Journal: Journal of neuroinflammation

Article Title: Single-cell RNA sequencing unveils Lrg1's role in cerebral ischemia‒reperfusion injury by modulating various cells.

doi: 10.1186/s12974-023-02941-4

Figure Lengend Snippet: Fig. 3 Single-cell transcriptomic data demonstrate that the impact of Lrg1 knockout on various cellular components in brain tissue of cerebral ischemia‒reperfusion mice. A A schematic representation of the experimental design employed in this study is depicted. B UMAP plots of 99,991 cells from 11 mice, including 14 cell types. C Heatmap of gene expression across different cell types. D Boxplot displaying cell purity for cell types. E Bar graph showing the number of differentially expressed genes for different cell types. F Bar graph showing the proportion of genes with unique changes in a single cell type among differentially upregulated or downregulated genes in MCAO/R + Lrg1−/− mice compared to MCAO/R + WT mice

Article Snippet: Lrg1 knockout mice Lrg1 knockout mice were obtained from Cyagen Biosciences Corporation (strain number: C57BL/6N-Lrg1em1cyagen, strain name: KOCMP-76905-Lrg1-B6N‒VA).

Techniques: Knock-Out, Gene Expression

Journal: Cancers

Article Title: Biomarker Panel for the Diagnosis of Pancreatic Ductal Adenocarcinoma

doi: 10.3390/cancers12061443

Figure Lengend Snippet: Comparison of 11 candidate markers concentration between PDAC samples and normal control samples. Among the 11 markers (ApoA1, CA125, CA19-9, CRP, CYFRA21.1, LRG1, CEA, ApoA2, TTR, B2M, and D.Dimer), 10 biomarkers except B2M showed statistical differences between PDAC and normal controls.

Article Snippet: LRG-1 was measured by an enzyme-linked immunosorbent assay (ELISA) using recombinant LRG1 protein and anti-Human LRG1 antibody (R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, Concentration Assay, Control

Journal: Experimental lung research

Article Title: Leucine rich α2 glycoprotein 1 derived from malignant pleural mesothelioma cells facilitates macrophage M2 phenotypes.

doi: 10.1080/01902148.2024.2380988

Figure Lengend Snippet: Figure 2. LRG1 was a potential candidate. Ten-fold concentrated supernatants from Met5A, Meso1, Meso2 or H2452 were subjected for 10% SDS-PAGE, and the gel was silver-stained and then analyzed by Mass Spectrometry. (A) Image of the silver-stained gel; (B) The Venn graph of Mass Spectrometry results; (C) ELISA analysis of LRG1 in the 10-fold concentrated supernatants of Met5A, Meso1, Meso2, and H2452 cells. Statistical significance was analyzed from three independent experiments by the two-tailed Student’s t test. ** and *** indicate P values <0.01 and 0.001, respectively.

Article Snippet: The culture media were then replaced by 0.5 mL of 9:1 mixture of fresh X-VIVO 15 and the 10-fold concentrated supernatants from Met5A, Meso1, Meso2 or H2452, or replaced by 0.5 mL fresh X-VIVO 15 containing 16 ng/mL recombinant human LRG1 (R&D systems, Minneapolis).

Techniques: SDS Page, Staining, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Experimental lung research

Article Title: Leucine rich α2 glycoprotein 1 derived from malignant pleural mesothelioma cells facilitates macrophage M2 phenotypes.

doi: 10.1080/01902148.2024.2380988

Figure Lengend Snippet: Figure 3. LRG1 elicited dose-and time-dependent responses in THP-1 cells. THP-1 cells were stimulated with 50 ng/mL PMA for 12 h to allow initial activation and cell attachment. For observation of a dose-dependent response, the THP-1 cells were then cultured in fresh serum-free X-VIVO 15 containing different doses of recombinant human LRG1 for 24 h; For observation of a time-dependent response, the PMA-activated cells were cultured in fresh X-VIVO 15 containing 16 ng/mL of recombinant human LRG1 for different time. The culture media were collected for ELISA analysis of cytokines, and the cells were used for qPCR. (A) qRT-PCR analysis of the expression of IL-10, TGF-β and CD206 in THP-1 cells stimulated with different doses of LRG1 (0, 2, 4, 8, 16 and 32 ng/mL); (B) qRT-PCR analysis of the expression of IL-10, TGF-β and CD206 in THP-1 cells stimulated with 16 ng/mL LRG1 for different time (0, 12, 24 and 48 h); (C and D) ELISA detection of IL-10 and TGF-β in the superna tants of THP-1 cells stimulated with 16 ng/mL for 24 h. Statistical significance was analyzed from three independent experiments by One way ANOVA. * and **, versus the 0 ng/mL or 0 h groups, indicate P values <0.05 and 0.01, respectively.

