Journal: bioRxiv

Article Title: Physiological perfusion of human vasculature reveals a YAP/TAZ-Apelin switch linking intraluminal flow to endothelial state transitions and vessel remodeling

doi: 10.64898/2026.03.21.713033

Figure Lengend Snippet: (A) T1 endothelial cells on Day 4 plotted for APLN and APLNR expressions. (B) Rank plot of all analyzed genes and their correlation coefficient with APLN (left). Correlation of APLN expression with gene set scoring for YAP/TAZ targets (center). Overlay of scores onto the scatter plot of APLN and APLNR expressions (right). Expression values are from T1 endothelial cells on Day 4. (C) qPCR expression data of APLN and APLNR from monolayer endothelial cells treated with IAG933 (2 µM; 24 hrs; 3 independent experiments; 2-tailed unpaired student’s t-test). (D, E) Mapping tip cell and stalk-like clusters from the human tumor angiogenesis atlas of Goveia et al . onto T1 endothelial cells on Day 4, with (E) additionally showing RNA velocity streamlines. (F) Gene set scoring of T1 endothelial cells on Day 4 for Apelin up-regulated genes (Park et al ., ). (G) Negative correlation between gene set scores for YAP/TAZ targets and Apelin up-regulated genes. (H) Box plot summaries of (G). (A, B, G) Spearman’s correlation coefficient. (A, B, D-F) Axes show log-normalized expression values of APLN and APLNR denoised using Markov Affinity-based Graph Imputation of Cells. (I) Skeleton visualizations of vascular beds grown using low or high flow on Day 4. (J) Quantification of (I) (11 devices per condition across 6 independent experiments). Data is a subset of (L) and (N). (K) Skeleton visualizations of low/high flow vascular beds on Day 4 treated with IAG933 (2 µM). (L) Quantification of (K) (6 devices per condition across 3 independent experiments). (M) Skeleton visualizations of low/high flow vascular beds on Day 4 treated with ML221 (10 µM). (N) Quantification of (M) (5-6 devices per condition across 3 independent experiments). (J, L, N) 2-tailed unpaired Welch’s t-test. (C, J, L, N) * p < 0.05; ** p < 0.01; *** p < 0.001. Error bars show SEM.

Article Snippet: IAG933 (Piramal Pharma Solutions, kindly provided by Methvin Isaac, Ontario Institute for Cancer Research) was used at 2 μM, ML221 (MedChemExpress) was used at 10 μM, human recombinant BMP (R&D Systems) was used at 10 ng/mL, and Axitinib (MedChemExpress) was used at 100 nM.

Techniques: Expressing

Journal: Theranostics

Article Title: Exercise ameliorates osteopenia in mice via intestinal microbial-mediated bile acid metabolism pathway

doi: 10.7150/thno.104186

Figure Lengend Snippet: FMT from exercised mice activated apelin signaling pathway. (A) PCA plot of samples from TranspCtrl and TranspExer groups (n = 3 per group). (B) Volcano plot of DEGs. (C) Enrichment analysis of upregulated DEGs in the TranspExer group. (D) Heatmap of expression levels of genes associated with the apelin signaling pathway. (E) Relative mRNA expression levels of Apln , Aplnr , Adcy4 , and Adcy5 . (F) Serum apelin concentration. (G) Representative apelin-stained IHC sections. (H) Relative mRNA expression levels of Pparg , Cebpa , Fabp4 , and Lpl . (I) Representative H&E sections depicting adipose tissue. (J-K) Quantitative analysis of J) number of adipocytes and K) adipocytes area. (L) Representative µCT images of tibias in TranspCtrl and TranspExer mice treated with AAV-Ctrl or AAV-Apln. (M-N) Trabecular bone microarchitecture showing BMD and Tb. N. Graphs show mean ± SEM (n = 6 per group), with statistical significance determined by two-tailed student t test in E-F, H, and J-K, and one-way ANOVA followed by Bonferroni's multiple comparisons test in M-N. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The apelin signaling pathway was suppressed using the apelin receptor antagonist ML221 (TargetMol, USA), wherein mice were administered ML221 (150 μg/kg) via tail vein injections three times a week for 8 weeks .

