Journal: Biomedicines

Article Title: Functional Characterization of the SCN5A p.D372H Variant Associated with Brugada Syndrome

doi: 10.3390/biomedicines14030582

Figure Lengend Snippet: The SCN5A p.D372H variant reduces Nav1.5 expression through decreased mRNA abundance and stability, independent of plasmid DNA levels. ( A ) Relative SCN5A mRNA expression measured by RT-qPCR. Transcript levels were significantly reduced in the D372H group compared with WT (**** p < 0.0001). Co-expression of WT and D372H (WT+MUT) also resulted in a significant decrease in SCN5A mRNA expression compared with WT (**** p < 0.0001). Data were normalized to WT. ( B ) Quantification of intracellular SCN5A plasmid DNA levels. No significant differences were observed among WT, WT+MUT, and MUT groups (ns), indicating comparable transfection efficiency. ( C ) SCN5A mRNA decay following transcriptional inhibition with actinomycin D. Relative mRNA remaining was normalized to the 0-h time point. The D372H group exhibited accelerated mRNA decay compared with WT, indicating reduced transcript stability. ( D ) Representative Western blot images showing total Nav1.5 protein expression in BLANK, CONTROL, WT, WT+MUT, and MUT groups. Molecular weight markers are indicated in kilodaltons (kDa). The predicted molecular weight of native Nav1.5 is approximately 227 kDa; however, in this study SCN5A was expressed as a GFP-tagged fusion protein, resulting in an apparent band migrating above 245 kDa. Tubulin (50 kDa) was used as a loading control. ( E ) Quantitative analysis of Nav1.5 protein expression normalized to Tubulin. Protein levels were significantly reduced in the D372H group compared with WT (* p < 0.05). The WT+MUT group also showed decreased expression compared with WT (** p < 0.01). Data are presented as mean ± SD from at least three independent experiments.

Article Snippet: The membrane was then incubated with primary antibodies against Nav1.5 (1:1000, Cat# 23016-1-AP, Proteintech, Wuhan, China) and Tubulin (1:000, Cat# A03989-1, Boster, Wuhan, China) to detect the target protein and serve as a loading control, respectively.

Techniques: Variant Assay, Expressing, Plasmid Preparation, Quantitative RT-PCR, Transfection, Inhibition, Western Blot, Control, Molecular Weight

Journal: Lupus science & medicine

Article Title: Proteomics uncovers ICAM2 (CD102) as a novel serum biomarker of proliferative lupus nephritis.

doi: 10.1136/lupus-2024-001446

Figure Lengend Snippet: Figure 2 ELISA validation of three serum biomarkers using an independent cohort and their correlation with clinical indices. (A–C) A cohort of 80 serum samples: 30 healthy controls (HC, green) and 50 patients with lupus nephritis (LN, red) were tested by ELISA for the levels of ICAM2, TIMP1 and THBS1. (D) The discriminatory abilities of the three serum markers were assessed in distinguishing patients with LN from HCs using receiver operating characteristic (ROC) curve analysis. (E–K) The correlation of ELISA-assayed serum ICAM2 levels with clinical indices in LN. r denotes Spearman’s correlation coefficient. ****p<0.0001. AUC, area under the curve.

Article Snippet: Serum samples for TIMP1 (CUSABIO, Catalogue No CSB- E08003h, Wuhan, China) and THBS1 (CUSABIO, Catalogue No CSB- E08763h, Wuhan, China) were diluted 1:100 and 1:400, respectively, whereas the ICAM2 (BOSTER, Catalogue No EK0999, Peachtree Corners, Georgia) ELISA assays used serum at 1:5 dilution.

Techniques: Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: Lupus science & medicine

Article Title: Proteomics uncovers ICAM2 (CD102) as a novel serum biomarker of proliferative lupus nephritis.

doi: 10.1136/lupus-2024-001446

Figure Lengend Snippet: Figure 3 Serum ICAM2 levels in lupus nephritis (LN) classes and their association with renal histology. (A) Serum ICAM2 was significantly elevated in patients with LN III (±V) and LN IV (±V) when compared with healthy controls (HC) or LN V. (B) Serum ICAM2 outperformed C3, C4 or anti-dsDNA antibody in discriminating LN V from LN III (±V) or IV (±V). Values in the plot indicate areas under the curve. (C–H) The correlation of serum ICAM2 levels with particular pathological attributes of the activity index. r denotes Spearman’s correlation coefficient. *p<0.05; **p<0.01; ****p<0.0001. AUC, area under the curve; ROC, receiver operating characteristic.

Article Snippet: Serum samples for TIMP1 (CUSABIO, Catalogue No CSB- E08003h, Wuhan, China) and THBS1 (CUSABIO, Catalogue No CSB- E08763h, Wuhan, China) were diluted 1:100 and 1:400, respectively, whereas the ICAM2 (BOSTER, Catalogue No EK0999, Peachtree Corners, Georgia) ELISA assays used serum at 1:5 dilution.

Techniques: Activity Assay

Journal: Lupus science & medicine

Article Title: Proteomics uncovers ICAM2 (CD102) as a novel serum biomarker of proliferative lupus nephritis.

doi: 10.1136/lupus-2024-001446

Figure Lengend Snippet: Figure 4 Expression of ICAM2 within the renal tissues in lupus nephritis (LN). (A) Immunohistochemical analysis of ICAM2 protein expression was performed in kidney sections. The representative images were shown in paracancer controls and LN class III, IV and V patients. (B, C) ICAM2 expression in LN glomeruli and tubulointerstitium from Nephroseq database. *p<0.05; ****p<0.0001. HC, healthy control; MLN, membranous lupus nephritis (class V); PLN, proliferative lupus nephritis (class III (±V) or IV (±V)).