Article Snippet: The culture media were then replaced by 0.5 mL of 9:1 mixture of fresh X-VIVO 15 and the 10-fold concentrated supernatants from Met5A, Meso1, Meso2 or H2452, or replaced by 0.5 mL fresh X-VIVO 15 containing 16 ng/mL recombinant human LRG1 (R&D systems, Minneapolis).

Techniques: Activation Assay, Cell Attachment Assay, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

Journal: Experimental lung research

Article Title: Leucine rich α2 glycoprotein 1 derived from malignant pleural mesothelioma cells facilitates macrophage M2 phenotypes.

doi: 10.1080/01902148.2024.2380988

Figure Lengend Snippet: Figure 4. LRG1 enhanced M2 phenotypes via TGF-β receptor. THP-1 cells were activated with 50 ng/mL PMA and then stimulated with 16 ng/mL of recombinant human LRG1 in the presence or absence of 5 µm of the TGF-β receptor inhibitor SB-431542 for 24 h. The culture media were used for ELISA detection and the cells were used for western blot and immuno fluorescence. (A and B) ELISA detection of IL-10 and TGF-β in the supernatants of THP-1 cells; (C) Representative images of immunofluorescence staining of CD206-positive THP-1 cells; (D) Percentage of CD206-positive THP-1 cells; (E) western blot analysis of p-Smad2, Smad2, p-Smad3, and Smad3. GAPDH was used as an internal control; (F) The optical intensity of the bands analyzed using Image-Pro Plus. Statistical significance was analyzed from three independent experiments by the two-tailed Student’s t test. *, ** and ***indicate P values <0.05, 0.01 and 0.001; respectively, versus the control, while #, ## and ### indicate P values<0.05, 0.01 and 0.001; respectively, versus the LRG1 group.

Article Snippet: The culture media were then replaced by 0.5 mL of 9:1 mixture of fresh X-VIVO 15 and the 10-fold concentrated supernatants from Met5A, Meso1, Meso2 or H2452, or replaced by 0.5 mL fresh X-VIVO 15 containing 16 ng/mL recombinant human LRG1 (R&D systems, Minneapolis).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, Immunofluorescence, Staining, Control, Two Tailed Test

Journal: Journal of Cancer

Article Title: LRG1 Suppresses Migration and Invasion of Esophageal Squamous Cell Carcinoma by Modulating Epithelial to Mesenchymal Transition

doi: 10.7150/jca.36189

Figure Lengend Snippet: Expression levels of LRG1 in patients with ESCC and select ESCC cell lines. (A) Comparison of the mRNA levels of LRG1 in tumor and adjacent normal tissue samples from 51 patients with ESCC. The analysis was done with dataset GSE23400 from the GEO database, and the mRNA levels were reflected by the median centered intensity of the original profiling signal. *** indicates P < 0.001. (B) The mRNA levels of LRG1 in select ESCC cell lines as revealed by real-time PCR. Three randomly chosen ESCC cell lines (TE1, KYSE30 and EC109) were tested along with the normal esophageal tissue cell HET-1A and the hepatoma line HepG2 as positive control. The relative mRNA levels of LRG1 in HET-1A was set as 1. (C) The protein levels of LRG1 in the select ESCC cell lines as revealed by Western blot. β-actin was used as loading control.

Article Snippet: Three siRNAs against LRG1 (SR314597) were purchased from OriGene (USA), The siRNA oligo sequences (sense strand) are the following: #1: 5'-GCAACCCGCUUAACAAAUAAUCCTG-3'; #2: 5'-GCUACAUCUAGAAGGCAACAAAUTG-3'; #3: 5'-GCCUAAGCUCCAAGAAUUGCACC-3').

