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: 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: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 4 PARIS expression leads to p53 activation via PARIS Y137 phosphorylation-dependent epigenetic repression of MDM4. (A) Representative immunoblots examining the expression of MDM4, pS15-p53, p53 and FLAG (PARIS) in SH-SY5Y cells transfected with FLAG-PARIS wild-type or a Y137F mutant (48 h) using the indicated antibodies. b-Actin serves as an internal loading control. (B–D) Relative expression levels of MDM4 (B), p53 (C) and pS15-p53 (D) in the indicated experimental groups from A normalized to the internal loading control (b-actin; n = 3 per group). (E) Quantification of the relative expression of MDM4 mRNA in SH-SY5Y cells transfected (48 h) with mock or FLAG-PARIS and treated with TSA (300 nM, 42 h) deter- mined by RT-qPCR (normalized to internal GAPDH loading control; n = 3 per group). (F) Representative immunoblots of FLAG (PARIS) and MDM4 ex- pression in SH-SY5Y cells transfected (48 h) with mock or FLAG-PARIS and treated with TSA (300 nM, 42 h). (G) Quantification of the relative expression of MDM4 protein in the experimental groups in F normalized to b-actin (n = 3 per group). (H) A schematic diagram depicting the promoter structures of human MDM4 (hMDM4). IRS1, IRS2 and IRS3 motifs are indicated (top). Anti-acetyl-histone and anti-FLAG ChIP analysis of putative IRS (motif 1, 2 and 3) within the MDM4 promoter region in SH-SY5Y cells transfected with mock, or FLAG-PARIS wild-type (48 h, bottom) with or without the HDAC inhibitor TSA (300 nM, 42 h). The non-IRS region within the MDM4 promoter (Ctrl motif) was used as a negative control. Samples immuno- precipitated using either anti-histone antibodies or rabbit IgG were included as experimental controls in ChIP assays. (I) Quantification of relative ace- tylated histone enrichment on the indicated motifs located within MDM4 promoter determined by PCR amplification of ChIPed DNA in H (n = 3 per group). Data are expressed as mean SEM. *P 5 0.05, **P 5 0.01 and ***P 5 0.001 and statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Western Blot, Transfection, Mutagenesis, Control, Quantitative RT-PCR, Negative Control

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 5 Pharmacological inhibition of c-Abl activation restores behaviour/motor deficits and dopaminergic degeneration and prevents MDM4 repres- sion and p53 activation in mice with AAV-PARIS injection. (A) Representative exploratory paths from an open field test of mice that underwent AAV- Con or AAV-PARIS stereotaxic nigral injections (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). (B) Anxiety assessment of each experimental mouse group by examining the percentage of exploration time in the border versus the sum of the centre and periphery zones (n = 9 AAV-Con-injected mice, n = 10 AAV-Con-injected mice + nilotinib, and n = 8 AAV-PARIS-injected mice). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of vertical pole. (D) Motor coordination of each experimental mouse group used in B determined by the latency to fall in an accelerating rotarod test. (E) Representative TH immunohistochem- ical staining with Nissl counterstain of substantia nigra from mice that underwent AAV-Con or AAV-PARIS stereotaxic nigral injections (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). The substantia nigra and ventral tegmental area regions are indicated by dot- ted yellow and white lines, respectively. Scale bar = 500 mm. (F) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) in the indicated mouse groups (n = 4 AAV-Con-injected mice + DMSO, n = 5 AAV-Con-injected mice + nilotinib and n = 4 AAV-PARIS-injected mice). (G) Representative TUNEL assay images of ventral midbrain from mice that underwent stereotaxic nigral injection of AAV-Con or AAV-PARIS (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). The coronal brain sections were counterstained with DAPI. (H) Quantification of the percentage of TUNEL-labelled cells in AAV-Con- or AAV-PARIS-injected ventral midbrain regions from mice with or without nilotinib treatment (n = 16 sections from four mice per group). (I) Representative immunoblots examining pY137-PARIS, PARIS, c-Abl, pY245-c-Abl, MDM4, pS15-p53 and p53 expression in the ventral midbrain of AAV-Con- or AAV-PARIS-injected mice with or without nilotinib treatment using the indicated antibodies. (J) Quantification of the relative expression of pY137-PARIS, PARIS, c-Abl, pY245-c-Abl MDM4, pS15-p53 and p53 proteins nor- malized to b-actin (n = 5 AAV-Con-injected mice and n = 4 AAV-PARIS-injected mice). Data are expressed as mean SEM. Statistical analyses was per- formed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. **P 5 0.01 and ***P 5 0.001. DMSO = dimethyl sulphoxide; WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Inhibition, Activation Assay, Injection, Staining, TUNEL Assay, Western Blot, Expressing, Two Tailed Test

