Journal: Physiological Genomics

Article Title: Circular RNAs in rat models of cardiovascular and renal diseases

doi: 10.1152/physiolgenomics.00064.2017

Figure Lengend Snippet: Box plots show the distribution of expression levels for all samples in the circular (circ)RNA microarray data set. R-1, R-2, and R-3 are 3 individual Dahl salt-resistant rats (R). S-1, S-2, and S-3 are 3 individual Dahl salt-sensitive rats (S). WKY-1, WKY-2, and WKY-3 are 3 individual Wistar Kyoto rats (WKY). SHR-1, SHR-2, and SHR-3 are 3 individual spontaneously hypertensive rats (SHR).

Article Snippet: Arraystar Rat Circular RNA Microarray ( www.arraystar.com ) was used for microarray experiments.

Techniques: Expressing, Microarray

Journal: Physiological Genomics

Article Title: Circular RNAs in rat models of cardiovascular and renal diseases

doi: 10.1152/physiolgenomics.00064.2017

Figure Lengend Snippet: Differentially expressed circRNAs between normotensive and hypertensive rats. The figure represents the number of circRNAs differentially expressed according to the microarray analysis. Based on the location and direction on the genome, circRNAs are subcategorized into exonic, intronic, antisense, sense overlapping, and intergenic. S vs. R up indicates circRNAs were upregulated in Dahl salt-sensitive rats (S) compared with Dahl salt-resistant rats (R). S vs. R down indicates circRNAs were downregulated in S compared with R. SHR vs. WKY up indicates circRNAs were upregulated in spontaneously hypertensive rats (SHR) compared with Wistar Kyoto rats (WKY). SHR vs. WKY down indicates circRNAs were downregulated in SHR compared with WKY.

Article Snippet: Arraystar Rat Circular RNA Microarray ( www.arraystar.com ) was used for microarray experiments.

Techniques: Microarray

Journal: Physiological Genomics

Article Title: Circular RNAs in rat models of cardiovascular and renal diseases

doi: 10.1152/physiolgenomics.00064.2017

Figure Lengend Snippet: Validation of differentially expressed circRNAs in the microarray study by quantitative RT-PCR. R, Dahl salt-resistant rat; S, Dahl salt-sensitive rat; WKY, Wistar Kyoto rat; SHR, spontaneously hypertensive rat. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Arraystar Rat Circular RNA Microarray ( www.arraystar.com ) was used for microarray experiments.

Techniques: Biomarker Discovery, Microarray, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Identification of circRNA and mRNA expression profiles and functional networks of vascular tissue in lipopolysaccharide‐induced sepsis

doi: 10.1111/jcmm.15424

Figure Lengend Snippet: Validation of circRNA and mRNA expression. A, The relative expression levels of selected circRNAs, as detected by RT‐qPCR. B, The relative expression levels of selected mRNAs, as detected by RT‐qPCR. β‐actin was used as a housekeeping gene for normalizing changes in specific gene expression. * P < .05 and ** P < .01 vs control group, n = 5 per group. RT‐PCR, real‐time quantitative polymerase chain reaction

Article Snippet: The labelled cRNAs were hybridized onto the Arraystar Rat circRNA Array (8 × 15K; Arraystar).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Physiological Genomics

Article Title: Circular RNAs in rat models of cardiovascular and renal diseases

doi: 10.1152/physiolgenomics.00064.2017

Figure Lengend Snippet: Heat maps of differentially expressed circRNAs between normotensive and hypertensive rats. A: Dahl salt-sensitive rats (S) vs. Dahl salt-resistant rats (R). B: spontaneously hypertensive rats (SHR) vs. Wistar Kyoto rats (WKY). The differentially expressed circRNAs were selected with a P value cut-off 0.05 and a fold-change cut-off 1.5. CircRNA expression level is shown as a function of color, with lower expression in green and higher expression in red. R-1, R-2, and R-3 are 3 individual R rats. S-1, S-2, and S-3 are 3 individual S rats. WKY-1, WKY-2, and WKY-3 are 3 individual WKY rats. SHR-1, SHR-2, and SHR-3 are 3 individual SHR rats.

Article Snippet: The labeled cRNAs were hybridized onto the Arraystar Rat CircRNA Array (8x15K) and incubated for 17 h at 65°C in an Agilent hybridization oven.