Techniques: Expressing, Concentration Assay, Staining, Two Tailed Test

Journal: Theranostics

Article Title: Exercise ameliorates osteopenia in mice via intestinal microbial-mediated bile acid metabolism pathway

doi: 10.7150/thno.104186

Figure Lengend Snippet: Exercise-matched FMT reshaped the gut metabolite profiles in OVX mice. (A) PCA plot. (B) Volcano plot of differential metabolites. (C) Enrichment analysis of upregulated metabolites in the TranspExer group. (D) Heatmap of differential metabolites between the TranspCtrl and TranspExer groups. (E) Flowchart of the gavage procedure for key metabolites in OVX mice. (F) Representative µCT images of tibias from mice in Sham, Ctrl, Tau, and UDCA groups. (G) Representative H&E-stained, OCN-stained, and TRAP-stained sections. Black arrows indicating osteoclasts. (H-I) Quantitative results of N. OBs/BS and N. OCs/BS. (J) Relative RNA expression levels of genes Apln and Aplnr . Graphs show mean ± SEM (n = 6 per group), with statistical significance determined by one-way ANOVA followed by Bonferroni's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The apelin signaling pathway was suppressed using the apelin receptor antagonist ML221 (TargetMol, USA), wherein mice were administered ML221 (150 μg/kg) via tail vein injections three times a week for 8 weeks .

Techniques: Staining, RNA Expression

Journal: Theranostics

Article Title: Exercise ameliorates osteopenia in mice via intestinal microbial-mediated bile acid metabolism pathway

doi: 10.7150/thno.104186

Figure Lengend Snippet: Exercise-matched FMT and key metabolites ameliorated age-related bone loss. (A) Flowchart of FMT administration in aged mice. (B) Representative µCT images of tibias from aged mice in the TranspCtrl group and the TranspExer group. (C-E) Trabecular bone microarchitecture showing C) BMD, D) BV/TV, and E) Tb. N. (F) Ct. Th statistical results. (G-H) Biomechanical results of G) yield point and H) ultimate force. (I) Representative H&E-stained, OCN-stained, and TRAP-stained sections from the TranspCtrl group and the TranspExer group. Black arrows indicating osteoclasts. (J) Quantitative results of N. OBs/BS. (K) Relative mRNA expression levels of genes Apln and Aplnr . (L) Flowchart of metabolites supplementation in aged mice. (M) Representative µCT images of tibias from aged mice in the AgeCtrl, AgeTau, and AgeUDCA groups. (N-P) Trabecular bone microarchitecture showing N) BMD, O) BV/TV, and P) Tb. N. (Q) Representative H&E-stained, OCN-stained, and TRAP-stained sections from the AgeCtrl, AgeTau, and AgeUDCA groups. Black arrows indicating osteoclasts. (R) Quantitative results of N. OBs/BS. (S) Relative mRNA expression levels of genes Apln and Aplnr . Graphs show mean ± SEM (n = 6 per group), with statistical significance determined by two-tailed student t test in C-H and J-K, and one-way ANOVA followed by Bonferroni's multiple comparisons test in N-S. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The apelin signaling pathway was suppressed using the apelin receptor antagonist ML221 (TargetMol, USA), wherein mice were administered ML221 (150 μg/kg) via tail vein injections three times a week for 8 weeks .

Techniques: Staining, Expressing, Two Tailed Test

Journal: Theranostics

Article Title: APLN promotes hepatocellular carcinoma through activating PI3K/Akt pathway and is a druggable target

