Journal: MedComm

Article Title: Novel aspect of neprilysin in kidney fibrosis via ACSL4‐mediated ferroptosis of tubular epithelial cells

doi: 10.1002/mco2.330

Figure Lengend Snippet: Genetic knockdown of neprilysin improved the fibrotic and ferroptotic phenotype in TGFβ1‐induced TCMK‐1 cells. (A and B) The knockdown efficiency of NEP siRNA (siNEP) in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (C) Representative Western blot images and quantitative analysis of NEP expression in TCMK‐1 cells. (D) RT‐PCR was performed to detect the mRNA level of Fn, α‐SMA, and Col Iα1. (E) RT‐PCR was performed to detect the mRNA level of ACSL4, GPX4, and Ptgs2 (COX2). (F) Western blot analysis was performed to detect protein expression of Fn, α‐SMA, and Col Iα1. (G) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (H) The accumulation of lipid peroxidation in TCMK‐1 cells was analyzed by BODIPY C11 staining (×200, scale bar = 50 μm). NS p > 0.05, versus control. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, versus siNC. ### p < 0.001, ## p < 0.01, # p < 0.05, versus siNC+TGF‐β1. siNC, negative control.

Article Snippet: Primary antibodies against NEP (1:200, 18008‐1‐AP; Proteintech, USA), α‐SMA (1:100, ET1607‐43; Huabio, China), and ACSL4 (1:200, 81196‐1‐RR; Proteintech) were used in the study.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Staining, Control, Negative Control

Journal: MedComm

Article Title: Novel aspect of neprilysin in kidney fibrosis via ACSL4‐mediated ferroptosis of tubular epithelial cells

doi: 10.1002/mco2.330

Figure Lengend Snippet: Neprilysin aggravated fibrosis by ACSL4‐mediated ferroptosis in TGF‐β1‐induced TCMK‐1 cells. (A and B) The efficiency of NEP overexpression in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (C and D) The overexpression efficiency of ACSL4 in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (E and F) The knockdown efficiency of ACSL4 siRNA in TCMK‐1 cells evaluated by RT‐PCR and Western blot analysis. (G) RT‐PCR was performed to detect the mRNA expression of ACSL4, GPX4, and Ptgs2 (COX2). (H) Western blot analysis was performed to detect protein expression of ACSL4, GPX4, and COX2. (J) RT‐PCR was performed to detect the mRNA expression of Fn, α‐SMA, and Col Iα1. (I) Western blot analysis was performed to detect protein expression of Fn, α‐SMA, and Col Iα1. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, versus oeNC or siNC or oeNC+siNC. #### p < 0.0001, ### p < 0.001, ## p < 0.01, # p < 0.05, versus oeNC+siNC+TGF‐β1. $$$$ p < 0.0001, $$ p < 0.01, $ p < 0.05, versus oeNEP+siNC+TGF‐β1. siNC, silencing negative control; siNEP, NEP siRNA; siACSL4, ACSL4 siRNA; oeNC, overexpression negative control; oeNEP, overexpression of NEP; oeACSL4, overexpression of ACSL4.

Article Snippet: Primary antibodies against NEP (1:200, 18008‐1‐AP; Proteintech, USA), α‐SMA (1:100, ET1607‐43; Huabio, China), and ACSL4 (1:200, 81196‐1‐RR; Proteintech) were used in the study.

Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Knockdown, Expressing, Negative Control

Journal: Annals of Translational Medicine

Article Title: Cinacalcet attenuated bone loss via inhibiting parathyroid hormone-induced endothelial-to-adipocyte transition in chronic kidney disease rats

doi: 10.21037/atm.2019.06.44

Figure Lengend Snippet: CINA treatment inhibits the EndMT in CKD rats. (A,B) Representative western blot analysis and quantification of levels of the CD31, FSP1, α-SMA, CD10, and CD44 proteins in the different groups of rats, as indicated. The data are presented as the mean ± SD. n=4 for each group. *, P<0.05 vs. the CTL group; #, P<0.05 vs. the CKD group. (C) Representative images of immunofluorescence staining for CD31, FSP1, and CD44 in the bone marrow of rats (upper panel: green for CD31, red for FSP1; lower panel: red for CD31, green for CD44). White arrows indicate colocalization between CD31 and either FSP1 or CD44. DAPI (blue) labels the nuclei. Scale bar, 50 µm. CTL, control; CKD, chronic kidney disease; CINA, cinacalcet; SD, standard deviation.

Article Snippet: PVDF membranes were incubated overnight with primary antibodies against CD31 (sc-376764, Santa Cruz, 1:1,000 dilution), FSP1 (ab-197896, Abcam, dilution), α-SMA (ab-7817, Abcam, 1:1,000 dilution), CD10 (NBP2-15771, Novus Biologicals, 1:1,000 dilution), CD44 (ab-157107, Abcam, 1:1,000 dilution), C/EBP-α (sc-365318, Santa Cruz, 1:1,000 dilution), or PPAR-γ (ab-209350, Abcam, 1:1,000 dilution) followed by horseradish peroxidase-labeled secondary IgG (Santa Cruz).