Techniques: Expressing

Journal: Physiological Genomics

Article Title: Circular RNAs in rat models of cardiovascular and renal diseases

doi: 10.1152/physiolgenomics.00064.2017

Figure Lengend Snippet: Targeted circRNA-micro (mi)RNA-gene network and functional pathway analysis for rno_circRNA_006016. A: the circRNA-miRNA-gene network based on bioinformatics analysis. Only top 30 miRNA target genes for each miRNA are shown in the network. B: the top 10 significant Gene Ontology for Biological Processes (GO:BP) were predicted by using all the 5 miRNA target genes. C: the top 10 significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were predicted by using all 5 miRNA target genes. “Enrichment Score” = “−log10(P value)” and P value < 0.05 was considered as statistically significant.

Article Snippet: The labeled cRNAs were hybridized onto the Arraystar Rat CircRNA Array (8x15K) and incubated for 17 h at 65°C in an Agilent hybridization oven.

Techniques: Functional Assay

Journal: Science Advances

Article Title: Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress

doi: 10.1126/sciadv.add9789

Figure Lengend Snippet: ( A ) Representative images showing the distribution of FKBP12 in entorhinal cortex of mouse brain. Scale bars, 50 μm. ( B ) Quantification of MAP-2 and FKBP12 fluorescence intensity in entorhinal cortex of PS19 mice brain in comparison to age-matched wild type (WT). Data are expressed as means ± SEM. N = 5. Statistics by unpaired t test, *** P < 0.005. ( C ) Representative Western blot showing high–molecular weight (HMW) tau aggregation in PS19 mice brain lysate and the decrease of FKBP12 in comparison to wild type. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( D ) Quantification of the tau-5 band intensity showing the robust increase of the ratio between high–molecular weight tau to monomeric tau. ( E ) Quantification of the FKBP12 band intensity. ( F ) Representative Western blot images showing the expression level of phosphorylated tau (CP13 and PHF-1), FKBP12, and neuronal markers [postsynaptic density 95 (PSD-95) and MAP-2] in the brain lysate of 3-, 6-, and 9-month-old PS19 mice, respectively. ( G ) Quantification of the phosphorylated tau (CP13 and PHF-1) band intensity. Result was normalized by internal control of corresponding GAPDH band intensity. ( H ) Quantification of FKBP12 Western blot (WB) band intensity normalized by GAPDH and MAP-2, respectively. Statistics by one-way analysis of variance (ANOVA), post hoc multiple comparisons test by Tukey’s test. ( I ) Quantification for the band intensity of neuronal markers including PSD-95 and MAP-2, normalized by GAPDH. Data are expressed as means ± SEM. N = 3. Statistics by two-way ANOVA, and post hoc multiple comparisons test by Tukey’s. ** P < 0.01, *** P < 0.005, and **** P < 0.001.

Article Snippet: The primary antibodies used in this study for immunofluorescence labeling are as follows: CP-13 (mouse; provided by P. Davies, Northwell), 1:300; TOMA2 (Anti-Tau oligomer clone 2, mouse,provided by Rakez Kayed laboratory), 1:200; FKBP12 (rat; R&D Systems, catalog no. MAB3777), 1:500.

Techniques: Fluorescence, Comparison, Western Blot, High Molecular Weight, Expressing, Control

Journal: Science Advances

Article Title: Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress

doi: 10.1126/sciadv.add9789

Figure Lengend Snippet: ( A ) Representative images of tau phosphorylation (CP13 antibody, red) and FKBP12 translocation in primary cortical neurons after induction of tau aggregation by oligomeric S1p fraction. Scale bars, 20 μm. ( B ) Representative images showed the high expression level of FKBP12 (green) in axonal hillock/axon initial segment (labeled by anti–ankyrin-G antibody, bright blue) under basal conditions whereas FKBP12 translocated to soma and dendrites when neurons bear tau aggregation. Scale bars, 20 μm. ( C ) Representative images showed the spatial colocalization of FKBP12 and aggregated Tau in the neurons after 24 hours of oTau seeding. Scale bars, 5 μm. DAPI, 4′,6-diamidino-2-phenylindole. ( D ) Quantification of FKBP12 intensity in the axon hillock of the neurons at 3 and 24 hours of S1p treatment, respectively. Data are expressed as means ± SEM. N = 10. Statistics by two-way ANOVA, post hoc multiple comparisons test by Fisher’s least significant difference (LSD). * P < 0.05, *** P < 0.005, and **** P < 0.001. ( E and F ) Quantification of granular intensity of CP13-labeled tau aggregates (E) and FKBP12 (F) in neurons at 3 and 24 hours of S1p treatment, respectively. Data are expressed as means ± SEM. N = 10. Statistics by two-way ANOVA, and post hoc multiple comparisons test by Fisher’s LSD. **** P < 0.001.

Article Snippet: The primary antibodies used in this study for immunofluorescence labeling are as follows: CP-13 (mouse; provided by P. Davies, Northwell), 1:300; TOMA2 (Anti-Tau oligomer clone 2, mouse,provided by Rakez Kayed laboratory), 1:200; FKBP12 (rat; R&D Systems, catalog no. MAB3777), 1:500.

Techniques: Phospho-proteomics, Translocation Assay, Expressing, Labeling

Journal: Science Advances

Article Title: Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress

doi: 10.1126/sciadv.add9789

Figure Lengend Snippet: ( A ) Representative blot image showed the elevation of FKBP12 in neurons transduced with FKBP12 AAV9. ( B ) Quantification of FKBP12 in neuronal lysate overexpressed with AAV9 vector. Data are expressed as means ± SEM. N = 3. Statistics by unpaired t test, **** P < 0.001. ( C ) Representative fluorescence-labeled images showed that overexpression of FKBP12 (green) preserved the integrity of neurons (labeled by MAP-2, magenta) and reduced the granular intensity of tau (labeled by CP13, red). Neurons were fixed at 24 hours after oTau stimulation. Scale bars, 20 μm. ( D ) Quantification of CP13 granular intensity as shown in (C). * P < 0.05, *** P < 0.005, and **** P < 0.001. ctrl, control. ( E ) Quantification of neuronal dendritic length labeled by MAP-2 as shown in (C). Data were collected from five independent experiments, expressed as means ± SEM. * P < 0.05, *** P < 0.005, **** P < 0.001. ( F ) Representative blot image showed the expression level of FKBP12, phosphorylated tau detected by CP13 (Tau phos Ser 202 ) and cleaved caspase 3 in neurons transduced with FKBP12 AAV9 and/or stressed by tau oligomers. GAPDH is examined as the internal control. ( G and H ) Quantification of CP13 and cleaved caspase 3 band intensity. Data were normalized by GAPDH, shown as % of basal condition without FKBP12 overexpression and oTau stress, and expressed as means ± SEM. N = 3. ** P < 0.01and **** P < 0.001. ( I ) Representative fluorescence colabeling images showed that FKBP12 knock down by small interfering RNA (siRNA) accelerated and potentiated the neuronal apoptosis (by cleaved caspase 3, magenta) induced by tau oligomers. ( J and K ) Quantification of MC1 (red) and cleaved caspase 3 fluorescence intensity as shown in (F), respectively. Data were collected from five independent experiments, expressed as means ± SEM. Statistics by two-way ANOVA, and post hoc multiple comparisons test by Fisher’s LSD. *** P < 0.005 and **** P < 0.001.

Article Snippet: The primary antibodies used in this study for immunofluorescence labeling are as follows: CP-13 (mouse; provided by P. Davies, Northwell), 1:300; TOMA2 (Anti-Tau oligomer clone 2, mouse,provided by Rakez Kayed laboratory), 1:200; FKBP12 (rat; R&D Systems, catalog no. MAB3777), 1:500.

Techniques: Transduction, Plasmid Preparation, Fluorescence, Labeling, Over Expression, Control, Expressing, Knockdown, Small Interfering RNA

Journal: Science Advances

Article Title: Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress

doi: 10.1126/sciadv.add9789

Figure Lengend Snippet: ( A ) 2D 1 H- 15 N HSQC spectra of tau in the absence (blue) or fivefold excess (orange) of FKBP12. The FKBP12-induced broadening of V393 is highlighted in the inset. ppm, parts per million. ( B and C ) Changes in the intensities (B) and chemical shift perturbations (CSPs) (C) of cross peaks in the HSQC spectra of tau upon addition of twofold (black) and fivefold (orange) excess of FKBP12. The sequence similarity of the two interaction sites of tau is displayed in the inset in (B). The domain organization of tau is shown on above. ( D ) Selected region of the 2D 1 H- 13 C HSQC spectrum of tau. The Cα-Hα assignments of 391 EIVYK 395 and 308 IVY 310 are marked. Assignments of Q307 and K311 are not available. ( E ) Superposition of the preferred solution conformations of the two tau sequences 307 QIVYK 311 (purple) and 391 EIVYK 395 (cyan) to which FKBP12 binds.

Article Snippet: The primary antibodies used in this study for immunofluorescence labeling are as follows: CP-13 (mouse; provided by P. Davies, Northwell), 1:300; TOMA2 (Anti-Tau oligomer clone 2, mouse,provided by Rakez Kayed laboratory), 1:200; FKBP12 (rat; R&D Systems, catalog no. MAB3777), 1:500.

Techniques: Sequencing

Journal: Science Advances

Article Title: Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress

doi: 10.1126/sciadv.add9789

Figure Lengend Snippet: ( A ) Schematic representation of FKBP12 (orange) interacting with the disordered tau protein (cyan). ( B and C ) Ensemble of complex structures predicted by AlphaFold2 for the interaction of FKBP12 with 307 QIVYK 311 (B) and 391 EIVYK 395 (C) of tau. The three (B) and four (C) most similar peptide conformations (from five calculated models) are shown. ( D and E ) Residue-specific predicted alignment error plots generated by AlphaFold2 for one of the representative complex structures shown in (B) and (C), respectively. Because FKBP12 and the tau sequences were connected by a 300-residue poly-glycine linker for the AlphaFold2 calculations, FKBP12 residues are found at positions 1 to 108 and the tau sequences at positions 409 to 419. ( F to H ) Structure of 391 EIVYK 395 of tau (cyan) bound to the hydrophobic surface of FKBP12 as predicted by AlphaFold2 . The electrostatic surface potential of FKBP12 is displayed in (F) with positive and negative charges shown in blue and red, respectively. The interaction interface is highlighted in (G) and (H). The hydrophobic interaction of V393 of tau with F100, W60, V56, and Y27 of FKBP12 is shown in (G). The anion-π interaction between E391 of tau and F47 of FKBP12 is marked by dashed lines in (H).

Article Snippet: The primary antibodies used in this study for immunofluorescence labeling are as follows: CP-13 (mouse; provided by P. Davies, Northwell), 1:300; TOMA2 (Anti-Tau oligomer clone 2, mouse,provided by Rakez Kayed laboratory), 1:200; FKBP12 (rat; R&D Systems, catalog no. MAB3777), 1:500.

Techniques: Residue, Generated

Journal: Science Advances

Article Title: Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress

doi: 10.1126/sciadv.add9789

Figure Lengend Snippet: ( A and B ) Contacts between Y310 (A) or Y394 (B) of tau with F47 and E55 of FKBP12. ( C ) 2D 1 H- 15 N HSQC spectra of cAbl-phosphorylated tau in the absence (green) or fivefold excess (brick red) of FKBP12. The NMR signals of V393 and pY394 are highlighted in the inset. ( D ) Changes in the intensities (top) and CSPs (bottom) of cross peaks in the HSQC spectra of cAbl-phosphorylated tau upon addition of twofold (gray) or fivefold (brick red) excess of FKBP12. The domain organization of tau is shown on top.

Article Snippet: The primary antibodies used in this study for immunofluorescence labeling are as follows: CP-13 (mouse; provided by P. Davies, Northwell), 1:300; TOMA2 (Anti-Tau oligomer clone 2, mouse,provided by Rakez Kayed laboratory), 1:200; FKBP12 (rat; R&D Systems, catalog no. MAB3777), 1:500.

Techniques:

Journal: Science Advances

Article Title: Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress

doi: 10.1126/sciadv.add9789

Figure Lengend Snippet: ( A ) Aggregation kinetics of 25 μM tau either in the absence (blue) or presence (red) of 2% preformed tau seeds. Aggregation kinetics of 1:1 (green), 1:2 (purple), and 1:5 (yellow) mixture of tau:FKBP12 in the presence of 2% preformed tau seeds. N = 3 per condition. a.u., arbitrary units. ( B ) Half time of aggregation (Tm) of de novo aggregated tau (blue) or seeded tau in the absence (red) or presence of equimolar (green), twofold (purple), or fivefold (yellow) molar excess of FKBP12. * P ([tau + 2% tau seeds] versus {[tau + FKBP12 (1:1)] + 2% tau seeds}) = 0.0104, * P ([tau + 2% tau seeds] versus {[tau + FKBP12 (1:2)] + 2% tau seeds}) = 0.0137, *** P = 0.0005. ( C ) Span of ThT intensity in the aggregation curves of de novo aggregated tau (blue) or seeded tau in the absence (red) or presence of equimolar (green), twofold (purple), or fivefold (yellow) molar excess of FKBP12. **** P < 0.0001. ( D ) SDS-PAGE gel of the monomeric tau protein and the supernatant (SN) of the seeded tau fibrils (after centrifugation) either in the absence or presence of equimolar, twofold, or fivefold excess of FKBP12. The fibril samples were taken after the 4 days of aggregation, as shown in (A). ( E ) The amount of protein aggregated. The % of aggregated proteins was determined by dividing the intensity of the SN to the monomeric tau. ( F ) CD spectra of tau fibrils formed without (blue) and with fivefold FKBP12 excess (orange). ( G and H ) Negative-stain EM image of tau fibril (G) + fivefold FKBP12 excess (H). Scale bars, 200 nm. ( I ) SDS-PAGE gel of tau fibrils without and with fivefold FKBP12 excess. Data are represented as means ± SD of n = 3 samples. Statistical analyses were performed by one-way ANOVA.

Article Snippet: The primary antibodies used in this study for immunofluorescence labeling are as follows: CP-13 (mouse; provided by P. Davies, Northwell), 1:300; TOMA2 (Anti-Tau oligomer clone 2, mouse,provided by Rakez Kayed laboratory), 1:200; FKBP12 (rat; R&D Systems, catalog no. MAB3777), 1:500.

Techniques: SDS Page, Centrifugation, Circular Dichroism, Staining

Journal: Science Advances

Article Title: Chaperoning of specific tau structure by immunophilin FKBP12 regulates the neuronal resilience to extracellular stress

doi: 10.1126/sciadv.add9789

Figure Lengend Snippet: ( A ) Experimental schematic for AstTau describing the work flow of 2D culture, oTau seeding, 3D assembloid, FKBP12 lentivirus transduction, and analysis at indicated data collection time points [week 1 (WK-1), WK-2, and WK-3]. ( B ) Representative images showing accumulation of phosphorylated tau (red, labeled by CP13, pTau Ser 202 ) in oTau seeded asteroids and the reduction of pTau in FKBP12 overexpression conditions. The neurons were labeled by MAP-2 (violet), and astrocytes were labeled by glial fibrillary acidic protein (GFAP) (blue). Scale bars, 50 μm. ( C and D ) Quantification of FKBP12 and CP13-labeled fluorescence intensity normalized to the corresponding MAP-2 intensity as shown in (B). Data obtained from five independent asteroids. Error bars = SEM. Two-way ANOVA with Tukey’s multiple comparisons test was performed; * P < 0.01, *** P < 0.005, and **** P < 0.0001. ( E ) Representative images showing reduction in misfolding tau upon FKBP12 overexpression in brain assembloids after 3 weeks of oTau seeding. The misfolded tau was labeled with MC1 (red), neurons were labeled with Tuj1 (violet), and astrocytes were labeled with S100β (green) and DAPI used to label nuclei. Scale bars, 50 μm. ( F ) Quantification of MC1 labeled fluorescence intensity with normalization to corresponding levels of Tuj1. Data obtained from five independent asteroids. Error bars = SEM. Two-way ANOVA with Tukey’s multiple comparisons test was performed; * P < 0.01 and **** P < 0.0001. ( G ) Representative images showing toxic tau oligomers labeled with TOMA2 antibody (red) and degenerating neurons labeled with Fluoro-Jade B (green) in neuron-astrocyte assembloids with exposure to oTau and/or treated with FKBP12 overexpression for 3 weeks. Neurons were labeled with Tuj1 (violet) and FKBP12 labeled with antibody in bright blue. Scale bars, 50 μm. ( H and I ) Quantification of TOMA2 and Fluoro-Jade B labeled fluorescence intensity normalized to the corresponding Tuj1 intensity. Data obtained from five independent asteroids. Error bars = SEM. Two-way ANOVA with Tukey’s multiple comparisons test was performed; * P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.0001.

Article Snippet: The primary antibodies used in this study for immunofluorescence labeling are as follows: CP-13 (mouse; provided by P. Davies, Northwell), 1:300; TOMA2 (Anti-Tau oligomer clone 2, mouse,provided by Rakez Kayed laboratory), 1:200; FKBP12 (rat; R&D Systems, catalog no. MAB3777), 1:500.

Techniques: Transduction, Labeling, Over Expression, Fluorescence