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 6 Pharmacological inhibition of c-Abl activity in in vivo adult parkin knockout mice prevents motor dysfunction and dopaminergic neurode- generation with concomitant blocking of PARIS phosphorylation and p53 activation. (A) Representative exploratory paths from an open-field test of 6-month-old wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) nigrally injected with AAV-GFPCre (3 m) and treated with the c-Abl in- hibitor nilotinib (200 mg nilotinib per 1 kg diet, p.o. for 2 months) or standard chow diet (chow). (B) Anxiety assessment of each experimental mouse group examining the percentage of exploration time in the border versus the sum of the centre and periphery zones (n = 4 mice per group). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of the vertical pole (n = 4 mice per group). (D) Representative TH immunohistochemical staining of substantia nigra from wild-type or homozygous floxed parkin mice (parkinfl/fl) with intranigral injection of AAV-GFPCre with or without nilotinib treatment (200 mg nilotinib per 1 kg diet, p.o. for 2 months). Scale bar = 500 mm. (E) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) of the indicated mouse groups (n = 4 mice per group). (F) Representative TUNEL assay images of ventral midbrain from wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) that expe- rienced stereotaxic nigral injection of AAV-GFPCre with or without nilotinib treatment (200 mg nilotinib per 1 kg diet, p.o. for 2 months). The coronal brain sections were counterstained with DAPI. Magnified images are shown in the bottom panel. (G) Quantification of the percentage of TUNEL- labelled cells in AAV-GFPCre-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice with or without nilotinib treatment (n = 16 sections from four mice per group). (H) Representative immunofluorescence images examining the expression of pY245-c-Abl in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treat- ment. (I) Quantification of the relative pY245-c-Abl fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). (J) Representative immunofluorescence images examining the expression of pY137-PARIS in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treatment. (K) Quantification of the relative pY137-PARIS fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). (L) Representative immunofluorescence images examining the expression of pS15-p53 in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treatment. (M) Quantification of the relative pS15-p53 fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). Data are expressed as mean SEM. Statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. ***P 5 0.001. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Inhibition, Activity Assay, In Vivo, Knock-Out, Blocking Assay, Phospho-proteomics, Activation Assay, Injection, Immunohistochemical staining, Staining, TUNEL Assay, Expressing, Two Tailed Test

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 7 Y137F-PARIS-mediated suppression c-Abl-PARIS pathway rescues MDM4 repression, blocks p53 activation and prevents development of motor deficits and dopamine neuron loss in parkin knockout mice. (A) Representative exploratory paths from an open field test of 6-month-old wild- type littermate or homozygous floxed parkin mice (parkinfl/fl) nigrally injected with AAV-GFPCre (3 months) AAV-PARIS-Y137F (3 months, phospho- deficient mutant PARIS). (B) Anxiety assessment of each experimental mouse group examining the percentage of exploration time in the border ver- sus the sum of the centre and periphery zones (n = 8 mice per group). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of the vertical pole (n = 8 mice per group). (D) Representative TH immunohistochemical staining of substantia nigra from wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) with intranigral injection of AAV-GFPCre AAV-PARIS-Y137F. Scale bar = 500 mm. (E) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) of the indicated mouse groups (n = 4 mice per group). (F) Representative TUNEL assay images of ventral midbrain from wild-type littermate or homozygous floxed parkin mice (par- kinfl/fl) that experienced stereotaxic nigral injection of AAV-GFPCre AAV-PARIS-Y137F. The coronal brain sections were counterstained with DAPI. Merged images are shown in the bottom panel. (G) Quantification of the percentage of TUNEL-labelled cells in AAV-GFPCre-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice AAV-PARIS-Y137F (n = 16 sections from four mice per group). (H) Representative immunoblots examining the expression of pY245-c-Abl, c-Abl, pY137-PARIS, PARIS, MDM4, pS15-p53, and parkin in the AAV-GFPCre AAV-PARIS-Y137F-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice using the indicated antibodies. b-Actin serves as an internal loading control. (I) Quantification of the relative expression of pY245-c-Abl, c-Abl, pY137-PARIS, PARIS, MDM4, pS15-p53 and parkin proteins normalized to b-actin in the indicated experimental groups (n = 4 mice per group). Data are expressed as mean SEM. Statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. ***P 5 0.001. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Activation Assay, Knock-Out, Injection, Mutagenesis, Immunohistochemical staining, Staining, TUNEL Assay, Western Blot, Expressing, Control, Two Tailed Test

