Journal: Cell Reports

Article Title: Critical Role of Type III Interferon in Controlling SARS-CoV-2 Infection in Human Intestinal Epithelial Cells

doi: 10.1016/j.celrep.2020.107863

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal antibody against SARS-CoV NP (Sino biologicals MM05), mouse monoclonal against J2 (scions), mouse monoclonal against E-cadherin (BD Transductions #610181), mouse monoclonal anti-SYP (Santa Cruz Biotechnology sc-17750), and rabbit polyclonal anti-Mucin-2 (Santa Cruz Biotechnology# sc-15334) were used at 1:500 for immunofluorescence.

Techniques: Virus, Recombinant, SYBR Green Assay, RNA Extraction, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Software

Journal: bioRxiv

Article Title: CRISPRi-based screen of Autism Spectrum Disorder risk genes in microglia uncovers roles of ADNP in microglia endocytosis and uptake of synaptic material

doi: 10.1101/2024.06.01.596962

Figure Lengend Snippet: A. Experimental and analytical strategy for measuring iTF-Microglia uptake of synaptic material from iPSC-derived neurons (iNeurons) using flow cytometry. (Top) iNeurons are engineered to express synaptophysin linked to the acid-tolerant green fluorescent protein, Gamillus. iTF-Microglia are engineered to express a nuclear blue fluorescent protein. (Bottom) After coculture and measurement using flow cytometry, iTF-Microglia are identified by their blue fluorescence, and the amount of green fluorescence from uptake of synaptophysin-Gamillus is measured within this iTF-Microglia population. B. Representative micrographs of iNeurons expressing synaptophysin-Gamillus (green) costained with an antibody against a presynaptic marker, vGlut1 (magenta, top), or a post-synaptic marker, Homer (magenta, bottom). C. Correlation analysis of synaptophysin-Gamillus with vGlut1 or Homer (N = 3 fields of view from 3 wells, bars represent mean +/− standard deviation). D,E. Time lapse images of iNeurons expressing synaptophysin-Gamillus (green) and iTF-Microglia expressing a membrane marker, Lck-mApple (gray). D. The yellow arrow highlights a synaptophysin-Gamillus punctum that is taken up into a phagocytic cup and beginning to be trafficked toward the iTF-Microglia’s soma. E. The yellow arrow highlights a synaptophysin-Gamillus puncta inside a phagocytic cup that is being trafficked toward the iTF-Microglia’s soma. F. Representative micrographs of iNeurons engineered to express cytosolic Gamillus or synaptophysin-Gamillus. Nuclei are marked by Hoechst 33342 and displayed in blue. Gamillus is displayed in gray. G. Expression of the Gamillus protein measured by total fluorescence intensity per field of view (n = 5 wells, bars represent mean +/− standard deviation, Tukey’s multiple comparisons test). H. Uptake of Gamillus by iTF-Microglia in coculture measured by flow cytometry (n = 3 wells, bars represent mean +/− standard deviation, Tukey’s multiple comparisons test).

Article Snippet: pOT020 was prepared by PCR amplifying Gamillus (addgene plasmid #124837) and synaptophysin (addgene plasmid #119738) and ligating them into a lentivirus backboneusing Gibson assembly as per the manufacturer’s instructions.

Techniques: Derivative Assay, Flow Cytometry, Fluorescence, Expressing, Marker, Standard Deviation, Membrane

Journal: bioRxiv

Article Title: CRISPRi-based screen of Autism Spectrum Disorder risk genes in microglia uncovers roles of ADNP in microglia endocytosis and uptake of synaptic material

doi: 10.1101/2024.06.01.596962

Figure Lengend Snippet: A. iTF-Microglia and iNeuron differentiation and coculture strategy. B. Heatmap showing relative gene expression from RNA sequencing of neuronal genes, microglial genes, complement components, and microglial activation markers by iTF-Microglia (M) and iNeurons (N) in monoculture and coculture preparations (N = 2 biological replicates). C. Gamillus expression constructs for cytosolic Gamillus (top) and synaptophysin linked to Gamillus (bottom). D. Representative micrographs iNeuron monocultures (left) and iTF-Microglia and iNeuron coculture (right) stained with the dead cell indicator, TO-PRO-3 (magenta), and nuclear stain, Hoechst 33342 (green). E. (Left) Percent live cells based on nuclear TO-PRO-3 intensity (N = 4 fields of view (FOV) for 5 wells, bars represent mean +/− standard deviation). (Right) Total cell count per field of view based on nuclear Hoechst segmentations (N = 4 fields of view for 5 wells, bars represent mean +/− standard deviation).

