Journal: medRxiv

Article Title: COVID-19 Diagnostic Testing For All - Using Non-Dilutive Saliva Sample Collection, Stabilization and Ambient Transport Devices

doi: 10.1101/2021.01.20.20243782

Figure Lengend Snippet: Gamma-irradiated SARS-CoV-2 virus (BEI Resources) at 2 genome equivalents/uL was spiked in 1mL of saliva kept either in non-GTR STM (“Saliva”) (open circles) or GTR-STM devices (open diamonds), extracted with MagMAX Viral RNA Kit (ThermoFisher) and RT-PCR performed with CDC’s N1 primer. The pass/fail criteria set at 35.7 CT is 3 CT values more than the average CT value of the “Saliva” only samples. “Saliva” samples without GTR-STM gave a mean CT of 32.4 CT (Std Dev, ±0.3), and Saliva Samples in GTR-STM gave a mean CT of 32.7 CT (Std Dev, ±0.2). Study setup Experimental Sample : A contrived GTR-STM sample (n=9) was prepared by spiking 1mL of saliva with 2 genome equivalents/uL of -gamma-irradiated SARS-CoV-2 virus. Control sample : A contrived non-GTR STM (“Saliva”) (n=3) sample was prepared by spiking 1mL of saliva with 2 genome equivalents/uL of gamma-irradiated SARS-CoV-2 virus. Sample Extraction :RNA was extracted from 200uL of sample from both control and stressed samples following manufacturer’s instructions for MagMAX Viral RNA (ThermoFisher) manual protocol and eluted with 50uL of elution buffer. Quantification : Amplify 5uL of extracted RNA from each sample in triplicates with TaqPath master mix (ThermoFisher) and CDC’s N1 Primer (IDT).

Article Snippet: GTR-STM was tested by extraction with either QIAamp Viral RNA Mini Kit (Qiagen, cat #52906) or MagMAX Viral RNA Isolation Kit (ThermoFisher, cat #AM1939) and compared with either PBS or neat saliva as controls.

Techniques: Irradiation, Reverse Transcription Polymerase Chain Reaction

Journal: medRxiv

Article Title: COVID-19 Diagnostic Testing For All - Using Non-Dilutive Saliva Sample Collection, Stabilization and Ambient Transport Devices

doi: 10.1101/2021.01.20.20243782

Figure Lengend Snippet: Heat-inactivated SARS-CoV-2 virus RNA (BEI Resources) at 500 genome equivalents/uL was spiked into 1mL of saliva kept either in GTR-STM collection devices or non-GTR-STM (“Saliva”) tubes, and stored at 25°C for 36 days. Matched spiked control saliva samples in both kinds of tubes were stored at -80°C. The pass/fail criteria are set at 32 CT value. 200uL of the sample is used at each time point to extract viral RNA with the MagMAX kit with a final elution volume of 50uL. The CT value of the viral RNA extracted with MagMAX viral RNA kit is normalized to input volume of 200uL (volume of sample used for RNA extraction). All GTR-STM samples gave excellent recoveries when compared to their matched -80°C control. Both the -80°C and the 25°C samples for the non-GTR-STM (“Saliva”) is above the pass/fail line even in the -80°C control samples indicating that the viral RNA is degraded by the Rnase in the short time (less than half hour) that the saliva sample is defrosting before RNA extraction is performed. Study setup Experimental Samples : 1 mL aliquots of saliva contrived with SARS-CoV-2 at 500 genome equivalents/uL (BEI Resources) were placed into either GTR-STM devices or non-GTR-STM (“Saliva”) tubes and stored at ambient (25°C) for up to 36 days. Control sample : 1 mL aliquots of saliva contrived with SARS-CoV-2 at 500 genome equivalents/uL (BEI Resources) were placed into either GTR-STM devices or non-GTR-STM (“Saliva”) tubes and stored at -80°C for up to 36 days. Sample Extraction : RNA extracted from 200uL of experimental and control samples with MagMAX viral RNA kit at days 10, 15, 20, 25, and 36 and eluted in 50uL of elution buffer. Quantification : 5uL of RNA was quantified with CDC’s SARS-CoV-2 RT-qPCR assay for N1 primer.