doi: 10.7150/thno.34713

Figure Lengend Snippet: APLN activates PI3K/Akt signaling. (A) Nine cancer pathway- related firefly luciferase reporter constructs were co-transfected with renilla luciferase reporter (internal control) in HKCI-10 cells. (B) BYL-719 (4uM) and GDC-0941 (1uM), two specific PI3K/Akt Inhibitors, were added to HKCI-2 or HKCI-10 cells. Control cells were treated with an equivalent dilution of DMSO. MTT assays were performed. (C) Upregulation of APLN increased phospho-PI3K p85, phospho-Akt, phospho-GSK3β and Cyclin D1 expression by western blot analysis. (D) APLN knockdown decreased the expression of phospho-PI3K p85, phospho-Akt, phospho-GSK3β and Cyclin D1 by western blot analysis. (E) Top panel showed APLN overexpression was associated with the increased expression of phospho-Akt and phospho-GSK3β in human HCC specimens. Bottom panel demonstrated positive correlation of APLN with PI3K-AKT pathway in TCGA-LIHC cohort by gene set enrichment analysis. (F) HKCI-2 and HKCI-10 cells were serum starved overnight and stimulated with 1uM Apelin-13 for indicated time. For ML221 treatment, HKCI-2 and HKCI-10 cells were pretreated with 50µM ML221 for 30min before Apelin-13 stimulation. Cell lysates were blotted with indicated antibodies. The images are representative of two independent experiments. Error bars in A and B represent means ± sd from three independent experiments, (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Specific APLN receptor inhibitor ML221 was purchased from R&D Systems (Minneapolis, MN) .

Techniques: Luciferase, Construct, Transfection, Control, Expressing, Western Blot, Knockdown, Over Expression

Journal: Theranostics

Article Title: APLN promotes hepatocellular carcinoma through activating PI3K/Akt pathway and is a druggable target

doi: 10.7150/thno.34713

Figure Lengend Snippet: APLN-APLNR is a potential therapeutic target of HCC. ( A ) One immortalized hepatocyte cell lines (LO2) and four HCC cell lines (HepG2 PLC5, HKCI-2 and HKCI-10) were treated with ML221 and cell viability was assessed by MTT assay. Top panel: Cells were treated with 100 µM ML221. The colors represent different time points and the diameter indicates relative cell viability. Bottom panel: Cells were treated with different concentrations of ML221 for three days. The colors represent different ML221 concentration and the diameter indicates relative cell viability. ( B ) ML221 treatment significantly decreased colony numbers in HKCI-2 and HKCI-10 cells in a dose-dependent manner. ( C ) HKCI-2 and HKCI-10 cells were treated with 50 µM ML221 for 24h. Cell lysates were then blotted with indicated antibody. ( D ) ML221 (50 µM) was added to HKCI-2 or HKCI-10 cells. Cell viability was assessed by MTT assay. ( E and F ) Intraperitoneal injection of ML221 (10 mg/kg, every other day) significantly inhibited the growth of subcutaneous PLC5 xenografts (N = 4) ( E ) and HepG2 xenografts (N = 10) ( F ), as evidenced by reductions in tumor volume and weight. ML221 treatment had no effect on body weight. ( G ) Representative images of Ki67-positive (Top panel) and TUNEL-positive cells (Bottom panel). At least 4 fields per slide and 2 slides per tumor were counted at 200 × magnification. ( H ) Western blot analysis of PLC5 xenografts (Top panel) and HepG2 xenografts (Bottom panel) confirmed that ML221 treatment inhibited PI3K/Akt signaling, as evidenced by the decreased expression of phospho-Akt. Error bars in B and D represent means ± sd from three independent experiments, (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Specific APLN receptor inhibitor ML221 was purchased from R&D Systems (Minneapolis, MN) .

Techniques: MTT Assay, Concentration Assay, Injection, TUNEL Assay, Western Blot, Expressing

Journal: Development (Cambridge, England)

Article Title: Thymic epithelial organoids mediate T-cell development.

