Journal: BioMed Research International

Article Title: LRG1 May Accelerate the Progression of ccRCC via the TGF- β Pathway

doi: 10.1155/2020/1285068

Figure Lengend Snippet: The differential expression of LRG1 in ccRCC patients. (a) LRG1 is upregulated in primary tumor ( p < 0.001). (b) LRG1 expression of male patients is significantly different from male and normal contols ( p < 0.001). (c) LRG1 expression was significantly different between the normal control group and the 41-60 ( p = 0.0024) and 61-80-year-old subgroups ( p < 0.0001). (d) Caucasian group is the most differential expressed group compared with normal controls ( p < 0.0001). (e) LRG1 expression levels were significantly different between the normal control group and different ccRCC stage subgroups ( p < 0.0001). (f) All grades except grade 1 were significantly different from that in normal controls ( p < 0.05). ∗ ccRCC: clear cell renal cell carcinoma; TPM: transcript per million.

Article Snippet: The cytokines used in the study were carrier-free recombinant human LRG1 (R&D, 7890-LR).

Techniques: Quantitative Proteomics, Expressing, Control

Journal: BioMed Research International

Article Title: LRG1 May Accelerate the Progression of ccRCC via the TGF- β Pathway

doi: 10.1155/2020/1285068

Figure Lengend Snippet: Survival curves and differential methylation and expression of LRG1 promoter in ccRCC patients. (a) Low LRG1 expression indicates a prolonged patient survival time. The FPKM cutoff value of high expression and low expression is 1.19. (b) The methylation level of LRG1 gene has a strong negative correlation (corr = 0.677) with the expression of LRG1.

Article Snippet: The cytokines used in the study were carrier-free recombinant human LRG1 (R&D, 7890-LR).

Techniques: Methylation, Expressing

Journal: BioMed Research International

Article Title: LRG1 May Accelerate the Progression of ccRCC via the TGF- β Pathway

doi: 10.1155/2020/1285068

Figure Lengend Snippet: Promoter methylation level of LRG1 gene in ccRCC and subgroups. (a) Promoter methylation level of LRG1 gene is significantly downregulated compared with normal controls. (b) Methylation level of LRG1 gene in male and female patients is decreased compared with normal patients ( p < 0.0001). (c) Methylation level of LRG1 gene in different ages has significant differences compared with normal controls ( p < 0.0001). (d) Methylation level of LRG1 gene in different races has significant differences compared with normal controls ( p < 0.001). (e) Methylation level of all of the stages are downregulated than normal controls ( p < 0.001). (f) Methylation level of all of the grads are downregulated than normal controls ( p < 0.0001). (g) Methylation level of metastatic ccRCC is lower than nonmetastatic, but both of them are significantly downregulated than normal controls ( p < 0.001).

Article Snippet: The cytokines used in the study were carrier-free recombinant human LRG1 (R&D, 7890-LR).

Techniques: Methylation

Journal: BioMed Research International

Article Title: LRG1 May Accelerate the Progression of ccRCC via the TGF- β Pathway

doi: 10.1155/2020/1285068

Figure Lengend Snippet: Expression and methylation level in carcinoma and paracarcinoma samples of ccRCC. (a) mRNA expression of LRG1 in carcinoma and paracarcinoma tissues ( p < 0.01, n = 6) detected by qPCR. (b, c) Protein expression of LRG1 in carcinoma and paracarcinoma tissues ( p < 0.01, n = 6) detected by western blot. (d) LRG1 methylation level of CpG1 and CpG2 is downregulated in ccRCC tissue ( p < 0.0001).

Article Snippet: The cytokines used in the study were carrier-free recombinant human LRG1 (R&D, 7890-LR).

Techniques: Expressing, Methylation, Western Blot

Journal: BioMed Research International

Article Title: LRG1 May Accelerate the Progression of ccRCC via the TGF- β Pathway

doi: 10.1155/2020/1285068

Figure Lengend Snippet: Expression of TGFB1 in ccRCC patients. (a) Expression of TGFB1 in ccRCC patients is upregulated ( p < 0.001). (b) TGFB1 is upregulated in 789-O cells after stimulated with LRG1 ( p < 0.001) but not with heat-denatured LRG1. (c) TGFB1 is downregulated in 789-O cells after LRG1 knockdown ( p < 0.001).