Article Snippet: GTR-STM was tested by extraction with either QIAamp Viral RNA Mini Kit (Qiagen, cat #52906) or MagMAX Viral RNA Isolation Kit (ThermoFisher, cat #AM1939) and compared with either PBS or neat saliva as controls.

Techniques: RNA Extraction, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of generating D50 MCT8-COs and obtaining single-cell suspension for sc-RNA seq analysis. B . UMAP plot showing the cell clusters identified by principal component analysis in the indicated COs C . UMAP plots showing the distribution of markers for NPCs and neurons. D . Dot plot showing the relative expression levels of the gene markers used to identify each cell population. E . UMAP plot showing the cell types identified by manual curation in control COs. F . Dot plot showing the relative expression levels of SLC16A2, THRB, and THRA in each cell population of the dorsal projection trajectory. G . Interpretation of sequential gene expression changes during dorsal projection trajectory progression. H . UMAP plot showing the cell types identified by manual curation in MCT8-COs. I . Histogram of the relative number of cells in control and MCT8-COs. J . Volcano plots showing the distribution of differentially expressed genes in MCT8-deficient vs. control COs. iPSCs, induced pluripotent stem cells; COs, cortical organoids; NPCs, neural precursor cells; IPCs, intermediate precursor cells; DPT, dorsal projection trajectory; UMAP, Uniform Manifold Approximation and Projection. The X 2 test for multiple comparisons and pairwise cell proportions. **P < 0.01; ***P < 0.0001. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 0.26.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Suspension, RNA Sequencing, Expressing, Control, Gene Expression