Journal: Nature communications

Article Title: A child with perinatal HIV infection and long-term sustained virological control following antiretroviral treatment cessation.

doi: 10.1038/s41467-019-08311-0

Figure Lengend Snippet: Fig. 3 HIV-specific responses and immune response capability of the case at 9.5 years of age. a Detection of HIV-specific antibodies at 9.5 years of age by western blot. The case antibody profile is compared with controls that are a high positive, low positive and HIV-negative. HIV proteins corresponding to bands in the blots are shown in the left grey-shaded block; the case profile was positive for the core proteins indicated in pink. b Quantitation of HIV- specific antibodies by multiplex bead array for all isotypes and subclasses (indicated on the left side—IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2) against gp41, Gag, RT, gp120, Nef, Tat, Vpu, peptide Vpu9 and V1V2 scaffold antigens (indicated at the top). Results are expressed as mean fluorescence intensities (MFI)), the colour key shows ranges of MFI according to colour intensity (the darker the more HIV-specific antibody detected). A result is considered positive if above the cut-off (mean ± 3 SD) determined from eight adult uninfected controls. Vpu9 amino acid sequence: STMVDMGHLRLLDVNDL. c Proportions of natural killer (NK) cells that respond to anti-CD16 antibody, and CD4+ and CD8+ T cells that respond to staphylococcal enterotoxin B (SEB) in a whole blood intracellular cytokine (ICC) assay that measures induction of interferon-γ (IFN-γ) and interleukin-2 (IL-2). HIV-uninfected adult reference values for comparison (n = 21; median % and range)—natural killer (NK) anti-CD16%: 37.92 (12–67.6), CD4 SEB%: 6.04 (0.25–11.91), CD8 SEB%: 5.82 (0.18–18.94). d A weak positive CD4+ T cell response to Gag (0.116%) in the absence of a detectable CD8+ T cell response to Gag (<0.1%; 0.023%). UN: addition of costimulatory antibodies anti-CD28 and anti-CD49d, no stimulation with peptides

Article Snippet: HIV-specific antibody isotypes were detected by adding 50 μL per well at 2 μgmL−1, R-phycoerthyrinconjugated mouse anti-human IgG1 to IgG4 (Cat. Nos 9052-09, 9070-09, 9210-09, 9200-09, respectively, Southern Biotech, USA), mouse anti-human IgM (Cat. No. 9020-09, Southern Biotech), mouse anti-human IgA1 (Cat. No. 9130-09, Southern Biotech) or mouse anti-human IgA2 (Cat. No. 9140-09, Southern Biotech) with shaking (in the dark at room temperature) followed by three washes.

Techniques: Western Blot, Blocking Assay, Quantitation Assay, Multiplex Assay, Sequencing, Immunocytochemistry, Comparison

Journal: Experimental and therapeutic medicine

Article Title: MicroRNA-146a inhibits NF-κB activation and pro-inflammatory cytokine production by regulating IRAK1 expression in THP-1 cells.

doi: 10.3892/etm.2019.7881

Figure Lengend Snippet: Figure 1. Effects of miR‑146a modulation on the expression of IRAK1 mRNA levels. THP‑1 cells were transfected with a miR‑146a mimic or inhibitor. Reverse transcription‑quantitative PCR was performed to measure the rela tive expression levels of (A) miR‑146a and (B) IRAK1 mRNA, which were normalized to their respective control genes. Experiments were performed with six replicates for each group. **P<0.01 vs. corresponding NC groups. miR, microRNA; IRAK1, interleukin‑1 receptor‑associated kinase 1; NC, negative control.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against anti-IRAK1 (cat. no. SC-5288; dilution, 1:400), anti-NF-κB p65 (cat. no. SC-8008; dilution, 1:400), anti-β-actin (cat. no. SC-47778; dilution, 1:1,000) (all from Santa Cruz Biotechnology, Inc.) and anti-histone H3 (cat. no. 4499; dilution, 1:2,000; Cell Signaling Technology, Inc.) at 4 ̊C overnight.