Article Snippet: The cytokines used in the study were carrier-free recombinant human LRG1 (R&D, 7890-LR).

Techniques: Expressing, Knockdown

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: bioRxiv

Article Title: LRG1 is a novel ligand of HER3 and promotes metastatic colorectal cancer growth

doi: 10.1101/2023.02.18.529070

Figure Lengend Snippet: (A) Schematics of CM fractionation by FPLC. (B) Western blotting showed P-HER3 levels in CRC cells after 30 minutes treating with EC-9 CM fractions. (C) Schematics of HER3-ECD with His tags pulling down soluble factors from EC-9 CM. Green, HER3-ECD. Red, EC-secreted factor(s) that bind to HER3-ECD. (D) Western blotting showed P-HER3 levels in CRC cells after 30 minutes treating with EC-9 CM after beads or ECD depletion, or with soluble factors eluted from beads or ECD. Ctrl beads: Ni-NAT beads only without ECD. Ctrl ECD: HER2-ECD. (E) The MTT assay showed CRC cell viability after 72 hours incubating with CM. (F) Western blotting showed P-HER3 levels in CRC cells after 30 minutes treating with LRG1 peptides. (G) The MTT assay showed CRC cell viability after 72 hours treating with CM or LRG1 (250 ng/ml). (H) Western blotting showed soluble LRG1 in CM from EC-1 and -9 cells. (I) Western blotting showed LRG1 pulled down by HER3-ECD. (J) SPR assay determined LRG1 and HER3-ECD binding affinity. K D =K on /K off , 1:1 fit model. In (E) and (G), data are shown as Mean +/-SEM, ***p<0.001, **p<0.01, *p<0.05 by unpaired two-tailed t -test.

Article Snippet: For LRG1 immuno-depletion by antibodies, 5 μg control IgG or anti-LRG1 antibodies (both from R&D Systems) were added to 500 μ l of 3X CM (1000 μ g total proteins) from un-modified ECs After rocking overnight at 4 ° C, a 50 μ l mixture of protein A/G PLUS-Agarose beads was added to CM and rocked at 4°C for 1 hour.

Techniques: Fractionation, Western Blot, MTT Assay, SPR Assay, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: LRG1 is a novel ligand of HER3 and promotes metastatic colorectal cancer growth

doi: 10.1101/2023.02.18.529070

Figure Lengend Snippet: (A) Western blotting showed P-HER3 levels in CRC cells after 30 minutes treating with EC-1 CM with or without beads or ECD depletion, or with soluble factors eluted from beads or ECD. (B) Western blotting showed P-HER3 and P-SMAD3 levels in CRC cells with functional TGFβ receptors after 30 minutes treating with LRG1 or TGFβ peptides.

Article Snippet: For LRG1 immuno-depletion by antibodies, 5 μg control IgG or anti-LRG1 antibodies (both from R&D Systems) were added to 500 μ l of 3X CM (1000 μ g total proteins) from un-modified ECs After rocking overnight at 4 ° C, a 50 μ l mixture of protein A/G PLUS-Agarose beads was added to CM and rocked at 4°C for 1 hour.

Techniques: Western Blot, Functional Assay

Journal: bioRxiv

Article Title: LRG1 is a novel ligand of HER3 and promotes metastatic colorectal cancer growth

doi: 10.1101/2023.02.18.529070

Figure Lengend Snippet: (A) Western blotting showed LRG1 levels in CM from ECs with control or LRG1-specific siRNAs. (B) Western blotting showed P-HER3 levels in CRC cells after 30 minutes treating with CM from CRC, or from EC-9 with control or LRG1-specific siRNAs. (C) The MTT assay showed CRC cell viability after 72 hours incubating with CM. (D) Western blotting showed LRG1 levels on pull-down beads with control or LRG1 antibodies, or in EC-9 CM after immuno-depletion by control or LRG1 antibodies. (E) Western blotting showed P-HER3 levels in CRC cells after 30 minutes treating with CRC CM or EC-9 CM after immuno-depletion by control or LRG1 antibodies. (F) The MTT assay showed CRC cell viability after 72 hours incubating with CM. (G) Tumor volumes of subQ xenografts treated by CRC CM, complete EC-9 CM, or LRG1-depleted EC-9 CM (n=8 mice/group). For (C) and (F), data are shown as Mean +/-SEM, **p<0.01, *p<0.05 by unpaired two-tailed t -test. For (G), data are Mean -/+ SD, **p<0.01, *p<0.05 by Wilcoxon rank-sum test.