Article Snippet: Serum samples for TIMP1 (CUSABIO, Catalogue No CSB- E08003h, Wuhan, China) and THBS1 (CUSABIO, Catalogue No CSB- E08763h, Wuhan, China) were diluted 1:100 and 1:400, respectively, whereas the ICAM2 (BOSTER, Catalogue No EK0999, Peachtree Corners, Georgia) ELISA assays used serum at 1:5 dilution.

Techniques: Expressing, Immunohistochemical staining, Control

Journal: Lupus science & medicine

Article Title: Proteomics uncovers ICAM2 (CD102) as a novel serum biomarker of proliferative lupus nephritis.

doi: 10.1136/lupus-2024-001446

Figure Lengend Snippet: Figure 5 Downregulation of ICAM2 inhibited the activation of PI3K/Akt pathway and reduced the injury of human renal glomerular endothelial cells (HRGECs) induced by immunoglobulin G (IgG) extracted from lupus nephritis (LN) serum. (A) Representative immunofluorescence images of the costaining of ICAM2 (green) and CD31 (red) in control and patients with LN. Scar bar=10 µm. (B, C) Western blotting and quantitative analysis of ICAM2 and VCAM1 in HRGECs (n=3). (D–F) Western blotting and quantitative analysis of ICAM2 and VCAM1 in HRGECs after ICAM2 knockdown (n=3). (G–K) Western blotting and quantitative analysis of Akt, p-Akt, PI3K and p-PI3K in HRGECs after ICAM2 knockdown (n=3). *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Serum samples for TIMP1 (CUSABIO, Catalogue No CSB- E08003h, Wuhan, China) and THBS1 (CUSABIO, Catalogue No CSB- E08763h, Wuhan, China) were diluted 1:100 and 1:400, respectively, whereas the ICAM2 (BOSTER, Catalogue No EK0999, Peachtree Corners, Georgia) ELISA assays used serum at 1:5 dilution.

Techniques: Activation Assay, Immunofluorescence, Control, Western Blot, Knockdown

Journal: Nature Communications

Article Title: Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1

doi: 10.1038/s41467-017-01738-3

Figure Lengend Snippet: Pericytes control endothelial behavior in angiogenesis. a Confocal images showing proliferating cells (EdU, blue), IB4 (red) and EC nuclei (Erg1, green) in the P6 control and DTR iPC retinal angiogenic front, as indicated. Images on the right show higher magnification of boxed insets in left column. Dashed lines outline the angiogenic leading vessel from where the sprouts emerge (second column) or vessel shape (fourth column) at the edge of the vascular plexus. Scale bar, 100 µm (left panels) and 50 µm (higher magnifications). b – d Quantitation of total b and regional EC proliferation c as well as EC density d within the peripheral vascular plexus of P6 control and DTR iPC retinas. Error bars, s.e.m. p -values, Student’s t -test. NS, not statistically significant. e High magnification of IB4 (red) and Erg1 (green) stained sprouts highlighting the accumulation of EC nuclei in P6 DTR iPC but not control sprouts. Scale bar, 50 µm. f Confocal images showing IB4 (red), NG2+ pericytes (green) and vessel lumen (ICAM2, white) in the control and DTR iPC angiogenic front of P6 retinas. Images on the right show higher magnification of boxed insets in the second column. Note that IB4 signal covers a larger area than the apical ICAM2 immunostaining (arrowheads in third column). Scale bar, 100 µm (left panels) and 50 µm (higher magnifications)

Article Snippet: Moreover, the following primary antibodies were used: rabbit anti-collagen type IV (colIV, 1:100, Millipore, AB756P), rat monoclonal anti-laminin α5 (LAMA5, 1:50, clone 4G A2 11, kindly provided by Prof. Dr. Lydia Sorokin, Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Germany), goat anti-PDGFRβ (1:100, R&D, AF1042), rat anti-PDGFRβ (1:100, eBioscience, 14–1402), rabbit anti-desmin (1:100, Abcam, ab15200), rabbit anti-NG2 (1:200, Millipore, AB5320), rabbit anti-Erg1 (1:100, Abcam, ab110639), rat anti-ICAM2 (1:100, Pharmingen, 553326), rabbit anti-VEGF (1:100, Santa Cruz, sc-152), rat anti-VEGFR2 (1:100, Pharmingen, 555307), rabbit anti-VEGFR2 (1:100, Cell Signaling, 2479), goat anti-VEGR3 (1:100, R&D, AF743), goat anti-Tie2 (1:100, R&D, AF762), goat anti-Endocan (Esm1, 1:100, R&D, AF1999), goat anti-Dll4 (1:100, R&D, AF1389), mouse anti-αSMA-Cy3 (1:200, Sigma, C6198), rabbit anti-fibronectin (1:100, Sigma, F3648), rat anti-VE-cadherin (1:100, BD Bioscience, 555289), rabbit anti-Claudin-5 (1:100, Zymed, 341600), rat anti-integrin β1 (1:100, BD Biosciences, 553715), rabbit anti-Zonula occludens 1 (ZO1, 1:100, Invitrogen, 617300), chicken anti-green fluorescent protein (GFP, 1:300, AVES Labs, Inc., GFP-1020) and rabbit anti-GFP-Alexa Fluor 488 (1:400, Invitrogen, A21311).

Techniques: Quantitation Assay, Staining, Immunostaining