Techniques: Expressing, Transfection, Control, Negative Control

Journal: Experimental and therapeutic medicine

Article Title: MicroRNA-146a inhibits NF-κB activation and pro-inflammatory cytokine production by regulating IRAK1 expression in THP-1 cells.

doi: 10.3892/etm.2019.7881

Figure Lengend Snippet: Figure 2. Effects of miR‑146a modulation on IRAK1 protein levels. THP‑1 cells were transfected with either a miR‑146a mimic or inhibitor. Western blot analysis was performed to measure IRAK1 protein levels relative to the internal reference protein, β‑actin. Three independent experiments were performed. (A) Representative image of the western blot analysis. (B) Relative quantification of IRAK1 expression. **P<0.01 vs. corresponding NC groups. miR, microRNA, IRAK1, interleukin‑1 receptor‑associated kinase 1; NC, negative control.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against anti-IRAK1 (cat. no. SC-5288; dilution, 1:400), anti-NF-κB p65 (cat. no. SC-8008; dilution, 1:400), anti-β-actin (cat. no. SC-47778; dilution, 1:1,000) (all from Santa Cruz Biotechnology, Inc.) and anti-histone H3 (cat. no. 4499; dilution, 1:2,000; Cell Signaling Technology, Inc.) at 4 ̊C overnight.

Techniques: Transfection, Western Blot, Quantitative Proteomics, Expressing, Negative Control

Journal: Experimental and therapeutic medicine

Article Title: MicroRNA-146a inhibits NF-κB activation and pro-inflammatory cytokine production by regulating IRAK1 expression in THP-1 cells.

doi: 10.3892/etm.2019.7881

Figure Lengend Snippet: Figure 3. Inhibitory effects of miR‑146a on IRAK1 expression and NF‑κB activation under inflammatory conditions. THP‑1 cells were treated with LPS following transfection with either a miR‑146a mimic or inhibitor to acti vate the inflammatory signaling response. Western blot analysis was used to measure relative protein expression levels of IRAK1 and nuclear NF‑κB p65 subunit, normalized to their respective internal references. Three independent experiments were performed. (A) Representative images of the western blot analysis. Relative protein expression levels of (B) IRAK1 and (C) the nuclear p65 subunit. ##P<0.01 vs. blank control and **P<0.01 vs. corresponding NC groups. miR, microRNA; IRAK1, interleukin‑1 receptor‑associated kinase 1; NF‑κB, nuclear factor κB; LPS, lipopolysaccharide; NC, negative control.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against anti-IRAK1 (cat. no. SC-5288; dilution, 1:400), anti-NF-κB p65 (cat. no. SC-8008; dilution, 1:400), anti-β-actin (cat. no. SC-47778; dilution, 1:1,000) (all from Santa Cruz Biotechnology, Inc.) and anti-histone H3 (cat. no. 4499; dilution, 1:2,000; Cell Signaling Technology, Inc.) at 4 ̊C overnight.

Techniques: Expressing, Activation Assay, Transfection, Western Blot, Control, Negative Control

Journal: Experimental and therapeutic medicine

Article Title: MicroRNA-146a inhibits NF-κB activation and pro-inflammatory cytokine production by regulating IRAK1 expression in THP-1 cells.

doi: 10.3892/etm.2019.7881

Figure Lengend Snippet: Figure 5. miR‑146a interacts directly with the IRAK1 3'‑UTR. A luciferase reporter assay was performed to investigate the potential interaction between miR‑146a and the IRAK1 3'‑UTR. (A) Potential miR‑146a target sites in the 3'‑UTR of the IRAK1 sequence, as revealed using TargetScan analysis. (B) Luciferase activity in cells co‑transfected with miR‑146a mimics or mimics NC and the wild‑type 3'‑UTR sequence, relative to the blank control. (C) The mutant sequence of the IRAK1 3'‑UTR. (D) Luciferase activity in cells co‑transfected with miR‑146a mimics or mimics NC and the mutant 3'‑UTR sequence, relative to the blank control. Experiments were performed with 3 replicates for each group. **P<0.01 vs. blank control. miR, microRNA; IRAK1, interleukin‑1 receptor‑associated kinase 1; 3'‑UTR, 3'‑untranslated region; NC, negative control.

Article Snippet: Subsequently, the membranes were incubated with primary antibodies against anti-IRAK1 (cat. no. SC-5288; dilution, 1:400), anti-NF-κB p65 (cat. no. SC-8008; dilution, 1:400), anti-β-actin (cat. no. SC-47778; dilution, 1:1,000) (all from Santa Cruz Biotechnology, Inc.) and anti-histone H3 (cat. no. 4499; dilution, 1:2,000; Cell Signaling Technology, Inc.) at 4 ̊C overnight.

Techniques: Luciferase, Reporter Assay, Sequencing, Activity Assay, Control, Mutagenesis, Negative Control