ± SD apical length of superficial neural plate cells per embryo, n of cells = 75 in each half of the neural plate, N of embryos = 4. **** p < 0.0001, two-tailed paired t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Immunoprecipitation, Western Blot, Control, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a Two-cell stage Xenopus laevis embryos were bilaterally microinjected with hEEA1-GFP and membrane mCherry mRNAs and unilaterally microinjected with 9.9 pmol CD2AP-MO (CD2AP KD) per blastomere along with fluorescent tracer and allowed to develop until they reached early neural plate stages (stage 13-14), when they were time-lapse imaged with an acquisition rate of 1 frame/6 min. Image is maximum intensity projection of single time frame. Dashed line indicates border between morpholino-injected and wild-type (WT) neural plate. Inset shows neural plate injected side showing tracer in blue. Graphs show distribution between both halves of the neural plate (in %) of the number of EEA1-GFP vesicles and area fraction of labeled endosomes per neural plate cell surface. Two-tailed paired t -test, n = 28 cells analyzed in each group from N = 5 embryos per group. Scale bar, 20 μm. b Two-cell stage Xenopus laevis embryos were bilaterally microinjected with 7.4 pmol Control-MO (Control) or CD2AP-MO (CD2AP KD) per blastomere and allowed to grow until they reached mid-neural plate stages (stage 15–17) when neural plate was dissected and processed for Western blot assays. Image is an example of Western blot assay. Graph shows individual and mean ± SD percent of optical density (OD) for C-cadherin immunoblot band normalized with GAPDH protein band OD and compared to controls. Two-tailed ratio t -test, n = 28 and 24 neural plates for Control and CD2AP KD groups, respectively, N = 5 independent experiments. In ( a , b ), * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Membrane, Injection, Labeling, Two Tailed Test, Control, Western Blot

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: Two-cell stage Xenopus laevis embryos were microinjected with 14.8 pmol Control-morpholino (MO, Control, a , b ), 3.2 pmol FOLR1-MO (FOLR1 KD, a ) or 14.8 pmol CD2AP-MO2 (CD2AP KD, b ) per embryo and incubated with saline or proteasome and lysosome inhibitors at the end of gastrulation (stage 12) until neural plate stages (stage 17) when they were processed for Western blot assays. Images are examples of Western blot assays. Graphs show individual and mean ± SD percent of optical density (OD) for CD2AP ( a ) or FOLR1 ( b ) immunoblot band normalized with GAPDH protein band OD and compared to controls. In ( a ), n = 34 and 42 neural plates for Control and FOLR1 KD, respectively, N = 7 independent experiments; n = 20 and 26 neural plates for Control+inhibitors and FOLR1 KD+inhibitors groups, respectively, N = 3 independent experiments. In ( b ) n = 16 and 20 for Control and CD2AP KD groups, respectively and n = 16 and 18 neural plates for Control+inhibitors and CD2AP KD+inhibitors, respectively, N = 3 independent experiments. In ( a , b ) * p < 0.05, *** p < 0.001, ns: not significant, two-tailed ratio t -test. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: Control, Incubation, Saline, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Noncanonical function of folate through folate receptor 1 during neural tube formation