Techniques: Expressing, Comparison, Real-time Polymerase Chain Reaction, Positive Control, Western Blot, Control

Journal: Journal of Cancer

Article Title: LRG1 Suppresses Migration and Invasion of Esophageal Squamous Cell Carcinoma by Modulating Epithelial to Mesenchymal Transition

doi: 10.7150/jca.36189

Figure Lengend Snippet: Effect of LRG1 knockdown on ESCC cell migration and invasion. (A) Western blot analysis of LRG1 protein levels in KYSE30 cells after transfection of 3 different siRNAs of LRG1 or a scramble negative control (NC). β-actin was used as loading control. NC or siLRG1 #2 transfected cells were starved for 12 h before wound-healing or transwell assay. (B) For wound-healing, cells were manually scratched using a pipette tip, washed and maintained in serum-free culture medium. Pictures were taken at 0 h and 24 h after scratching (left), and the wound closure ratio (cell migration distance at 24 h divided by the gap distance at 0 h) was obtained for comparison (right). Data were representative of three independent experiments and shown as mean ± SD. ** indicates P < 0.01. (C) For transwell assay, cells were added into the upper chamber in serum-free culture medium. The chamber was then placed in a well of 24-well plate that was filled with FBS-containing complete medium. 36 hours later, migrated cells were fixed and stained with DAPI for imaging. The invasion assay was performed similarly except that matrigel coated chambers was used (left). The number of migrated or invaded cells between the two groups were compared (right). Data were representative of three independent experiments and shown as mean ± SD.* indicates P < 0.05, ** indicates P < 0.01.

Article Snippet: Three siRNAs against LRG1 (SR314597) were purchased from OriGene (USA), The siRNA oligo sequences (sense strand) are the following: #1: 5'-GCAACCCGCUUAACAAAUAAUCCTG-3'; #2: 5'-GCUACAUCUAGAAGGCAACAAAUTG-3'; #3: 5'-GCCUAAGCUCCAAGAAUUGCACC-3').

Techniques: Knockdown, Migration, Western Blot, Transfection, Negative Control, Control, Transwell Assay, Transferring, Comparison, Staining, Imaging, Invasion Assay

Journal: Journal of Cancer

Article Title: LRG1 Suppresses Migration and Invasion of Esophageal Squamous Cell Carcinoma by Modulating Epithelial to Mesenchymal Transition

doi: 10.7150/jca.36189

Figure Lengend Snippet: Effect of LRG1 overexpression on ESCC cell migration and invasion. (A) Western blot analysis of LRG1 protein levels in EC109 cells after transfection of LRG1-overexpressing plasmid (pCMV-LRG1) or the empty vector. β-actin was used as loading control. Vector or pCMV-LRG1 transfected cells were starved in serum free medium for 12 h before wound-healing or transwell assay. (B) For wound-healing, cells were manually scratched using a pipette tip, washed and maintained in serum-free culture medium. Pictures were taken at 0 h and 24 h after scratching (left), and the wound closure ratio (cell migration distance at 24 h divided by the gap distance at 0 h) was obtained for comparison (right). Data were representative of three independent experiments and shown as mean ± SD.** indicates P < 0.01. (C) For transwell assay, cells were added into the upper chamber in serum-free culture medium. The chamber was then placed in a well of 24-well plate that was filled with FBS-containing complete medium. 36 hours later, migrated cells were fixed and stained with DAPI for imaging. The invasion assay was performed similarly except that matrigel coated chambers was used (left). The number of migrated or invaded cells between the two groups were compared (right). Data were representative of three independent experiments and shown as mean ± SD.*** indicates P < 0.001.

Article Snippet: Three siRNAs against LRG1 (SR314597) were purchased from OriGene (USA), The siRNA oligo sequences (sense strand) are the following: #1: 5'-GCAACCCGCUUAACAAAUAAUCCTG-3'; #2: 5'-GCUACAUCUAGAAGGCAACAAAUTG-3'; #3: 5'-GCCUAAGCUCCAAGAAUUGCACC-3').

Techniques: Over Expression, Migration, Western Blot, Transfection, Plasmid Preparation, Control, Transwell Assay, Transferring, Comparison, Staining, Imaging, Invasion Assay

Journal: Journal of Cancer

Article Title: LRG1 Suppresses Migration and Invasion of Esophageal Squamous Cell Carcinoma by Modulating Epithelial to Mesenchymal Transition

doi: 10.7150/jca.36189

Figure Lengend Snippet: Effect of recombinant LRG1 on ESCC cell migration and invasion. EC109 cells were treated with recombinant human LRG1 at indicated concentration and starved in serum free medium for 12 h before wound-healing or transwell assay. (A) For wound-healing, cells were manually scratched using a pipette tip, washed and maintained in serum-free culture medium. Pictures were taken at 0 h and 24 h after scratching. (B) For transwell assay, cells were added into the upper chamber in serum-free culture medium. The chamber was then placed in a well of 24-well plate that was filled with FBS-containing complete medium. 36 hours later, migrated cells were fixed and stained with DAPI for imaging. The invasion assay was performed similarly except that matrigel coated chambers was used. Data were representative of three independent experiments.