Article Snippet: For LRG1 immuno-depletion by antibodies, 5 μg control IgG or anti-LRG1 antibodies (both from R&D Systems) were added to 500 μ l of 3X CM (1000 μ g total proteins) from un-modified ECs After rocking overnight at 4 ° C, a 50 μ l mixture of protein A/G PLUS-Agarose beads was added to CM and rocked at 4°C for 1 hour.

Techniques: Western Blot, Control, MTT Assay, Immunodepletion, Two Tailed Test

Journal: bioRxiv

Article Title: LRG1 is a novel ligand of HER3 and promotes metastatic colorectal cancer growth

doi: 10.1101/2023.02.18.529070

Figure Lengend Snippet: (A) Kaplan-Meier curve for overall survival of mice with MC-38 mCRC allografts in the liver of WT mice and LRG1 KO mice (n=5 mice/group). (B) Western blotting showed P-HER3 levels in CRC cells after 30 minutes treating with CM and the LRG1 neutralizing antibody 15C4. (C) The MTT assay showed CRC cell viability after 72 hours incubating with CM and 15C4. (D) HCP-1 subQ xenograft mean volume over time, and images and weights of harvested subQ xenograft from mice treated with CM and 15C4 (n=10 mice/group). (E) Representative images of P-HER3 (Y1289) IHC staining of HCP-1 xenografts. For (A) and (D), data are Mean -/+ SD, **p<0.01, *p<0.05 by Wilcoxon rank-sum test. For (C), data are shown as Mean +/-SEM, ***p<0.001, **p<0.01, *p<0.05 by unpaired two-tailed t -test.

Article Snippet: For LRG1 immuno-depletion by antibodies, 5 μg control IgG or anti-LRG1 antibodies (both from R&D Systems) were added to 500 μ l of 3X CM (1000 μ g total proteins) from un-modified ECs After rocking overnight at 4 ° C, a 50 μ l mixture of protein A/G PLUS-Agarose beads was added to CM and rocked at 4°C for 1 hour.

Techniques: Western Blot, MTT Assay, Immunohistochemistry, Two Tailed Test

Journal: bioRxiv

Article Title: LRG1 is a novel ligand of HER3 and promotes metastatic colorectal cancer growth

doi: 10.1101/2023.02.18.529070

Figure Lengend Snippet: (A, B) Principal component analysis and volcano plot were used for clustering of identified serine- and threonine-protein phosphorylation in CRC cells after 30 minutes culturing in CRC or EC-9 CM. (C) Western blotting showed P-eIF4B levels in CRC cells with or without siRNA of HER3 and after 30 minutes treating with or without LRG1. (D) Western blotting showed P-eIF4B levels in CRC cells after 30 minutes treating with CM and with or without the LRG1 neutralizing antibody 15C4. (E) The global protein synthesis assay showed the levels of de novo protein synthesis in CRC cells after 24 hours incubation with CM and 15C4. (F) The MTT assay showed CRC cell viability after 72 hours incubating with CM and an eIF4 inhibitor. (G) Representative images and quantifications of P-eIF4 IHC staining of HCP-1 xenografts treated by CM from CRC or EC-9, and with/without 15C4. For (E) and (F), data are shown as Mean +/-SEM, ***p<0.001, **p<0.01, *p<0.05.

Article Snippet: For LRG1 immuno-depletion by antibodies, 5 μg control IgG or anti-LRG1 antibodies (both from R&D Systems) were added to 500 μ l of 3X CM (1000 μ g total proteins) from un-modified ECs After rocking overnight at 4 ° C, a 50 μ l mixture of protein A/G PLUS-Agarose beads was added to CM and rocked at 4°C for 1 hour.