doi: 10.1242/dev.202853

Figure Lengend Snippet: Fig. 2. TECs cultured as organoids show in vitro functionality when reaggregated with T-cell progenitors. (A) Workflow to generate organoid reaggregate fetal thymic organ cultures (ORFTOCs). (B) Flow cytometry plot showing T-cell development in D6 ORFTOCs. (C) Proportion of DP thymocytes at D6 in ORFTOCs and controls [fetal thymic organ cultures (FTOCs) and reaggregates containing only MEFs and the EpCAM-depleted cells from thymic lobes]. **P=0.0047; ns, not significant (P>0.05) (Kruskal–Wallis test with Dunn’s multiple comparison test; n=9, 16 and 9 for ORFTOCs, FTOCs and reaggregates from MEFs+EpCAM-depleted cells, respectively, from 9 independent experiments). (D,E) Flow cytometry plots. (D) T-cell development at D6 in controls (left: FTOCs; right: reaggregates from MEFs+EpCAM-depleted cells). (E) Absence of a CD45– Lineage−(Lin−) population in control reaggregates containing only TECs cultured as organoids and MEFs. Gating strategy is indicated on the left for all flow cytometry plots. (F) Proportion of CD3ɛ-positive, TCRβ-positive cells in ORFTOCs and FTOCs at D6, D13 and D20. **P=0.0021, ***P=0.0003, ****P<0.0001; ns, not significant (P>0.05) (two-way ANOVA with Tukey’s multiple comparison test; n=9, 6, 3, 16, 8 and 5 for D6 ORFTOCs, D13 ORFTOCs, D20 ORFTOCs, D6 FTOCs, D13 FTOCs and D20 FTOCs, respectively, from at least 3 independent experiments). All graphs represent individual datapoints with mean±s.d. (G) RNAscope for Foxn1 in a D13 ORFTOC. Nuclei are counterstained using Spectral DAPI (grey). (H-J) Immunofluorescence images. (H) KRT8 (blue) and MHCII (green) staining in a D13 ORFTOC (left) and a D13 FTOC (right). (I) Medullary cells (UEA1 reactivity; magenta) in a D13 ORFTOC. (J) Epithelial cells (EpCAM; blue), medullary cells (UEA1 reactivity; bright pink) and T cells (CD3ɛ; amber) in a D13 ORFTOC. DAPI stains nuclei (grey). ORFTOCs were formed with TECs cultured as organoids originating from E16.5 thymi, with the EpCAM-depleted fraction of cells from E13.5 thymi and with MEFs. FTOCs were from E13.5 thymi. Scale bars: 100 µm (G-I); 10 μm (J).

Article Snippet: The cells were incubated for 20 min with the following antibodies: Ter119-FITC (BioLegend, 116205; 1/800), Cd45R-FITC (BioLegend, 103205; 1/800), CD11b-FITC (Thermo Fisher Scientific, 11-0112-82; 1/800), Ly-6G-FITC (BioLegend, 108405; 1/800), Cd11C-FITC (BioLegend, 117306; 1/800), NK-1.1-FITC (BioLegend, 108705; 1/800) (together referred to as Lineage), CD44-PE (BioLegend, 103008; 1/160), CD69-APC (BioLegend, 104513; 1/160), CD4-BV605 (BioLegend, 100548; 1/40), CD3-PerCP/Cy5.5 (BioLegend, 100327; 1/160), CD8-PE/Cy7 (BioLegend, 100722; 1/160), CD25-BV711 (BioLegend, 102049; 1/160), CD45-AF700 (BioLegend, 103127; 1/160), TCRβ-BV421 (BioLegend, 109230; 1/80) and DAPI (Tocris, 4748, 0.5 μg/ml).

Techniques: Cell Culture, In Vitro, Flow Cytometry, Comparison, Control, RNAscope, Immunofluorescence, Staining

Journal: BMC Ophthalmology

Article Title: Protective effect of apelin-13 in lens epithelial cells via inhibiting oxidative stress-induced apoptosis

doi: 10.1186/s12886-024-03746-6

Figure Lengend Snippet: Apelin-13 prevented H 2 O 2 -induced cellular viability in HLE-B3 cells. Viability of HLE-B3 cells as assessed using CCK8 assay. HLE-B3 cells were treated with ML221 (10 µmol) for 1 h, preincubated with apelin-13 (0.1 µM) for 24 h, and subsequently treated with H 2 O 2 (200 µM) for 24 h in this experiment. Representative images of three replicated experiments were presented. The significance marked at the top of the columns refers to comparison with the control group. Data are shown as mean ± SD ( n = 3)

Article Snippet: HLEC-B3 cells were preincubated with different concentrations of apelin-13 (MCE, New Jersey, USA) and/or ML221 (Anaspec Inc, CA, USA) for 24 h, then treated with H 2 O 2 (KEHBIO, China).

Techniques: CCK-8 Assay, Comparison, Control