doi: 10.1038/s41467-024-45775-1

Figure Lengend Snippet: a – c Neural plate from mid neural plate stage Xenopus laevis embryos was dissected and dissociated cells plated in vitro. After 2 h, cells were loaded with the Ca 2+ sensor Fluo4-AM and time-lapse imaged. a Example of 1-h recording of neural plate cell Ca 2+ activity. b , c Folinic acid ( b , c ), folic acid ( c ) or vehicle was added to neural plate cells in culture during time-lapse imaging and the Ca 2+ response was recorded in the first minute post addition. b Example of acute transient elicited by 100 μM folinic acid. c Graph shows mean ± SEM folinic- or folic acid-responsive neural plate cells compared to total number of cells with spontaneous Ca 2+ transients in 30 min recording, N = 3 independent experiments. Two-tailed one sample t and Wilcoxon test. d Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA and grown until early and mid-neural plate stages when they were time-lapse imaged before and after addition of vehicle or 300 μM folinic acid. Image shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles. Graph shows individual and mean ± SD percent change in Ca 2+ transient frequency before and after addition of vehicle or folinic acid, n = 4, 5, 6 and 7 embryos for Early-Vehicle, Early-Folinic acid, Mid-Vehicle and Mid-Folinic acid groups, respectively. One-sample two-tailed t -test, compared to hypothetical value of 100%. e – h Two-cell stage embryos were bilaterally microinjected with GCaMP6s mRNA ( e – h ) and unilaterally microinjected with 9.9 pmol FOLR1-MO1 (FOLR1 KD1/KD) or 1.6 pmol FOLR1-MO2 (FOLR1 KD2) per blastomere ( e ) and grown until mid-neural plate stages when they were time-lapse imaged. Image in ( e ) shows example of embryo with cells exhibiting Ca 2+ transients indicated with circles in WT and FOLR1 KD1 halves. Graphs show individual Ca 2+ transient frequency (transients/5 min) in WT and FOLR1 KD1 or KD2 halves ( e , n = 6 embryos) and in WT embryos before and after addition of vehicle ( f , n = 7 embryos), 50 μM folic acid ( g , n = 6 embryos), Na + and voltage-gated Ca 2+ channel blockers (VGC block : 0.02% tricaine+10 μM nitrendipine+25 μM TTA-2, h , n = 5 embryos), or a mix of folic acid and VGC block ( h , n = 5 embryos). Two-tailed paired t -test ( e – g ) and 1-way ANOVA-Tukey multiple comparisons test ( h ). In ( c – h ), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. i Model of FOLR1 and CD2AP regulation of neural tube formation. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies used were: anti-phospho-tyr-4,8,10 CD2AP, 1:500 (Rockland, cat. # 600-401-J96), anti-phospho-tyr-674 c-Cbl, 1:500 (MyBioSource, cat. # MBS820886), anti-C-cadherin, 1:100 (Developmental Studies Hybridoma Bank, cat. # 6B6), anti-EEA1, 1:1000 (Origene, cat. # AB0006-200), anti-ubiquitin, 1:500 (Stress Marq, cat. # SPC-119B), anti-Rab7, 1:500 (Cell Signaling, cat. # 9367), anti-LAMP1, 1:500 (Abcam, cat. # ab24170), anti-GFP, 1:750 (Abcam, cat. # ab13970), anti-mCherry, 1:750 (Biorbyt, cat. # orb11618), anti-SOX2, 1:300 (Cat # AF2018, R&D Systems), anti-α-tubulin, 1:500 (Abcam, cat. # ab15246).

Techniques: In Vitro, Activity Assay, Imaging, Two Tailed Test, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Decreasing β-Catenin Leads to Altered Endothelial Morphology, Increased Barrier Permeability and Cognitive Impairment During Chronic Methamphetamine Exposure

doi: 10.3390/ijms26041514

Figure Lengend Snippet: Elisa for p-Tau, NEFL(Nfl), and GFAP in serum of mice administered METH for 2 or 4 weeks. ( A ) Elisa for p-Tau in serum of mice post-administered METH. ( B ) Elisa for Nfl in serum of mice post-administered METH. ( C ) Elisa for GFAP in serum of mice post-administered METH. Data are expressed as mean ± SEM. Statistical significance was assessed using one-way ANOVA and Tukey HSD test (for multiple comparison test). The statistical significance is shown as ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Phosphorylated Tau (p-Tau), glial fibrillary acidic protein (GFAP) and neurofilament light polypeptide (Nfl/NEFL) in mice serum were measured by ELISA kits (p-Tau: E-EL-M1289; GFAP: E-EL-M0554; NEFL: E-EL-M0817; Elabscience, Wuhan, China) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

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: Aging (Albany NY)

Article Title: Geniposide-mediated protection against amyloid deposition and behavioral impairment correlates with downregulation of mTOR signaling and enhanced autophagy in a mouse model of Alzheimer's disease

doi: 10.18632/aging.101759

Figure Lengend Snippet: Geniposide treatment decreases mTOR activation markers in brains of APP/PS1 mice. Hippocampal expression of Akt, mTOR, and 4E-BP1, and their respective phosphorylated forms was detected by western blot. The expression of p-Akt ( A ) and p-mTOR ( B ) was enhanced in APP/PS1 mice compared to WT, and geniposide attenuated this increase. The expression of p-4E-BP1 ( C ) in APP/PS1 mice was reduced compared to WT, and geniposide partly restored this decrease. Data are presented as mean ± SEM (n = 6). *** p < 0.001, ** p < 0.01, * p < 0.05 vs. WT; # p < 0.05 vs. APP/PS1 mice (one-way ANOVA, Tukey's Multiple Comparison Test). WT: wild-type mice. GP: geniposide.

Article Snippet: The membranes were blocked in 5% bovine serum albumin in TBST (Tris-buffered saline with 0.05% Tween-20) for 1h, and incubated overnight at 4°C with primary antibodies directed against: Akt (1:1,000), p-Akt (1:2,000), mTOR (1:1,000), p-mTOR (1:1,000), 4E-BP1 (1:1,000), or p-4E-BP1 (1:2,000), followed by incubation at 4°C for 2h with a goat-anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Boster, Wuhan, China).

Techniques: Activation Assay, Expressing, Western Blot, Comparison