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: Nature Communications

Article Title: Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models

doi: 10.1038/s41467-022-34990-3

Figure Lengend Snippet: a The expression level of Apln mRNA in TM4 cell under high glucose (HG) and insulin. Data are presented as means ± SEM. Unpaired two-tailed t test. Results of three independent experiments are shown ( n = 3). b Immunofluorescence of ACTB (red) co-stained with APLN (green) in TM4 cell under HG and insulin. Scale bar, 5 μm. c Quantitative analysis of APLN immunofluorescence level in ( b ). Box-and-whisker plots denote the maximum (top whisker), 75th (top edge of box), 25th (bottom edge of box) and minimum (bottom whisker) percentiles, and the median (line in box). Unpaired two-tailed t test. n = 100 cells examined over 3 independent experiments. d Immunofluorescence of ACTB (red) co-stained with HIF1A (green) in TM4 cell under HG. Scale bar, 5 μm. e Genome browser view of the HIF1A density in the Apln range in negative control (NC) and HG treated TM4 cell. f PCA analysis of metabolomic data in NC and APLN group. Four biological replicates were performed for each group. g Heatmap showing all differential metabolites in NC and APLN group. h The metabolic level of NAD+, carnitine and glutathione and Palmitelaidic Acid in NC and APLN group. unpaired two-tailed t test between sample groups. n = 4 biologically independent samples. Each box represents the median and the 25 and 75% quartiles, and the whiskers indicate 1.5 times of the interquartile range. i , Immunoblots of TJP1 and GJA1 in TM4 treated APLN and by combination of three metabolites in different concentrations. j Immunoblots of TJP1 and GJA1 in TM4 treated APLN and three different concentrations (200, 500, 1000 μM) of each metabolites separately. k Immunoblots of indicated proteins in stable knockdown APJ cell treated APLN or HG. l Immunoblots of indicated proteins in TM4 treated APLN or HG and rescued by ML221. m Immunoblots of indicated proteins in TM4 treated APLN or HG and rescued by F13A. n Immunoblots of AMPKα1 and p-AMPKα1 in TM4 treated APLN or different concentrations of Palmitelaidic Acid.

Article Snippet: Mouse in group 3 was injected with ML221 (20 mM, Selleck, S8695).

Techniques: Expressing, Two Tailed Test, Immunofluorescence, Staining, Whisker Assay, Negative Control, Western Blot, Knockdown

Journal: Nature Communications

Article Title: Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models

doi: 10.1038/s41467-022-34990-3

Figure Lengend Snippet: a Schematic illustration of the APLN or ML221 injection experiment in db/db . b Immunofluorescence of biotin (red) between indicated sample groups. Scale bar, 50 μm. c Biotin positive seminiferous tubules percentage in Control, APLN and ML221 injection group. Data are presented as means ± SEM. One-way ANOVA. Statistics were performed in five mouse testes each group ( n = 5). d Immunofluorescence of TJP1 and GJA1 (green) co-stained with VIM (red) in APLN injection and ML221 injection testicular paraffin sections. Scale bar, 10 μm. e Quantitative analysis of TJP1 and GJA1. Box-and-whisker plots denote the maximum (top whisker), 75th (top edge of box), 25th (bottom edge of box) and minimum (bottom whisker) percentiles, and the median (line in box). Statistics were performed in five mouse testes each group ( n = 5). One-way ANOVA was performed. f Schematic illustration of the ML221 injection experiment in db/db for IVF and ICSI. Mice were treated with ML221 at a dose of 10 mg per kg body weight per day. g Bright field diagram of testicular size in control and ML221 injection group. Scale bar, 2 mm. h H&E staining of testicular sections in control and ML221 injection group. Scale bar, 100 μm. i Sperm counts, sperm motility and testosterone level between control and ML221 injection group. PR: progressive motile, NP: non-progressive motile, IM: immotility. unpaired two-tailed t test was performed. j Brightfield diagram of 4-cell, morula, and blastocyst between control and ML221 injection group. Arrows indicated normal developing embryos. Scale bar, 200 μm. k The trilinear table shows all the embryo injection, two-cell embryos, blastocysts and live C-section-born mice data of IVF between control and ML221 injection group. l The trilinear table shows all the embryo injection, two-cell embryos, blastocysts and live C-section-born mice data of ICSI between control and ML221 injection group.

Article Snippet: Mouse in group 3 was injected with ML221 (20 mM, Selleck, S8695).

Techniques: Injection, Immunofluorescence, Control, Staining, Whisker Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting APLN/APJ restores blood-testis barrier and improves spermatogenesis in murine and human diabetic models

doi: 10.1038/s41467-022-34990-3

Figure Lengend Snippet: a Immunofluorescence of SOX9 (red) in human testis culture in Day 0 and Day 7 paraffin sections. Scale bar, 20 μm. The percentage of SOX9-positive cells was calculted on Day 0 and Day 7 separately. Mean ± SEM. ns, not significant, unpaired two-tailed t test. n = 3 human testis culture examined over 3 independent experiments . b Immunofluorescence of SYCP3 (green) and CREM (red) in human testis culture on Day 0 and Day 7 paraffin sections. Scale bar, 20 μm. The percentage of SYCP3-positive cells was counted. Mean ± SEM. Unpaired two-tailed t test. n = 3 human testis culture examined over 3 independent experiments . c Bright field diagram of human testis culture between different groups. Scale bar, 50 mm. d , e Immunofluorescence of TJP1 and GJA1 (green) and VIM (red) in human testis culture in Day 7 paraffin sections between indicated sample groups. n = 3 per group. Scale bar, 20 μm. Box-and-whisker plots denote the maximum (top whisker), 75th (top edge of box), 25th (bottom edge of box), and minimum (bottom whisker) percentiles, and the median (line in box). Quantitative analysis of TJP1. Two-tailed student’s t test was performed. f Immunofluorescence of TJP1 or GJA1 (green) and VIM (red) in diabetic patient testis culture in Day 7 paraffin sections between indicated sample groups. Scale bar, 50 μm. n = 3 per group. Box-and-whisker plots denote the maximum (top whisker), 75th (top edge of box), 25th (bottom edge of box) and minimum (bottom whisker) percentiles, and the median (line in box). Quantitative analysis of TJP1 and GJA1. Two-tailed student’s t test was performed. g Hypothetical Mechanism. Elevated blood glucose in diabetic patients directly leads to elevated ROS in Sertoli cells, which promotes HIF1A nuclear translocation and activates Apln expression. The excess of APLN disrupted the BTB-related genes by decreasing NAD+, carnitine, and glutathione. Blocking APLN/APJ with F13A and ML221 could significantly ameliorate the BTB damage and improve low sperm quality.

Article Snippet: Mouse in group 3 was injected with ML221 (20 mM, Selleck, S8695).

Techniques: Immunofluorescence, Two Tailed Test, Whisker Assay, Translocation Assay, Expressing, Blocking Assay

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