Article Snippet: pOT020 was prepared by PCR amplifying Gamillus (addgene plasmid #124837) and synaptophysin (addgene plasmid #119738) and ligating them into a lentivirus backboneusing Gibson assembly as per the manufacturer’s instructions.

Techniques: Gene Expression, RNA Sequencing, Activation Assay, Expressing, Construct, Staining, Standard Deviation, Cell Counting

Journal: Journal of neurophysiology

Article Title: Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant.

doi: 10.1152/jn.01097.2011

Figure Lengend Snippet: Fig. 2. Confocal images of abducens motor neurons after normal conditioning for 2 pairing sessions (C2) or conditioning following treatment with the glutamate receptor A4 (GluA4) subunit small-interfering RNA (tGluA4 siRNA). Images are unprocessed except that the contrast was increased for the illustration. After drawing the outline of the cell of interest by the investigator, the software breaks the original image into its individual color channels, revealing punctate staining for synaptophysin (Syn; green), GluA4 (red), or GluA1 (blue) for quantitative analysis. Original scale bar 10 m.

Article Snippet: The secondary antibodies were a Cy3-conjugated rabbit anti-goat IgG for GluA4, a DyLight 648-conjugated goat anti-rabbit IgG for GluA1, and a DyLight 488-conjugated goat anti-mouse IgG for synaptophysin (Jackson ImmunoResearch Laboratories, West Grove, PA), which were used to visualize the primary antibodies.

Techniques: Small Interfering RNA, Software, Staining

Journal: Journal of neurophysiology

Article Title: Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant.

doi: 10.1152/jn.01097.2011

Figure Lengend Snippet: Fig. 8. Synaptic localization of tGluA4- or tGluA1-contain- ing AMPARs after conditioning or treatment with siRNA and rescue plasmids. A: representative confocal images of abducens motor neurons from the different treatment groups showing punctate staining for synaptophysin (green), GluA1 AMPAR subunits (blue), and GluA4 (red). Double colocalization of GluA1 with synaptophysin (GluA1 Syn) is cyan, GluA4 with synaptophysin (GluA4 Syn) is yellow, and triple colocalization of GluA1 and GluA4 with synaptophysin (GluA1 GluA4 Syn) is white. Original scale bar 2 m. B: quantitative analysis of synaptophysin punctate staining and the colocalization of AMPAR sub- units with synaptophysin for the different treatment groups. *Significant differences from Ps2; #significant differences from C2.

Article Snippet: The secondary antibodies were a Cy3-conjugated rabbit anti-goat IgG for GluA4, a DyLight 648-conjugated goat anti-rabbit IgG for GluA1, and a DyLight 488-conjugated goat anti-mouse IgG for synaptophysin (Jackson ImmunoResearch Laboratories, West Grove, PA), which were used to visualize the primary antibodies.

Techniques: Staining

Journal: Journal of neurophysiology

Article Title: Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant.

doi: 10.1152/jn.01097.2011

Figure Lengend Snippet: Fig. 9. Synaptic localization of tGluA2/3-containing AMPARs after condition- ing or treatment with the siRNAs. Levels of synaptophysin punctate staining increased significantly in all of the groups that received paired stimulation compared with Ps2 (P 0.0001). Conditioning in normal medium or after treatment with either the tGluA4 or GluA1 siRNA did not alter the colocal- ization of these AMPARs with synaptophysin. Representative images of abducens motor neurons are also shown (synaptophysin, green; tGluA2/3, red; colocalization, yellow). *Significant differences from Ps2; scale bar 2 m.

Article Snippet: The secondary antibodies were a Cy3-conjugated rabbit anti-goat IgG for GluA4, a DyLight 648-conjugated goat anti-rabbit IgG for GluA1, and a DyLight 488-conjugated goat anti-mouse IgG for synaptophysin (Jackson ImmunoResearch Laboratories, West Grove, PA), which were used to visualize the primary antibodies.