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of obtaining spatial transcriptomic data. B . Image showing the cell segmentation on the indicated COs, based on transcript localization data. Scale bar: 100 µm. C . Heatmap showing the relative expression levels of the genes used to identify the indicated neural cell types. D . Heatmap of the differentially expressed genes in the indicated neural cells between control vs. MCT8-COs. E . Same as in B , except the cells are classified into the indicated types. F . Distribution of distances between the indicated cells and groups. G . Histogram of the distribution of cell densities. Considering a radius of 100 µm, if only one cell was in contact with another cell, it was considered as sparse density. If one cell was in contact with five or more cells, it was considered a dense density. H . Violin plots show the expression levels of the indicated genes, considering the indicated cellular densities. I. Ligand-receptor interaction between the indicated neural cells in Control and MCT8-deficient COs. Differentially expressed genes threshold: p-value < 0.05.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of generating NPCs from iPSCs and the subsequent differentiation of the NPCs into neurons. B . Brightfield and confocal fluorescence images showing iPSCs-derived NPCs stained for SOX2 (magenta), NESTIN (yellow), MCT8 (magenta), and Dapi (blue; nuclear). C . Relative mRNA levels of the indicated genes in Control and MCT8 NPCs after 1, 6, and 12 days of neurodifferentiation. D . Brightfield images showing NPCs after six days of neurodifferentiation. E . Principal component plot illustrating differences between control and MCT8-deficient NPCs. F . Volcano plots showing the distribution of differentially expressed genes in control vs. MCT8-NPCs; each point represents the average of 5 control and 5 MCT8-deficient samples of pooled NPCs for each transcript. G . Heatmap depicting the top 20 differentially expressed genes related to neurogenesis between control vs. MCT8-NPCs identified by bulk-RNA seq. H . Venn comparison of differentially expressed genes belonging to the gene set related to neurogenesis between control vs. MCT8-NPCs identified by bulk-RNA seq. (blue) and sc-RNA seq analysis (green) and between control vs. MCT8-IPCs identified by sc-RNA seq analysis (purple); common differentially expressed genes were identified (grey box). I . Brightfield images of control and MCT8-NPCs after twelve days of neurodifferentiation; TUJ1 staining in green and Dapi (blue; nuclear). J . The upper two panels are MCT8 staining in red, TUJ1 in green, and Dapi (blue); the lower panels are RBFOX3 staining in green, NEUROD1 in red, and Dapi in blue. K . MCT8-NPCs after being treated with 60nM T3 during the twelve days of neurodifferentiation. TUJ1 staining is green, and SOX2 is red. L . SOX2 staining in red and Dapi in blue on the indicated cells and treatments. Scale bars: B: 50 µm; D, I, J: 100 µm. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 1.5 in the Partek Flow platform. Expression values are mean ± SD of n = 3–6 RNA samples, each of them consisting of 2 pooled 6-well plates of NPCs from either control or MCT8-NPCs; Two-tailed Student’s test for comparing D2 deiodination and relative mRNA expression between D1, D6 and D12 of neurodifferentiation; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Fluorescence, Derivative Assay, Staining, Control, RNA Sequencing, Comparison, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of treating iPSCs-derived NPCs with radioactive T4 I125 to measure T3 I125 production. B . Representative chromatograms of the medium after control and MCT8-NPCs were incubated with T4 I125 for 24 hours. C . Quantitation of the DIO2 deiodination in control and MCT8 NPCs; n = 5 DIO2 assays. D . Volcano plots showing the distribution of differentially expressed genes in control + 1nM T4 vs. control NPCs; each point represents the average of five control + 1nM T4 and five control samples of pooled NPCs for each transcript. E . Interpretation of the findings in A-D . F . Schematic of the generation and timing of COs generation, starting with iPSCs to a culture of embryoid bodies, followed by neural induction, neuroepithelial bud expansion, and maturation. G . Quantitation of DIO2 deiodination in control COs during their first 20 days in culture. n = 4 DIO2 assays per timepoint, each consisting of 4 pooled COs from control COs. H . Relative SOX2 mRNA levels in control COs during their first 20 days in culture. Expression values are mean ± SD of n = 3–6 RNA samples, each of them consisting of 4 pooled COs from control COs; I . Schematic representation of the experiment: COs are treated with 1nM T4 from D7 to D50 and then dissociated into a single-cell suspension for sc-RNA seq. J . UMAP plot showing the cell types identified. K . Histogram of the relative number of cells in T4-COs and control COs. L . Histograms of the relative number of cells in clusters of NPCs. The identification number of each cell cluster is indicated at the bottom right corner of each rectangle. M . Volcano plots showing the distribution of differentially expressed genes in T4-CO vs. control COs. H . Gene set enrichment analysis reveals gene ontology terms enriched in T4 TX COs. O . Histograms of the relative number of cells undergoing the indicated cell cycle phase in clusters one and nine of control and NPCs. Two-tailed Student’s test for comparing DIO2 deiodination in iPSC-derived NPCs; ***P < 0.001. Differentially expressed genes thresholds: p-value < 0.05, and Average Log 2 Fold-Change of 0.26.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Derivative Assay, Control, Incubation, Quantitation Assay, Expressing, Suspension, RNA Sequencing, Two Tailed Test

Journal: bioRxiv

Article Title: Thyroid hormone promotes fetal neurogenesis

doi: 10.1101/2025.05.14.654075

Figure Lengend Snippet: A . Schematic representation of the protocol to isolate mNPCs, propagate them in culture, and differentiate into neurons. B, C. Brightfield images showing representative neurospheres containing mNPCs ( B ) and the mNPCs cultured in collagen-coated plasticware ( C ). D-F . Confocal images showing mNPCs expressing Nestin (green) and Sox2 (red) ( D ), and Mct8 (red) ( E ). The insets in E depict two dividing mNPCs that exhibited higher intensity of Mct8 immunofluorescence. F . Quantitation of the DIO2 deiodination in mNPCs; n = 6 DIO2 assays. G. Brightfield images showing representative mNPCs after two days of neurodifferentiation. H - J . Relative mRNA levels of the indicated genes in mNPCs under the indicated conditions; n = 4. K . Brightfield images showing representative mNPCs after four days of neurodifferentiation. Note the neuronal process extension. L . Tuj1+ cells under the indicated conditions and the quantitation of the percentage of Tuj1+ cells. Values are the mean ± SD of 5 replicates. Scale bars: B: 300 µm, C-L: 25 µm. Two-tailed Student’s test for comparing DIO2 deiodination in iPSC-derived NPCs; **P < 0.01; ***P < 0.001.