Techniques: Phospho-proteomics, Western Blot, Incubation, MTT Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: LRG1 is a novel ligand of HER3 and promotes metastatic colorectal cancer growth

doi: 10.1101/2023.02.18.529070

Figure Lengend Snippet: (A) Western blotting showed P-HER3, P-eIF4B, and P-AKT levels in CRC cells after 30 minutes treating with either LRG1 or neuregulins (NRGs). (B) Western blotting showed P-eIF4B levels in CRC cells after 30 minutes treating with LRG1 and different doses of AKT inhibitors (MK-2206). (C) Western blotting showed P-eIF4B levels in CRC cells after 30 minutes treating with LRG1 and different doses of IRS inhibitors (NT157), p110α inhibitors (BYL719), PDK1 inhibitors (BX795), and RSK inhibitors (BI-D1870). (D) The global protein synthesis assay showed the levels of de novo protein synthesis in CRC cells after 24 hours incubation with LRG1 and specific inhibitors. (E) Schematics of the signaling pathway. For (D), data are shown as Mean +/-SEM, **p<0.01, by unpaired two-tailed t -test.

Article Snippet: For LRG1 immuno-depletion by antibodies, 5 μg control IgG or anti-LRG1 antibodies (both from R&D Systems) were added to 500 μ l of 3X CM (1000 μ g total proteins) from un-modified ECs After rocking overnight at 4 ° C, a 50 μ l mixture of protein A/G PLUS-Agarose beads was added to CM and rocked at 4°C for 1 hour.

Techniques: Western Blot, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: LRG1 is a novel ligand of HER3 and promotes metastatic colorectal cancer growth

doi: 10.1101/2023.02.18.529070

Figure Lengend Snippet: (A) Western blotting showed P-eIF4B levels in HCT116 cells after 30 minutes treating with LRG1 and different doses of IRS inhibitors (NT157), p110α inhibitors (BYL719), PDK1 inhibitors (BX795), and RSK inhibitors (BI-D1870). (B) Western blotting showed P-HER3 and P-eIF4B levels in CRC cells after 30 minutes treating with LRG1 and different doses of p110β inhibitors (TGX-221). (C) The global protein synthesis assay showed the levels of de novo protein synthesis in CRC cells after 24 hours incubation with LRG1 and p110β inhibitors, mean +/-SEM, **p<0.01, by unpaired two-tailed t -test.

Article Snippet: For LRG1 immuno-depletion by antibodies, 5 μg control IgG or anti-LRG1 antibodies (both from R&D Systems) were added to 500 μ l of 3X CM (1000 μ g total proteins) from un-modified ECs After rocking overnight at 4 ° C, a 50 μ l mixture of protein A/G PLUS-Agarose beads was added to CM and rocked at 4°C for 1 hour.

Techniques: Western Blot, Incubation, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: LRG1 modulates epithelial-mesenchymal transition and angiogenesis in colorectal cancer via HIF-1α activation

doi: 10.1186/s13046-016-0306-2

Figure Lengend Snippet: LRG1 was overexpressed in CRC tissues. a Overexpression of LRG1 mRNA in CRC tissues than normal tissues based on the microarray data from GSE20916 and GSE20842. b RT-PCR analysis of LRG1 mRNA expression in 30 pairs of CRC and adjacent non-tumor tissues. The expression levels of LRG1 were normalized to those of β-actin ( P < 0.0001, paired t test). c Representative immunohistochemical staining of LRG1 in human samples of normal tissue, early CRC tissue, and advanced CRC tissue (Original magnification: ×200 and 400×). Scale bar represents 50 μm

Article Snippet: Recombinant LRG1 was purchased from R&D Systems and added into the culture medium at concentration of 50–1000 ng/ml for indicated time before harvesting.