Techniques: Staining

Journal: Translational neurodegeneration

Article Title: Intestine-derived α-synuclein initiates and aggravates pathogenesis of Parkinson's disease in Drosophila.

doi: 10.1186/s40035-022-00318-w

Figure Lengend Snippet: Fig. 3 Intestinal overexpression of α-syn causes alterations of transcription profiles and microbiota composition. a Activation of intestinal immunity by intestinal α-syn. Antimicrobial peptide gene expression in the middle intestine of 28-day-old Drosophila using qPCR. Dpt, Diptericin; Attc, Attacin; Drs, Drosocin. n = 4. b Volcano plot comparing gene expression profiles of esgTS > Syn and esgTS > GFP in fly intestines. Red dots depict genes highly upregulated in esgTS > Syn intestines (log2 fold change < 1; adjusted P < 0.01), and green dots depict genes significantly downregulated in esgTS > Syn intestines (log2 fold change < 1; adjusted P < 0.01). Blue dots depict genes without significant alteration. c Gene ontology (GO) analysis of the significantly altered genes in esgTS > Syn intestines. The top 20 GO terms for the 2231 upregulated genes are shown in red. The heights of the columns represent the alteration level of gene expression in this GO term. d Supervised hierarchical clustering of genes based on RNA-Seq scores. The relative expression levels of clustered genes are shown on the right on a scale of -1.5 (downregulated) to 1.5 (upregulated)

Article Snippet: Primary antibodies were α-syn (Novus Biologicals, 1:1,000, Centennial, CO), phospho-histone 3 (Millipore, 1:1,000, Burlington, MA), Dlg (DHSB, 1:50, Iowa City, IA) and phospho-Jun N-terminal Kinase (phospho-JNK) (Millipore, 1:200), while nuclear DNA was detected by DAPI (Thermo Fisher).

Techniques: Over Expression, Activation Assay, Gene Expression, RNA Sequencing, Expressing

Journal: Translational neurodegeneration

Article Title: Intestine-derived α-synuclein initiates and aggravates pathogenesis of Parkinson's disease in Drosophila.

doi: 10.1186/s40035-022-00318-w

Figure Lengend Snippet: Fig. 5 Dysbiosis aggravates the pathology of Parkinson’s disease in the intestine. a Bacterial depletion attenuated the impairment of lifespan of intestinal Syn flies. Germ-free (GF) flies were generated with fly diet with antibiotics cocktail (carbenicillin, metronidazole, and tetracyclin). Adult flies were cultured as described in Fig. 1a, and lifespan curves were recorded. b Bacterial depletion alleviated the age-dependent DA neuron loss induced by intestinal α-syn. c Antibiotics improved the progressive locomotor deficits induced by intestinal α-syn. d Antibiotics inhibited the intestinal α-syn-induced dysplasia of midgut. Green, GFP; blue, DNA; Scale bars, 2 μm. e Bacterial depletion decreased the number of phospho-H3-positive cells in midgut. f Bacterial depletion attenuated the interruption of intercellular junction. Red, anti-Dlg; blue, DNA; Scale bars, 0.5 μm. Mean ± SEM; P-values for survival curves were calculated using log-rank tests (using total fly numbers), and for category graphs using one-way ANOVA with Bonferroni multiple-comparison test. *P < 0.05; **P < 0.01; ***P < 0.001. CR: conventionally reared; PH3: phospho-H3

Article Snippet: Primary antibodies were α-syn (Novus Biologicals, 1:1,000, Centennial, CO), phospho-histone 3 (Millipore, 1:1,000, Burlington, MA), Dlg (DHSB, 1:50, Iowa City, IA) and phospho-Jun N-terminal Kinase (phospho-JNK) (Millipore, 1:200), while nuclear DNA was detected by DAPI (Thermo Fisher).

Techniques: Generated, Cell Culture, Comparison

Journal: Scientific Reports

Article Title: Neuroprotective mitochondria targeted small molecule restores synapses and the distribution of synaptic mitochondria in the hippocampus of APP/PS1 mice

doi: 10.1038/s41598-025-90925-0

Figure Lengend Snippet: CP2 treatment improves gene expression related to synaptic function in brain tissue of APP/PS1 mice. ( a ) PCA based on the RNA-seq data generated on cortico-hippocampal brain tissue shows separated clusters of samples among three groups: NTG, green; APP/PS1 (AD), blue; APP/PS1 + CP2 (AD + CP2), orange. ( b – e ) Heat maps show changes in genes in pathways related to dendrite morphogenesis ( b ), regulation of axonal extension involved in axon guidance ( c ), synapse assembly ( d ) and synaptic transmission ( e ) after CP2 treatment in APP/PS1 mice. ( f ) Western blot analysis in the hippocampal tissue demonstrates increased levels of synaptophysin (Syn), post synaptic density 95 (PSD95), brain derived neurotrophic factor (BDNF), and sirtuin 3 (Sirt3) proteins in APP/PS1 mice after CP2 treatment. ( g ) Quantification of the Western blot from ( f ) All mice were 20 months of age treated with CP2 or vehicle for 12 months, n = 4–5 mice per group for RNA-seq and n = 6–8 per group for Western blot. Data are presented as mean ± S.E.M. A one-way ANOVA with Fisher`s LSD post-hoc test was used for statistical analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The original analyses of the RNAseq data and Western blots were reported in our previous publication and are repurposed here without major alterations.

Article Snippet: The following primary antibodies were used: Synaptophysin (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, cat. # 17750), BDNF (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, cat. # 546), PSD95 (1:1000, Cell Signaling Technology, cat. # 2507), Sirt3 (1:1000, Cell Signaling Technology, cat. # 5490), Tubulin (1:5000, Biovision, cat. # 3708).

Techniques: Gene Expression, RNA Sequencing, Generated, Transmission Assay, Western Blot, Derivative Assay

Journal: Cell reports

Article Title: Neurexin-3 defines synapse- and sex-dependent diversity of GABAergic inhibition in ventral subiculum

doi: 10.1016/j.celrep.2021.110098

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pAAV DJ hSyn-DIO LoxP -mGFP-T2A-Synaptophysin-mRuby , , Addgene, Plasmid #71760.

Techniques: Virus, Plasmid Preparation, Recombinant, RNA Sequencing, DNA Library Preparation, Software

Journal: Nature Communications

Article Title: Identification of the Notch ligand DLK1 as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma

doi: 10.1038/s41467-025-60649-w

Figure Lengend Snippet: A Immunoblot analysis of DLK1 and loading control (α-tubulin) proteins in CU-ACC1 cells with and without DLK1 KO. Four single-cell KO clones are shown. B Immunoblot analysis of NOTCH1 signaling, total NOTCH1 and NOTCH1 intracellular domain (ICD), NE marker synaptophysin (SYP), and loading control (α-tubulin) proteins with and without DLK1 KO in CU-ACC1 cells. Two single-cell KO clones are shown. C Correlation between NOTCH1 and DLK1 expression among TCGA ACC tumors. Pearson correlation coefficients with two-tailed p-values are shown. D DLK1 and NOTCH1 expression in TCGA ACC tumors and normal adrenals. Unpaired t tests were used to calculate two-tailed p -values. Error bar represents mean values ± 95% C.I. E SG3199 cytotoxicity in CU-ACC1 parental and DLK1 KO clones. Cells were treated with SG3199 for 3 days (data representative of n = 4 independent experiments). Error bars represent mean values ± S.E.M. F Flow cytometry histograms assessing ABCB1 in CU-ACC1 cells with and without DLK1 KO. G Immunoblot analysis of DLK1, total NOTCH1 and NOTCH1-ICD, SYP, and α-tubulin proteins in DLK1 + NCI-ACC40, DLK1 + NCI-ACC48 and DLK1 - ACC49 PDOs. H Flow cytometry histograms assessing ABCB1 in DLK1 negative NCI-ACC49 PDOs. I Immunoblot analysis of total NOTCH1 (to detect the NOTCH1-ICD plasmid expression), SYP, and α-tubulin proteins in CU-ACC1 cells with and without NOTCH1-ICD overexpression. J Flow cytometry histograms assessing ABCB1 in CU-ACC1 cells with and without N1ICD overexpression. K Correlation between NOTCH1 and ABCB1 expression among TCGA ACC tumors and ( L ) among GTEx normal adrenal tissues. Pearson correlation coefficients with two-tailed p-values are shown. M Single cell RNA-seq data of ABCB1 expression comparing high NOTCH1 to low NOTCH1 expressing cells from 18 ACC metastatic tumors. Unpaired t tests were used to calculate two-tailed p -values. N Model summarizing the findings of the current study. For immunoblots, experiments were performed two times with similar results. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies (1:1000) included DLK1 (CST, #2069), phospho-Histone H2A.X (Ser139) (Millipore, #05-636), cleaved caspase-3 (Asp175) (CST, #9661), cleaved PARP (Asp214) (CST, #9541), total NOTCH1 (CST, #3608), NOTCH1-ICD (CST, #4147), and SYP (CST, #36406).

Techniques: Western Blot, Control, Clone Assay, Marker, Expressing, Two Tailed Test, Flow Cytometry, Plasmid Preparation, Over Expression, RNA Sequencing

Journal: bioRxiv

Article Title: Development of Human Neuroblastomas in Mouse-Human Neural Crest Chimeras

doi: 10.1101/523795

Figure Lengend Snippet: ( A ) H&E and IHC comparing assay show that human NB tumors of chimeric mice express the typical NB markers Synaptophysin (SYP), Nestin (NES), Tyrosine hydroxylase (TH) and Chromogranin A (CgA), similarly to their expression found in NB samples of patient. hNCCS, which expressed oncogenes and were subcutaneously injected into immunocompromised mice to form xenograft tumors (left column) did not express these NB markers. (See IHC quantifications in Supplementary Figure 4 ; scale bars =100µm). ( B ) RNA-Seq of CHNB tumor samples (n=4) were separated in-silico into human and mouse reads to separate the tumor and hosts’-environment compartments (See material and methods). The analysis of RNA-Seq of the human-gene expression profile revealed that the human tumors in chimeric mice expressed a set of key genes normally associated with NB tumors ( ABCC1, BIRC5, CAMTA1, CCND1, DDX1, ENOS, IGF1R, KIF1B, KRAS, MAX, NES, NGFR, NME1, NRAS, PHOX2B, RAF1, SNW1, TH, TP53 and VEGFA ) with a significant correlation to expression in NB cell lines (Kelly and SHSY-5Y). linear regression p -value =0.011.

Article Snippet: For immunostainings, samples were blocked with 2% BSA and incubated with primary antibodies including Rabbit anti-ALK (1:200, Cell Signaling), Rabbit anti-MYCN (1:50, Cell Signaling), Rabbit anti-CD3 (1:300, Thermo Fisher Scientific), Rabbit anti-CD8a (1:300, Cell Signaling), Rabbit anti-FoxP3 (1:75, R&D systems), Mouse anti-Il2ra (CD25, 1:100, Novus), Rabbit anti-human-PD-L1 (1:300, Cell Signaling), Sheep anti-human-CD47 (1:100, R&D systems), Rat anti-mouse F4/80 (1:100, Thermo Fisher Scientific), Rabbit anti-γH2AX (1:300, Abcam), Rabbit anti-human-Ki67 (1:20, Thermo Fisher Scientific), Rabbit anti-Chromogranin A (1:500, Novus), Rabbit anti-Synaptophysin (1:200, Cell Signaling), Mouse anti-human-Nestin (1:300, Abcam), Rabbit anti-TH (1:500, PelFreez), Rabbit anti-Peripherin (1:500, Abcam), Rabbit anti-human neurofilament (1:100, 160kD, Abcam) and anti-eGFP (1:1000, Aves Labs) overnight at 4°C followed by appropriate secondary antibody incubation for 1-2h (Thermo Fisher Scientific).

Techniques: Expressing, Injection, RNA Sequencing Assay, In Silico

Journal: bioRxiv

Article Title: Development of Human Neuroblastomas in Mouse-Human Neural Crest Chimeras

doi: 10.1101/523795

Figure Lengend Snippet: ( A ) Tyrosine hydroxylase (TH), ( B ) Chromogranin A (CgA), ( C ) Nestin (NES), and ( D ) Synaptophysin (SYP), in samples of human NBs from chimeric mice (n=5), subcutaneous xenograft outgrowth of hNCCs (n=4), and NB samples of patients (Data presented as means, error bars represent SD, dots represent fields of view).

Article Snippet: For immunostainings, samples were blocked with 2% BSA and incubated with primary antibodies including Rabbit anti-ALK (1:200, Cell Signaling), Rabbit anti-MYCN (1:50, Cell Signaling), Rabbit anti-CD3 (1:300, Thermo Fisher Scientific), Rabbit anti-CD8a (1:300, Cell Signaling), Rabbit anti-FoxP3 (1:75, R&D systems), Mouse anti-Il2ra (CD25, 1:100, Novus), Rabbit anti-human-PD-L1 (1:300, Cell Signaling), Sheep anti-human-CD47 (1:100, R&D systems), Rat anti-mouse F4/80 (1:100, Thermo Fisher Scientific), Rabbit anti-γH2AX (1:300, Abcam), Rabbit anti-human-Ki67 (1:20, Thermo Fisher Scientific), Rabbit anti-Chromogranin A (1:500, Novus), Rabbit anti-Synaptophysin (1:200, Cell Signaling), Mouse anti-human-Nestin (1:300, Abcam), Rabbit anti-TH (1:500, PelFreez), Rabbit anti-Peripherin (1:500, Abcam), Rabbit anti-human neurofilament (1:100, 160kD, Abcam) and anti-eGFP (1:1000, Aves Labs) overnight at 4°C followed by appropriate secondary antibody incubation for 1-2h (Thermo Fisher Scientific).

Techniques:

Journal: Cell Death & Disease

Article Title: UHRF1 regulates AR ubiquitination to promote the loss of AR signaling and enzalutamide resistance in progression of prostate cancer

doi: 10.1038/s41419-026-08511-9

Figure Lengend Snippet: A Culture process and morphology of LNCaP-EnzR and C4-2-EnzR cell lines. B Enzalutamide IC50 determination of LNCaP and LNCaP-EnzR. C Enzalutamide IC50 determination for C4-2 and C4-2-EnzR. All cells were treated with increasing concentrations of enzalutamide for 7 days, then CCK8 was added for 2 h, and the absorbance at 450 nm was measured. D , E Western blot analysis of AR, UHRF1, SYP, and NSE expression levels in LNCaP and LNCaP-EnzR, C4-2 and C4-2-EnzR. F , G qPCR analysis of AR, UHRF1, SYP, and NSE expression levels in LNCaP, LNCaP-EnzR, C4-2, and C4-2-EnzR cells. H Principal component analysis of RNA-seq data from LNCaP, C4-2, their enzalutamide-resistant sublines, and NCI-H660 cells. I , J Effects of the negative control or UHRF1 knockdown on cell viability in LNCaP-EnzR and C4-2-EnzR. All cells were treated with increasing concentrations of enzalutamide for 7 days, then CCK8 was added for 2 h, and the absorbance at 450 nm was measured. K Effects of UHRF1 knockdown and enzalutamide on the viability of C4-2-EnzR cells. After transfection with vector or shUHRF1 plasmid, cells were re-inoculated, and cck8 was added to some cells every 24 h and incubated for 2 h, after which the absorbance at 450 nm was measured. L – M Colony formation analysis of UHRF1 knockdown and enzalutamide treatment in LNCaP-EnzR and C4-2-EnzR. After transfection with vector or shUHRF1 plasmid, cells were re-inoculated and treated with DMSO or enzalutamide for 14 days.

Article Snippet: After blocking with 5% milk in PBS containing 0.1% Tween-20 for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies: AR (Santa Cruz Biotechnology Inc., Dallas, TX, USA; Cat# sc-7305, 1:1000; Cell Signaling Technology, Danvers, MA, USA; Cat# 5153S, 1:1000), UHRF1 (Proteintech Group, Rosemont, IL, USA; Cat# 21402-1-AP, 1:1000), Ub (ZenBio Inc., Durham, NC, USA; Cat# 382766, 1:1000), NSE (Proteintech Group; Cat# 10149-1-AP, 1:1000), SYP (Proteintech Group; Cat# 17785-1-AP, 1:1000), Flag (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China; Cat# RA1003-01, 1:1000), and GAPDH (Santa Cruz Biotechnology Inc.; Cat# sc-47724, 1:1000) as a loading control.

Techniques: Western Blot, Expressing, RNA Sequencing, Negative Control, Knockdown, Transfection, Plasmid Preparation, Incubation

Journal: Cell Death & Disease

Article Title: UHRF1 regulates AR ubiquitination to promote the loss of AR signaling and enzalutamide resistance in progression of prostate cancer

doi: 10.1038/s41419-026-08511-9

Figure Lengend Snippet: A , B RT-qPCR results of mRNA levels of various lineage markers in LNCaP and C4-2 cells 14 days after transfection with the negative control or UHRF1 overexpression plasmid. C Quantitative PCR detection of mRNA levels of AR downstream genes KLK3 and TMPRSS2 following treatment with varying concentrations of dihydrotestosterone after transfection with the negative control or UHRF1 overexpression plasmid in C4-2. D Quantitative PCR detection of the mRNA levels of AR downstream genes KLK3 and TMPRSS2 following treatment with varying concentrations of dihydrotestosterone after negative control or UHRF1 knockdown in C4-2 cells. E Cell viability assay of C4-2 cells following UHRF1 knockdown with 5 μM enzalutamide treatment. F mRNA expression levels of AR, NMYC, SYP, NSE, and UHRF1 in C4-2 cells after 14 days of treatment with 5 μM enzalutamide following UHRF1 knockdown. G Volcano plot of differentially expressed genes in NC and UHRF1 knockdown cells ( P < 0.05, |log2FC | ≥ 1). H Heatmap of expression of some AR signaling, EMT, and NE & stem-like related genes. I GSEA results of hormone receptor binding gene set.

Article Snippet: After blocking with 5% milk in PBS containing 0.1% Tween-20 for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies: AR (Santa Cruz Biotechnology Inc., Dallas, TX, USA; Cat# sc-7305, 1:1000; Cell Signaling Technology, Danvers, MA, USA; Cat# 5153S, 1:1000), UHRF1 (Proteintech Group, Rosemont, IL, USA; Cat# 21402-1-AP, 1:1000), Ub (ZenBio Inc., Durham, NC, USA; Cat# 382766, 1:1000), NSE (Proteintech Group; Cat# 10149-1-AP, 1:1000), SYP (Proteintech Group; Cat# 17785-1-AP, 1:1000), Flag (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China; Cat# RA1003-01, 1:1000), and GAPDH (Santa Cruz Biotechnology Inc.; Cat# sc-47724, 1:1000) as a loading control.

Techniques: Quantitative RT-PCR, Transfection, Negative Control, Over Expression, Plasmid Preparation, Real-time Polymerase Chain Reaction, Knockdown, Viability Assay, Expressing, Binding Assay

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( A ) Log 2 -normalised expression of Th (tyrosine hydroxylase) , Syp (Synaptophysin), and Slc32a1 (vGAT) mRNA in DA neurons from single-cell RNA sequencing data . ( B ) Example confocal images of endogenous immunostaining for synaptophysin (green) and TH (magenta) on the left, and vGAT (blue) and TH (magenta) on the right. Both images were taken in the glomerular layer of the OB. Yellow arrowheads point to small clusters where TH and synaptophysin or TH and vGAT co-localise. Scalebars: 5 μm (main) and 0.5 μm (inset) for the images on the left, 4 μm (main) and 1 μm (inset) for the images on the right. ( C ) Strategy to label putative presynaptic release sites in individual DA neurons. ( D ) Example confocal image of a successfully labelled DA cell. Inset 1 reveals the TH+ DA identity of the neuron (cyan) and inset 2 highlights the Syn-mRuby puncta (magenta, black). Scalebars: 5 μm. ( E ) Example confocal image of a GFP+ (green), Syn-mRuby+ (magenta, black) neuronal process co-stained with vGAT (orange, black). Yellow arrowheads indicate examples where Syn-mRuby and vGAT puncta co-localise. The last panel shows the orthogonal views of the bottom punctum. Scalebars: 1 μm.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques: Expressing, RNA Sequencing, Immunostaining, Staining

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( A ) Example confocal image of a TRIM46-negative anaxonic DA neuron. Blue arrowheads point to examples of other TRIM46+ AISs (orange, black) in the same region which do not co-localise with this neuron’s GFP signal. Scalebars: 5 µm. ( B ) Snapshot of the same neuron in ( A ) showing Synaptophysin-mRuby puncta (magenta, black) on the dendrites. Yellow inset highlights a region of the neuron with multiple mRuby+ puncta within the GFP+ (green) processes (yellow arrows). Scalebars: 5 µm and 0.5 µm. ( C ) Example snapshots from three-dimensional (3D) dendritic reconstructions (green, GFP) and presynaptic puncta detection (magenta, Syn-mRuby) of anaxonic DA neurons. Note: these are not full dendritic reconstructions, but example dendrites. Dotted white circle represents the soma location. Scalebars: 5 µm.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques:

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( A ) Stitching of individual confocal stacks processed for maximum intensity projections of olfactory bulb (OB) DA neurons co-stained with tyrosine hydroxylase (TH) (green) and myelin basic protein (MBP, magenta). Yellow arrowheads point to myelinated parts of the axon, blue arrowheads show unmyelinated areas. Scalebar: 10 µm. ( B ) Example confocal images of a distal DA axon stained with GFP (green), TH (cyan), MBP (orange), and synaptophysin-mRuby (magenta, black). Yellow inset highlights the location of the presynaptic bouton. Yellow arrowheads point to co-localised regions, blue arrowheads show non-co-localisation. Scalebars: 2 µm and 1 µm. ( C ) Confocal image of an axon-bearing TRIM46+ DA neuron. Yellow arrowheads show co-localised staining for GFP (green) and TRIM46 (orange, black). Scalebars: 2 µm. ( D ) Soma area of axon-bearing and anaxonic DA neurons. Each dot shows one cell; lines show mean ± SEM; n=11 axon-bearing cells and n=9 anaxonic neurons from N=5 mice; unpaired t-test with Welch’s correction; ****, p<0.0001. ( E ) Snapshot of the same axon-bearing DA neuron shown in ( C ), co-stained with GFP (green) and synaptophysin-mRuby (magenta, black). Blue arrows show dendritic segments lacking mRuby label, despite the presence of clear mRuby+ puncta in neighbouring processes from a different GFP+ cell. Scalebars: 2 µm. ( F ) Dendritic puncta density in axon-bearing and anaxonic DA neurons. All conventions as in D ; n=11 axon-bearing cells and n=9 anaxonic neurons from N=5 mice; Mann-Whitney test; ***, p=0.0001.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques: Staining, MANN-WHITNEY

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: Neurons are stained with GFP (green), tyrosine hydroxylase (TH) (blue), myelin basic protein (MBP) (orange), and Synaptophysin-mRuby (black). Yellow arrows show co-localisation between the different channels; blue arrows show lack of co-localisation. Yellow inset highlights the location of the presynaptic bouton. Images at the bottom are zoomed-in snapshots from the images at the top, and arrows point at the synaptic puncta. Scalebars for axon #2: 2 μm and 1 μm; for axon #3, 10 μm and 5 μm; for axon #4, 1 μm and 0.5 μm; and for axon #5, 2 μm and 1 μm.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques: Staining

Journal: eLife

Article Title: Strikingly different neurotransmitter release strategies in dopaminergic subclasses

doi: 10.7554/eLife.105271

Figure Lengend Snippet: ( A ) Example confocal images showing soma size measurements in axon-bearing and anaxonic dopaminergic (DA) neurons. Axon-bearing (top) and anaxonic (bottom) cells labelled with GFP (green) and TRIM46 (orange). Yellow arrowheads point to the TRIM46-positive segment, indicating that the neuron has an axon. Red lines highlight the soma of the two neurons. Scalebars: 3 μm. ( B ) Maximum length of traced dendrites for axon-bearing (blue) and anaxonic (magenta) DA neurons. Each dot represents one cell, lines show mean ± SEM, n=11 axon-bearing neurons and n=9 anaxonic cells from N=4 mice, Welch’s t-test, p=0.63, n.s.=non-significant. ( C, D ) Dendritic mRuby puncta in a strongly over-expressing axon-bearing neuron. ( C ) Example confocal image with GFP (green) and TRIM46 (orange) labelling, revealing the axon-bearing identity of the neuron. Yellow arrowheads point to the GFP+/TRIM46+ co-localised zone. Scalebars: 2 mm. ( D ) Example confocal images showing synaptophysin-mRuby label (magenta, black) in the dendrites of the axon-bearing neuron from ( C ) (green). The levels of synaptophysin-mRuby look dramatically higher and with a less defined puncta profile than in all anaxonic DA cells – note levels of somatic expression compared to . Yellow arrowheads point to examples of detected puncta. Scalebars: 5 μm.

Article Snippet: For co-localisation analysis, the 3D centroid coordinates for the synaptophysin-mRuby puncta detected in Imaris were exported and analysed using a custom-written script in MATLAB.

Techniques: Expressing