Article Snippet: Three siRNAs against LRG1 (SR314597) were purchased from OriGene (USA), The siRNA oligo sequences (sense strand) are the following: #1: 5'-GCAACCCGCUUAACAAAUAAUCCTG-3'; #2: 5'-GCUACAUCUAGAAGGCAACAAAUTG-3'; #3: 5'-GCCUAAGCUCCAAGAAUUGCACC-3').

Techniques: Recombinant, Migration, Concentration Assay, Transwell Assay, Transferring, Staining, Imaging, Invasion Assay

Journal: Journal of Cancer

Article Title: LRG1 Suppresses Migration and Invasion of Esophageal Squamous Cell Carcinoma by Modulating Epithelial to Mesenchymal Transition

doi: 10.7150/jca.36189

Figure Lengend Snippet: Effect of LRG1 knockdown on the epithelial to mesenchymal transition (EMT) of ESCC cells. (A) Western blot analysis of protein expression of EMT marker genes upon LRG1 knockdown in KYSE30 cells with siRNA #2 and #3. β-actin was used as loading control. (B) Immunofluorescence staining of E-cadherin (top) and N-cadherin (bottom) in NC or siLRG1 #2 transfected cells. Data were representative of three independent experiments.

Article Snippet: Three siRNAs against LRG1 (SR314597) were purchased from OriGene (USA), The siRNA oligo sequences (sense strand) are the following: #1: 5'-GCAACCCGCUUAACAAAUAAUCCTG-3'; #2: 5'-GCUACAUCUAGAAGGCAACAAAUTG-3'; #3: 5'-GCCUAAGCUCCAAGAAUUGCACC-3').

Techniques: Knockdown, Western Blot, Expressing, Marker, Control, Immunofluorescence, Staining, Transfection

Journal: Journal of Cancer

Article Title: LRG1 Suppresses Migration and Invasion of Esophageal Squamous Cell Carcinoma by Modulating Epithelial to Mesenchymal Transition

doi: 10.7150/jca.36189

Figure Lengend Snippet: Effect of LRG1 overexpression on the epithelial to mesenchymal transition (EMT) of ESCC cells. (A) Western blot analysis of protein expression of EMT marker genes upon LRG1 overexpression in EC109 cells. β-actin was used as loading control. (B) Immunofluorescence staining of E-cadherin (top) and N-cadherin (bottom) in vector or pCMV-LRG1 transfected cells. Data were representative of three independent experiments.

Article Snippet: Three siRNAs against LRG1 (SR314597) were purchased from OriGene (USA), The siRNA oligo sequences (sense strand) are the following: #1: 5'-GCAACCCGCUUAACAAAUAAUCCTG-3'; #2: 5'-GCUACAUCUAGAAGGCAACAAAUTG-3'; #3: 5'-GCCUAAGCUCCAAGAAUUGCACC-3').

Techniques: Over Expression, Western Blot, Expressing, Marker, Control, Immunofluorescence, Staining, Plasmid Preparation, Transfection

Journal: Journal of Cancer

Article Title: LRG1 Suppresses Migration and Invasion of Esophageal Squamous Cell Carcinoma by Modulating Epithelial to Mesenchymal Transition

doi: 10.7150/jca.36189

Figure Lengend Snippet: Manipulation of LRG1 levels modulated the activation of TGFβ pathway. Shown were western blot analysis of protein expression of TGF-β1, SMAD2/3 and phosphorylated SMAD2/3 upon LRG1 knockdown in KYSE30 cells with siRNA #2 and #3 (left), or upon LRG1 overexpression in EC109 cells (right). β-actin was used as loading control. Data were representative of three independent experiments.

Article Snippet: Three siRNAs against LRG1 (SR314597) were purchased from OriGene (USA), The siRNA oligo sequences (sense strand) are the following: #1: 5'-GCAACCCGCUUAACAAAUAAUCCTG-3'; #2: 5'-GCUACAUCUAGAAGGCAACAAAUTG-3'; #3: 5'-GCCUAAGCUCCAAGAAUUGCACC-3').

Techniques: Activation Assay, Western Blot, Expressing, Knockdown, Over Expression, Control