Article Snippet: Mouse monoclonal anti-Nestin antibody 1:1000 (Biotechne; AF4320), rabbit polyclonal anti-MCT8 antibody 1:400 (Atlas antibodies; HPA003353), mouse monoclonal anti-Tuj1 1:1,000 (Bio-Techne; AF4320), anti-Sox2 1:1,000 (Abcam; 109186), guinea pig polyclonal anti-Rbfox3 (Millipore; ABN90).

Techniques: Cell Culture, Expressing, Immunofluorescence, Quantitation Assay, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: (a) top panel: Chemical structures of N-cadherin antagonists CRS-066; LCRF-0006 and analogues RB-192 and RB-028. Active side-chain or modified side-chain depicted by highlighted box. middle panel: Average cell adhesion index values of pre-ovulatory COCs (11h post-hCG) interacting with a fibronectin substrate in presence of vehicle or N-Cadherin antagonists CRS-066, LCRF-0006, and sidechain modified analogues RB-192 and RB-028 or vehicle at respective doses. Cell indices were determined using the xCELLigence Impedance system over 15h (n=3 independent experiments with 2 technical replicates per treatment). bottom panel: Mean ± SD adhesion index values at 6 h and IC 50 values of respective drugs calculated from dose-response curve and compared using one-way ANOVA. ( b and d) Representative confocal images of N-cadherin adherens junctions on SK-OV-3 cells treated with N-cadherin antagonists CRS-066 (0.1-1.0 μM), LCRF-0006 (36-360 μM) or vehicle for 24h. (N=3). Scale bar: 40μM.( c and e ) Quantification of N-cadherin adherens junctions. Mean of total pixel intensity in red channel, +/- SEM of at least 50 cells. N=3 independent experiments. Statistical analyses with two-tailed unpaired t-test. * denotes <0.01.( f ) Bright-field images of spheroid formation in 67NR mouse mammary cell line expressing ectopic Cdh2 were treated with CRS-066 or vehicle at respective time-points. Cells were seeded at 2000 cells per well, and formation of spheroids was assessed by imaging every hour for 6h. ( g ) Mean +/- SEM of spheroid area in 67NR-Cdh2 cells treated with either vehicle or increasing doses of CRS-066 (0-2 μM) over 6h.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Analogues, Modification, Two Tailed Test, Expressing, Imaging

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: ( a - c ) Time-course of Cdh2, ctnnb1 and Cdh1 mRNA expression in isolated granulosa cell (GCs) or Cumulus oocyte complexes (COCs) from mouse ovaries at indicated time-points after eCG and hCG stimulation of folliculogenesis and ovulation (N=3 animals per time point). Cdh2 and Ctnnb1 levels are high in GC and COC throughout folliculogenesis, with a transient drop in Cdh2 level 12 h after ovulation stimulus, while E-Cadherin was high in COCs and significantly reduced by ovulation stimulus. The levels shown of the indicated mRNAs were determined by TaqMan qPCR normalised to Rpl19. ( d ) Immunofluorescent staining of N-Cadherin, βcatenin and E-cadherin throughout ovarian folliculogenesis. Confocal images of mouse ovarian sections obtained from eCG primed mice and stained using anti N-Cadherin (left panel), anti-β-catenin (middle panel) and E-cadherin (right panel). DNA is counterstained with Hoechst. Arrows indicate presence of N-Cadherin and β-catenin at granulosa-granulosa cell junctions in secondary and antral follicle stages. Arrowheads indicate presence of N-cadherin and β-catenin at oocyte-cumulus interface. High magnification images show transzonal projections extending from cumulus cells and anchored to oocyte membrane Scale bar: 50 µM. ( e ) Whole-mount immunofluorescent staining showing co-localization of N-cadherin (green) and E-cadherin (red) at the oocyte plasma membrane in mouse COC from antral follicles of eCG primed mice. N-cadherin is also evident on cumulus cell surfaces and transzonal projections. Cumulus cell and oocyte nuclear DNA is counterstained with Hoechst. Scale bar: 50 µM. ( f ) Wholemount immunostaining shows loss of β-catenin and E-cadherin at the oocyte plasma membrane after treatment with CRS-066. COCs obtained from antral follicles of eCG primed mice and treated with CRS-066 or vehicle for 4h. COCs were fixed and stained with anti-E-cadherin (green) and anti-β-catenin (red). DNA was counterstained with Hoechst. Scale bar: 50 µM.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Expressing, Isolation, Staining, Membrane, Clinical Proteomics, Immunostaining

