Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Fibroblast growth factor 2 protects against renal ischaemia/reperfusion injury by attenuating mitochondrial damage and proinflammatory signalling

doi: 10.1111/jcmm.13203

Figure Lengend Snippet: FGF2 inhibits I/RI‐induced HMGB1 serum release and inflammatory response. Animals were divided into 5 groups ( n = 4), including sham‐operated control, I/RI group, and I/RI with FGF2 pre‐treatment or delayed treatment at 1 and 12 hrs, respectively, after reperfusion as indicated. The samples were collected at 48 hrs following reperfusion for Western blot, ELISA, Immunohistochemistry staining (IHC) and qRT‐PCR analysis as detailed below. ( A ) Western blot analysis to determine the expression of HMGB1 and TNFα in renal tissues with GAPDH as loading control. ( B ) ELISA assay was used to determine the levels of HMGB1 in the serum of animals receiving indicated treatments. ** P < 0.01 versus sham group, ## P < 0.01 versus I/R group. ( C ) IHC of kidney tissue sections for expression of HMGB1. Original magnification ×20. One representative area of renal tissue staining from 1 of 4 animals in each group is shown. (D) Real‐time PCR quantification of mRNA levels for KIM1, TLR2, TLR4, IL‐1α, IL‐6 and TNFα in the kidney, respectively. The result is normalized to GAPDH. The data are presented as mean ± S.E. ( n = 4). *** P < 0.001, ** P < 0.001 versus sham group; ### P < 0.001, # P < 0.05 versus I/R group.

Article Snippet: Rat HMGB1 ELISA Kit was purchased from CUSABIO (Hubei, China).

Techniques: Control, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Monoallelic expression of FL reduces FL production. (A) Semi-quantitative RT-PCR of FL transcripts in BM cells from C57BL/6 (B6), FL+/+ x RAG1-GFP/+, FL+/− x RAG1-GFP/+, or FL−/− x RAG1-GFP/+ mice. The cDNA was serially diluted 1:1, 1:3, and 1:9 for semi-quantitative analysis. Beta-actin was used as a loading control. Data are representative of two BM samples for each genotype. (B) Quantification of FL transcripts. Intensity data are the average of two BM samples for each genotype. (C) Concentration of FL (pg/mL) in the serum of C57Bl/6 (B6), FL+/−, and FL−/− mice as determined by ELISA. Data represents the mean ± S.D. (*p ≤ 0.0001) FL concentration in serum from ≥ 5 mice/genotype and 2 independent experiments. P-values were determined using the Students T-test.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: FL haploinsufficiency reduces LHP and RAG1 locus activation. (A) Flow cytometric analysis of LSK+ BM cells from RAG1-GFP x FL+/+, RAG1-GFP x FL+/−, and RAG1-GFP x FL−/− mice (top panels). Differential expression of Flt3 and VCAM-1 in LSK+ BM cells to discriminate HSC/MPP, GMLP, and LMPP (bottom panels). Boxed regions indicate Flt3hi GMLP. (B) Histogram of boxed region from (A) bottom panels, indicative of Flt3 expression in Flt3hi GMLP in different FL genotypes. (C) Flow cytometric analysis of LSK+ BM cells from RAG1-GFP+ x FL mice to distinguish HSC, MPP, and LHP. A littermate GFP- control was analyzed in each experiment to determine GFP+ gates (data not shown). (D) Flow cytometric analysis of Lin- BM cells to distinguish CLP: Lin- c-kitlo IL-7R+ (Top panels). CLP were further discriminated by Flt3 and Sca-1 expression (middle panels). GFP expression within Lin- c-kitlo IL-7R+ Flt3+ Sca-1+ CLP (bottom panels). GFP+ gates were determined by analysis of Lin- c-kitlo IL-7R- Flt3+ cells which do not express GFP (data not shown). (E) Flow cytometric analysis of BCP. Pre-Pro-B/Pro-B cells are B220+ CD43+, Pre-B cells are B220+ CD43-, and naïve/mature B cells are B220hi CD43-. Data are representative of ≥ 5 mice/genotype and ≥ 3 independent experiments.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: Activation Assay, Quantitative Proteomics, Expressing, Control

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Id1 does not rescue the lymphoid defect in FL−/− mice. (A) Flt3, tcfe2a, rag1, and ebf1 and (B) Scl and id1 were analyzed from FACS sorted GMLP and LMPP from C57Bl/6 (B6) and FL+/− mice by quantitative PCR. (A+B) Data are representative of 2 independent experiments with BM pooled from ≥ 5 mice/genotype. Error bars represent the mean ± SEM. (C) Flow cytometric analysis of BM from C57Bl/6 (B6), Id1−/−, FL−/−, and FL−/− Id1−/− mice stained with antibodies for lineage markers, c-kit, and Sca-1 to determine LSK+ cells (top panels). LSK+ cells stained with antibodies to Flt3 and VCAM-1 to distinguish HSC/MPP, GMLP, and LMPP (bottom panels). Data are representative of ≥ 3 mice/genotype and ≥ 3 independent experiments.

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: Real-time Polymerase Chain Reaction, Staining

Journal:

Article Title: Threshold levels of Flt3-ligand are required for the generation and survival of lymphoid progenitors and B cell precursors

doi: 10.1002/eji.201040710

Figure Lengend Snippet: Flt3 regulates the survival, but not the proliferation of LHP. (A-B) Flow cytometric analysis of bone marrow to examine BrdU incorporation 12 hours after 2 mg were injected i.p. into FL+/+ and FL−/− mice in c-kit+ Sca-1+ (SK+) (A) and c-kit+ Sca-1- (B). (A) Total BrdU incorporation in SK+ cells (top panels). BrdU incorporation in SK+ cells using Flt3 as additional criteria (bottom panels). (B) Total BrdU incorporation in c-kit+ Sca-1- cells (top panels). BrdU incorporation in c-kit+ Sca-1- cells using Flt3 as additional criteria (bottom panels). BrdU quadrants are set on the IgG isotype control for each genotype (data not shown). Data are representative of ≥ 3 mice/genotype and ≥ 3 independent experiments. (C-D) Annexin V staining and intracellular Mcl-1 expression in LSK+ cells (C) and CLP (D). FL+/+ represented by filled histogram, FL−/− indicated by solid line, and thin line depicts isotype control. (C) LSK+ cells were fractionated into Flt3lo and Flt3+hi subsets (top panels). Overlaid histograms depicting Annexin V staining in LSK+ Flt3lo and LSK+ Flt3+hi cells (middle panels). Overlaid histograms depicting intracellular Mcl-1 staining in LSK+ Flt3lo and LSK+ Flt3+hi cells (bottom panels). (D) CLP fractionated into three subsets: Flt3neg, Flt3lo, and Flt3hi (top panels). Overlaid histograms depicting Annexin V staining in CLP subsets (middle panels). Overlaid histograms depicting intracellular Mcl-1 staining in CLP subsets (bottom panels). Data are representative of 3 independent experiments (C+D).

Article Snippet: The serum concentration of FL was calculated by ELISA on serum harvested from FL+/+ , FL+/− , and FL−/− mice using mouse Flt3 Ligand Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per kit instructions.

Techniques: BrdU Incorporation Assay, Injection, Control, Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: iPSC-Derived Microglia for Modeling Human-Specific DAMP and PAMP Responses in the Context of Alzheimer’s Disease

doi: 10.3390/ijms21249668

Figure Lengend Snippet: Immune surveillance of PAMPs: ( a ) Human cytokine profiler assay of MGL-conditioned media (24 h) from UB068 and UB068_CHRFAM7A lines at baseline (left) and after 24 h of LPS stimulation (right). Note the robust increase in the IL-6 expression in response to the LPS (1 μg/mL) treatment in UB068_CHRFAM7A. ( b ) Densitometric analysis of NF-κB-independent (left) and NF-κB -dependent (right) cytokines in the LPS treated and untreated MGL cells. Note the difference in the baseline expression level of the NF-κB-dependent cytokines between the two lines. The LPS’s effect on the NF-κB-independent and NF-κB-dependent cytokines is depicted in the non-carrier (UB068) and carrier (UB068_CHRFAM7A) lines. NF-κB-independent cytokines demonstrated similar responses in both lines, while a marked NF-κB-dependent gain of function occurred in the carrier (UB068_CHRFAM7A line). Data are presented as mean ± SD. *** p < 0.001—difference in the baseline between the UB068 and UB068_CHRFAM7A lines; ** p < 0.01, *** p < 0.001 ( t -test)—difference in the cytokine expression between the LPS-treated and LPS-untreated MGL cells. ( c ) Quantitative analysis of the NF-κB binding in nuclear extracts of the MGL cells derived from the UB068 and UB068_CHRFAM7A lines treated with LPS (1 μg/mL, 3 h). Data are presented as mean ± SD. * p < 0.05 ( t -test). ( d ) Densitometric analysis of IL-6 in media of MGL cells treated with LPS (1 μg/mL, 24 h). Data are presented as mean ± SD. *** p < 0.001—difference in the IL-6 baseline between the UB068 and UB068_CHRFAM7A lines; * p < 0.05, *** p < 0.001 ( t -test)—difference in the IL-6 expression between the treated and untreated MGL cells. RT-qPCR ( e ) and ELISA ( f ) in the non-carrier (UB068) and carrier (UB068_CHRFAM7A and UB052) lines with pharmacological modulation. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001—difference in the LPS-induced inflammatory response compared to non-treated controls, with and without pharmacological modulation.

Article Snippet: The concentration of IL-6 in the cell culture media was quantified using an IL-6 ELISA kit (R&D Systems, Minneapolis, MN, USA), in keeping with the manufacturers’ protocols.

Techniques: Expressing, Binding Assay, Derivative Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 2. Expression of renalase in rats was increased after 6-OHDA treatment. (A) Detection of serum renalase concentration from the 2nd to 5th week after 6-OHDA application by ELISA kit (n = 6 rats per group). (B) qRT-PCR detection of relative renalase expression in NG, NTS, and heart tissues of Parkinson’s rats and sham group (4th week after 6-OHDA application, n = 5 rats per group). (C,D) Western blot assay showing relative protein levels of renalase in NG, NTS, and heart tissues of Parkinson’s rats and sham group (n = 4 rats per group). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. sham.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 3. PC12 cells transfected with RNLS overexpression plasmid (or empty vector control) continued to overexpress and metabolize DA. (A,B) Transient transfection of PC12 cells with RNLS overexpression plasmid (or empty vector control) expressing the protein. Western blot assay showing relative protein levels of RNLS in PC12 cells at different time points (n = 6). (C) qRT-PCR detection of relative RNLS at different time points after PC12 cells transfected with (n = 6). (D) Detection of relative dopamine (DA) concentration in PC12 cells at different time points by ELISA kit following RNLS overexpression or NC, showing altered DA levels (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Expressing, Western Blot, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 4. Increased levels of DA metabolites after RNLS overexpression of in PC12 cells. (A,B) Detection of DOPAL content at different time points in PC12 cells after RNLS overexpres- sion by high performance liquid chromatography (n = 3). (C) qRT-PCR detection of relative ALDH expression at different time points after PC12 cells were transfected with (n = 6). (D,E) Western blot analysis showing relative protein levels of ALDH at different time points after PC12 cells were transfected with (n = 6). Data are presented as mean ± SD, * p< 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Over Expression, High Performance Liquid Chromatography, Quantitative RT-PCR, Expressing, Transfection, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 6. α-Syn aggregated in PC12 cells transfected with RNLS for 96 h. (A,B) Representative graph of Tri-αS and higher molecular weight α-Syn aggregates in PC12 cells transfected with RNLS for 96 h (n = 6). (C(a,b)) Immunofluorescent photomicrographs of transfected cells (overexpressing RNLS protein for 96 h), cytoplasmic aggregates in the cell neurotic processes are highlighted by white arrows in the figure. (C(c,d)) Untreated control PC12 cells. Scale bar: 20 µm. Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Molecular Weight, Control

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 5. Changes in α-Syn content at different time points in RNLS-overexpressing PC12 cells. (A) qRT-PCR detection of relative α-Syn expression at different time points after PC12 cells trans- fected with RNLS (n = 6). (B) Representative image of immunofluorescence with enhanced α-Syn fluorescence after transfection of PC12 cells. Scale bar: 20 µm. (C) Immunohistochemical quantitative statistics (n = 60). (D–G) Western blot analysis showing relative protein levels of monomer of α-Syn (Mon-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). (E–H) Western blot analysis showing relative protein levels of dimer of α-Syn (Dim-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). (F–I) Western blot analysis showing relative protein levels of trimer of α-Syn (Tri-αS) at different time points after PC12 cells were transfected with RNLS (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Quantitative RT-PCR, Expressing, Immunofluorescence, Fluorescence, Transfection, Immunohistochemical staining, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 7. The content of tyrosine hydrolase (TH) decreased after PC12 cells were transfected with RNLS. (A) qRT-PCR detection of relative TH expression at different time points after PC12 cells transfected with RNLS (n = 6). (B,C) Western blot analysis showing relative protein levels of TH at different time points after PC12 cells were transfected withR NLS (n = 6). (D) Representative graph of immunofluorescent (IF) results of TH in PC12 cells at different time points administered with RNLS overexpression. Scale bar: 20 µm. (E) Immunohistochemical quantitative statistics (n = 60). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Over Expression, Immunohistochemical staining

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 8. Cell viability decreased after PC12 cells were transfected with RNLS. (A) Live/dead cell viability assay of cultured PC12 cells. The cells were transfected with plasmids and cultured for 24, 48, 72, or 96 h and then stained with the Calcein-AM/PI Double Staining Kit. The live and dead cells exhibited green and red fluorescence. Scale bar: 10 µm. (B) Immunohistochemical quantitative statistics (n = 3). (C) CCK-8 was used to determine PC12 cells viability following overexpression or NC for 24, 48, 72 or 96 h (n = 12). Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Transfection, Viability Assay, Cell Culture, Staining, Double Staining, Fluorescence, Immunohistochemical staining, CCK-8 Assay, Over Expression

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 9. Decreased expression of axonal transporters after RNLS overexpression in PC12 cells. (A) qRT-PCR detection of relative dynein (DYN) expression at different time points after PC12 cells transfected with RNLS (n = 6). (B,C) Western blot analysis showing relative protein levels of dynein at different time points after PC12 cells were transfected with RNLS (n = 6). (D) mRNA expression of relative kinesin in PC12 cells at different time points (n = 6). (E,F) Western blot analysis showing relative protein levels of KHC at different time points after PC12 cells were transfected with RNLS (n = 6). Data are presented as mean ± SD, * p < 0.05 and ** p < 0.01 vs. Ctrl.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Transfection, Western Blot

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 10. Ultrastructural damage after RNLS overexpression in PC12 cells. (A,B) The relative ∆Ψ m in PC12 cells for each treatment at different time points, as determined by JC-1 staining (n = 15). Data are presented as mean ± SD, ** p < 0.01 vs. Ctrl; scale bar: 20 µm; ∆Ψm: mitochondrial membrane potential. (C) Representative images of transmission microscopy at different time points after PC12 cells were transfected with RNLS. Scale bar: 1 µm and 2 µm, direct magnification: ×1200, ×2000 and ×4000, the red arrow indicates mitochondria.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Over Expression, Staining, Membrane, Transmission Assay, Microscopy, Transfection

Journal: Biomedicines

Article Title: Renalase Overexpression-Mediated Excessive Metabolism of Peripheral Dopamine, DOPAL Accumulation, and α-Synuclein Aggregation in Baroreflex Afferents Contribute to Neuronal Degeneration and Autonomic Dysfunction.

doi: 10.3390/biomedicines13051243

Figure Lengend Snippet: Figure 11. Numerous clues to PD peripheral pathology. (A) Vagal application of DOPAL (3,4- dihydroxyphenylacetaldehyde) to simulate PD-like autonomic dysfunction strongly suggests that DOPAL will likely induce the expression and aggregation of α-Syn/oligomers at the injection site, which will then be transported to the heart and the NG. Axonal transportation is likely to be impaired by DOPAL and DOPAL-mediated oligomer α-Syn [22]. (B) Typical PD model causes the RNLS up-regulation. (C) The pathological α-Syn protein fibers were injected into the mouse intestine, and it was found that the α-Syn protein eventually diffused into the substantia nigra pars compacta, where it degraded dopaminergic neurons. (D) Orthostatic hypotension (OH) has been linked to a higher risk of Parkinson’s disease in studies [48,49]. OH is a predictor of motor decline in individuals with early PD [50]. The experiment confirmed that the change in BP also occurred before the movement disorder in rats treated with 6-OHDA. (E) Current mechanisms and central nervous system origins of PD: MAO-mediated DA metabolism in the central nervous system. (F) The role of RNLS in peripheral etiology of PD.

Article Snippet: The concentration of RNLS was detected using the Rat RNLS ELISA Kit (CSB-EL002965RA, Cusabio, Wuhan, China) and the O.D. value was measured at 450 nm.

Techniques: Expressing, Injection