Techniques: Western Blot, Immunofluorescence, Staining, Control, Standard Deviation

Journal: Circulation Reports

Article Title: Soluble Neprilysin ― Cardiac Function and Outcome in Hypertrophic Cardiomyopathy ―

doi: 10.1253/circrep.cr-19-0034

Figure Lengend Snippet: Figure 1. Log soluble neprilysin (log sNEP) vs. log B-type natriuretic peptide (logBNP), log troponin I, left ventricular ejection fraction (LVEF) and right ventricular fractional area change (RV-FAC).

Article Snippet: Circulating plasma sNEP was measured on radioimmunoassay (ELH-Neprilysin-1 kit; RayBiotech, Norcross, GA, USA).

Techniques:

Journal: Circulation Reports

Article Title: Soluble Neprilysin ― Cardiac Function and Outcome in Hypertrophic Cardiomyopathy ―

doi: 10.1253/circrep.cr-19-0034

Figure Lengend Snippet: Figure 2. Kaplan-Meier analysis for cardiac event rate and all-cause mortality between the low and high soluble neprilysin (sNEP) groups.

Article Snippet: Circulating plasma sNEP was measured on radioimmunoassay (ELH-Neprilysin-1 kit; RayBiotech, Norcross, GA, USA).

Techniques:

Journal: Circulation Research

Article Title: Successive Action of Meprin A and Neprilysin Catabolizes B-Type Natriuretic Peptide

doi: 10.1161/circresaha.107.153585

Figure Lengend Snippet: Figure 2. Screening for peptidase(s) degrading BNP. A, Degra- dation of mBNP by kidney membrane of NEP-knockout mice in the presence of effective concentrations of peptidase inhibitors; values are shown as meansSEM (n5). B, Degradation of na- triuretic peptides by recombinant meprin (n3; SD within the

Article Snippet: Recombinant mouse NEP, meprin and subunits, and the cGMP kit were from R&D Systems (Wiesbaden, Germany).

Techniques: Membrane, Knock-Out, Recombinant

Journal: Circulation Research

Article Title: Successive Action of Meprin A and Neprilysin Catabolizes B-Type Natriuretic Peptide

doi: 10.1161/circresaha.107.153585

Figure Lengend Snippet: Figure 4. NEP participation in mBNP7-32 degradation. A, Deg- radation of natriuretic peptides by recombinant NEP (n3; SD within the points); degradation of mBNP (B) and simultaneous formation of mBNP7-32 (C) by wild-type mouse kidney mem- brane in the presence of no inhibitor (white; N), candoxatrilat (horizontally striped; C), actinonin (vertically striped; A), or candoxatrilat and actinonin (crosshatched; AC); control (black, CON) is mBNP without degradation; values are shown as meansSEM (n3). ns indicates not significant. #P0.005 vs no inhibitor.

Article Snippet: Recombinant mouse NEP, meprin and subunits, and the cGMP kit were from R&D Systems (Wiesbaden, Germany).

Techniques: Recombinant, Control

Journal: bioRxiv

Article Title: CITEseq analysis of non-small-cell lung cancer lesions reveals an axis of immune cell activation associated with tumor antigen load and TP53 mutations

doi: 10.1101/2020.07.16.207605

Figure Lengend Snippet: A , Expression of key genes discriminating scRNAseq clusters of monocytes and MΦ, grouped by cell type annotation. Heatmap shows the number of UMI per cell. Clusters are shown using an even number of randomly selected cells from each, drawing from 35 tumor and 32 nLung samples. Cells were downsampled to 2000 UMI/cell. B , Scatter plots showing normalized CITEseq CD10 and CD206 surface marker counts on AMΦ, MoMΦ, and CD14+ monocytes in nLung of a representative patient. C , IHC of CD10 staining AMΦ in the airspaces of nLung tissue. D-G , Gene module analysis of monocyte and MΦ clusters. Correlation of gene module expression across all monocytes and MΦ ( D ). Module groups illustrate groups of correlated modules which are expressed most specifically on AMΦ, MoMΦ, and monocytes (see G ). Five example genes from each module, ranked by correlation to the other genes in the module and colored by total expression in monocytes and MΦ ( E ), boxplots showing Log 2 FC of module expression among all monocytes and MΦ between patient matched tumor and nLung samples ( F ), and normalized average cluster expression of modules ( G ). H , Log 2 FC and expression level of gene sets that are mutually exclusively expressed in CD14+ monocytes, AMΦ, and MoMΦ (See ).

Article Snippet: Primary antibodies were: anti-human CD10 (200103, R&D systems), DC-Lamp (1010E1.01, Novus biologicals), pan-cytokeratin (AE1/AE3, Dako), PDPN (D@-40, Ventana), CD163 (10D6, Novus Biologicals) and PD-L1 (E1L3N, Cell Signaling Tech).

Techniques: Expressing, Marker, Staining