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: COCs from eCG primed mice were treated with LCRF-0006 (36-360 uM) or CRS-066 (0,1-1 uM) during in vitro maturation (EGF and FSH stimulated) and cumulus expansion was assessed after 12 h or gene expression assessed after 10 h IVM. ( a and d ) Representative bright-field images of COCs after 12 h IVM treated with LCRF-0006 or CRS-066. Scale bar: 10µm. ( b and e ) Mean ±SEM of Cumulus expansion indices from a and d n= >20 COCs per experiment. N=4 independent experiments, * = P<0.05, ** = P<0.01. ( c and f ) Effect of N-cadherin antagonist treatment during IVM (10 h) on the expression of key genes involved in COC expansion during IVM. Mean± SEM. N=3 independent experiments. Statistical testing with one-way ANOVA * P<0.05, **P<0.01. ( g and h ) Gene ontology enrichment of biological pathways and molecular functions of significantly differentially down-regulated genes identified in RNA-Seq analysis of COCs after CRS-066 (0.3 μM) treatment compared to vehicle treatment.. All data are presented as the ratio of CRS-066 over vehicle (N=3). ( I and j ) Gene set enrichment analysis plot (GSEA) demonstrating the upregulation of Ctnnb1 and Hippo signalling pathways in CRS-066 treated COCs versus vehicle treated COCs. Net enrichment score (NES) values are shown. N=3 independent biological replicates. ( k ) Heat map representing the relative expression profiles of transcripts involved in Wnt\β- catenin, Hippo\YAP and ovarian signalling axes.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: In Vitro, Gene Expression, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: ( a ) Ovulation rate of 21d old mice treated with CRS-066 (50mg/kg) or vehicle (7.5% DMSO in 0.9% saline). COCs in oviducts counted 16h after hCG injection. Graph represents mean ±SEM from N=6 animals; ***p<0001 (unpaired two-tailed t-test). ( b ) Histology of ovaries by Haematoxylin & Eosin staining. CL indicate corpus leuteum; arrows indicate trapped oocytes in CL. Scale bar: 100µm. ( c ) Hierarchical clustering of RNA-sequencing analysis results shows differentially expressed genes between CRS-066 and vehicle treated mice (N=6 mice per treatment). ( d and e ) Gene ontology enrichment of biological pathways (D) and molecular functions (E) of significantly differently down-regulated genes in CRS-066 treated mouse ovaries compared to vehicle treated ovaries. ( f and g ) Gene set enrichment analyses plot (GSEA) shows downregulation of Ctnnb1 and Hippo signalling pathways in CRS-066 treated mice ovaries compared to vehicle treated ovaries. Net enrichment score (NES) values are shown. ( h ) Heatmap representing the relative expression profiles of transcripts involved in Wnt\β-catenin, Hippo\YAP and ovarian signalling axes in CRS-066 treated ovaries compared to vehicle. N=3 biological replicates. ( i - k ) Relative mRNA expression of key genes involved in gonadotrophin signalling and oocyte function (i), COC expansion and ovulation (j) or folliculogenesis (k) h in ovaries treated with CRS-066 compared to vehicle treatment and determined by qRT-PCR. Bar graph show mean+/- SEM. N=6 ovaries from independent CRS or vehicle treated mice. Statistical testing with Student’s t-test; *P < 0.05; ** P<0.01; **** P <0.00001. ( l ) Follicle counts at primary, secondary, pre-antral, antral and ovulatory stages in ovaries from mice treated with either CRS-006 (50mg/kg) or vehicle control (7.5% DMSO). N=3 mice/treatment/time-point. ( m ) Representative follicle morphology H&E (left) section and N-cadherin immunofluorescence (right) section in mice treated with either CRS-066 or vehicle. H&E and immunoflourescence highlight disorganised granulosa cells organisation. Asterisks indicate loss of transzonal projections between oocyte and cumulus cells. Scale bar: 30µm. ( n ) Representative confocal immunofluorescent images of mouse ovaries stained with anti-cleaved caspase 3 and anti-Ki-67. Scale bar: 30µm. ( o ) Relative mRNA expression of key genes involved in oocyte growth and ovulation in CRS-066 or vehicle treated mice (N=3/ treatment) at either 44h post eCG or 11h post hCG.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Saline, Injection, Two Tailed Test, Staining, RNA Sequencing, Expressing, Quantitative RT-PCR, Control, Immunofluorescence

Journal: bioRxiv

Article Title: N-cadherin mechanosensing in ovarian follicles controls oocyte maturation and ovulation

doi: 10.1101/2023.10.06.561232

Figure Lengend Snippet: (a) qPCR analysis of relative mRNA expression of Cdh2, Areg, Ptgs2 and Cyp19a1 in ovaries of control ( Cdh2 Fl/+ ; Amhr2 Cre ) and granulosa-specific Cdh2 null mutants ( Cdh2 Fl/Fl ; Amhr2 Cre ), n=6 individual animals, *p<0.05. ( b-e ) Immunofluorescent analysis of N-cadherin protein in ovaries of control ( Cdh2 Fl/+ ; Amhr2 Cre ) and granulosa-specific Cdh2 null mutants ( Cdh2 Fl/Fl ; Amhr2 Cre ), showing mosaic depletion of N-cadherin in granulosa cells of mutant follicles. Arrows indicate mosaic regions with persistent N-cadherin. ( f ) Ovulation rate of 21d old mice with indicated control or granulosa-specific mutant genotypes. COCs in oviducts counted 16h after hCG injection. Graph represents mean ±SEM from N=4 and 7 animals respectively; **p<0.01 (unpaired two-tailed t-test). ( g ) Histology of ovaries by Haematoxylin & Eosin staining. Scale bar: 100µm.

Article Snippet: Sections were probed with primary antibodies against N-cadherin (BD Biosciences; Cat# 610920; 1:500); β-catenin (CST; Cat#8480; 1:500), E-cadherin (CST, Cat#14472; 1:500); a-tubulin (ThermoFisher; Cat#236-10501); Ki-67 (CST; Cat#9449; 1:1000) and Cleaved caspase 3 (CST; Cat#9661; 1:1000) diluted in 10% NGS and incubated O/N at at 4°C overnight in a humid chamber.

Techniques: Expressing, Control, Mutagenesis, Injection, Two Tailed Test, Staining

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 4. mTORC1 activation is an upstream event in palmitate induced ATF4 activation. A: AML12 cells were pretreated with or without Torin1 (0.25 mM) or rapamycin (Rapa at 50 nM) for 2 h before palmitate (0.4 mM) exposure for 16 h. Total protein was extracted. Protein abundance of ATF4 and actin were detected by Western blotting. The signal of ATF4 protein band was measured by densitometry and then divided by the sig- nal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.001 vs. control). B: AML12 were pretreated with Torin1 (0.25 mM) for 2 h before a 16-h palmitate (0.4 mM) exposure. Total RNA was extracted. ATF4 mRNA levels were detected by real time-qPCR. Data are expressed as means ± SD, n = 4 sep- arated experiments. Differences between the two groups were deter- mined using Student’s t test (P < 0.001 vs. control). ATF, activating transcription factor.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Quantitative Proteomics, Western Blot, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 5. mTORC1 activation contributes to palmi- tate-induced ER stress and NNMT upregulation. A and B: AML12 cells were pretreated with Torin1 (0.25 mM) for 2 h before the palmitate (0.4 mM) exposure for 16 h. Total RNA was extracted. The gene expres- sions of Xbp1, Xbp1s, and Xbp1u were quantified by real time-qPCR and Xbp1s/Xbp1u ratio calculated. Data are expressed as means ± SD, n = 4 different experiments. Differences between the two groups were determined using Student’s t test (P < 0.001vs. control). C: AML12 cells were pretreated with Tornin1 for 2 h before tunicamycin (10 μm) treat- ment for 16 h. Protein abundance of p-S6 and actin was detected by Western blotting. The signal of p-S6 protein band was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 5 separate experiments. Student’s t test was used for statistical evaluation (P < 0.01; P < 0.0001 vs. control). D: Ten- week-old male C57BL/6N mice were injected with tunicamycin (2 mg/kg body wt ip) or isovolumic vehi- cle (150 mM dextrose) and 16 h later livers were har- vested. Protein abundance of ATF4, p-S6 and actin was detected by Western blotting. E: AML12 cells were pretreated with Torin1 (0.25 mM) for 2 h before tunicamycin (10 μm) treatment for 16 h. Protein abun- dance of ATF4 was detected by Western blotting. F: AML12 cells were pretreated with Torin1 for 2 h before tunicamycin (10 μm) treatment for 16 h. Total RNA was extracted and NNMT gene expression quantified by real time-qPCR. All data were expressed as means ± SD, n = 4 separated experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001; P < 0.0001 vs. control). NNMT, nicotinamide N-methyl- transferase; XBP1, X-box binding protein 1.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Activation Assay, Control, Quantitative Proteomics, Western Blot, Injection, Gene Expression, Binding Assay

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 6. NNMT inhibition protects against palmitate-induced cell death. A: AML12 cells were pretreated with either JBSNF-000088 (20 mM) or II399 (20 mM) at the indicated concentrations for 4 h before palmitate (0.4 mM) exposure for 16 h. Cell viability was determined by LDH release mea- surement. Data are expressed as mean ± SD, n = 3 separated experi- ments. Bars with different character differ significantly (P < 0.05). B: AML12 cells were transfected with either scramble siRNA or NNMT siRNA for 24 h and treated with palmitate at 0.4 mM for 16 h. Cell death was determined by LDH release measurement. Data are expressed as means ± SD, n = 3 separated experiments. Differences between the two groups were determined using Student’s t test (P < 0.01; P < 0.001; P < 0.001 vs. control). NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Inhibition, Transfection, Control

Journal: American journal of physiology. Cell physiology

Article Title: Nicotinamide N-methyltransferase upregulation via the mTORC1-ATF4 pathway activation contributes to palmitate-induced lipotoxicity in hepatocytes.

doi: 10.1152/ajpcell.00195.2021

Figure Lengend Snippet: Figure 7. Protein kinase A (PKA) inhibition compro- mises the protective effect of NNMT inhibition against palmitate-induced cell death. A and B: AML12 cells were treated with either JBSNF-000088 (25 mM) or II399 (25 mM) for 6 h. Total proteins were isolated and PKA substrates detected by Western blotting. The signal of PKS substrates was measured by densitometry and then divided by the signal of its corresponding actin abundance in the same sample. Data are expressed as means ± SD, n = 3 separate experiments. Student’s t test was used for statistical evaluation (P < 0.05; P < 0.01 vs. untreated cells). C: AML12 cells were pretreated with either JBSNF-000088 (25 mM) or II399 (25 mM) at the pres- ence/absence of PKA inhibitor, either SQ22536 (200 mM) or H89 (10 mM) for 4 h before palmitate exposure for 16 h. Cell death was determined by LDH release. All data are expressed as means ± SD, n = 3 sepa- rated experiments. Differences between the two groups were determined using Student’s t test (P < 0.05; P < 0.01; P < 0.001 vs control). D: sche- matic illustration of the role and mechanism of NNMT upregulation in palmitate-induced hepatocyte lipotox- icity. The mTORC1-ATF4 pathway activation contrib- utes to palmitate-elicited NNMT upregulation and protein kinase A (PKA) activation contributes to NNMT inhibition-conferred protection against hepatolipotox- icity. NNMT, nicotinamide N-methyltransferase.

Article Snippet: Cells were grown at 80% confluence before the exposure of treatments in various experiments. siRNA Transfection Cultured AML12 hepatocytes were transfected with mouse ATF4 siRNA (Santa Cruz, sc-35113).

Techniques: Inhibition, Isolation, Western Blot, Control, Activation Assay