Techniques: Over Expression, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunohistochemical staining, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: LRG1 modulates epithelial-mesenchymal transition and angiogenesis in colorectal cancer via HIF-1α activation

doi: 10.1186/s13046-016-0306-2

Figure Lengend Snippet: Correlation between LRG1 expression and clinicopathological parameters of CRCs

Article Snippet: Recombinant LRG1 was purchased from R&D Systems and added into the culture medium at concentration of 50–1000 ng/ml for indicated time before harvesting.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: LRG1 modulates epithelial-mesenchymal transition and angiogenesis in colorectal cancer via HIF-1α activation

doi: 10.1186/s13046-016-0306-2

Figure Lengend Snippet: Knockdown of LRG1 repressed the invasion capacity of CRC cells. a RT-PCR and Western blot analyses showed effective downregulation of LRG1 following siRNA transfection. b HCT116 and SW480 cells transfected with LRG1 siRNA or control siRNA were seeded in the upper chamber. After 48 h, the cells invaded through the membrane were stained and counted in five random microscopic fields (200×). c LRG1- and control-siRNA transfected HCT116 and SW480 cells were wounded with pipette and wound closure percentage was quantified 48 h after scratch relative to that at 0 h. * P < 0.05 compared to the control cells. Scale bar represents 100 μm

Article Snippet: Recombinant LRG1 was purchased from R&D Systems and added into the culture medium at concentration of 50–1000 ng/ml for indicated time before harvesting.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Control, Membrane, Staining, Transferring

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: LRG1 modulates epithelial-mesenchymal transition and angiogenesis in colorectal cancer via HIF-1α activation

doi: 10.1186/s13046-016-0306-2

Figure Lengend Snippet: Knockdown of LRG1 prevents the mesenchymal transition. a and b Expression of EMT-associated markers in CRC cells with LRG1 siRNA or control siRNA were measured by RT-PCR and western blot assays. * P < 0.05 compared to the control cells

Article Snippet: Recombinant LRG1 was purchased from R&D Systems and added into the culture medium at concentration of 50–1000 ng/ml for indicated time before harvesting.

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

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: LRG1 modulates epithelial-mesenchymal transition and angiogenesis in colorectal cancer via HIF-1α activation

doi: 10.1186/s13046-016-0306-2

Figure Lengend Snippet: LRG1 promoted VEGF-A expression and angiogenesis. a HCT116 and SW480 cells were stimulated with rLRG1 (0–1000 ng/ml) for 24 h, and VEGF-A expression was quantified by RT-PCR and ELISA. b CRC cells were stimulated with rLRG1 (500 ng/ml) for indicated hours, and VEGF-A expression was quantified by RT-PCR and ELISA. c and d The medium of HCT116 cells incubated in the presence or absence of LRG1 (500 ng/ml) and LRG1 antibody (10 μg/ml) was collected and then applied to HUVECs for transwell migration assay and tube formation assay. e Effect of CM from LRG1-induced HCT116 cells on the aortic ring sprouting assay. Representative photos were shown and data were summarized from 3 independent experiments. *Compared to the control group, P < 0.05. Scale bar represents 100 μm

Article Snippet: Recombinant LRG1 was purchased from R&D Systems and added into the culture medium at concentration of 50–1000 ng/ml for indicated time before harvesting.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Transwell Migration Assay, Tube Formation Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: LRG1 modulates epithelial-mesenchymal transition and angiogenesis in colorectal cancer via HIF-1α activation

doi: 10.1186/s13046-016-0306-2

Figure Lengend Snippet: The role of HIF-1α in LRG1-induced EMT and VEGF-A expression. a HIF-1α mRNA expression in HCT116 and SW480 cells stimulated with indicated concentrations of LRG1 for 24 h was measured by RT-PCR. β-actin was used as the internal control. b The protein levels of HIF-1α, Twist1, E-cadherin, and N-cadherin in response to LRG1 in SW480 cells were measured by western blot. c HIF-1α expression was knocked down in siRNA-transfected SW480 cells at both mRNA and protein levels. d SW480 cells were transfected with HIF-1α or control siRNA for 24 h, and treated with or without rLRG1 (500 ng/ml) for additional 24 h. Secretion of VEGF-A from SW480 cells was quantified by ELISA. e HIF-1α, VEGF-A, E-cadherin, N-cadherin and Twist1 expressions were detected by western blot. f SW480 cells were harvested and applied for transwell invasion assays. The numbers of invasive cells (five random fields) were expressed as means ± SEM of three independent experiments. *Compared to the control group, P < 0.05. Scale bar represents 100 μm

Article Snippet: Recombinant LRG1 was purchased from R&D Systems and added into the culture medium at concentration of 50–1000 ng/ml for indicated time before harvesting.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay