Journal: The Journal of Biological Chemistry

Article Title: Carboxypeptidase M Is a Positive Allosteric Modulator of the Kinin B1 Receptor *

doi: 10.1074/jbc.M113.520791

Figure Lengend Snippet: CPM enhances B1R binding of its agonist at low concentration in primary human endothelial cells. HLMVEC were pretreated with 5 ng/ml IL-1β and 200 units/ml IFN-γ for 16 h (“cytokine-treated”) and then incubated with 50 μm CT peptide or 500 ng/ml CPM monoclonal antibody for 90 min. CPM expression (A) and activity (B) were determined. Inhibition by MGTA (dl-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid) was used as a positive control. C, cytokine-treated HLMVEC were then incubated with 50 μm CT peptide or 500 ng/ml CPM monoclonal antibody and 4 nm [3H]DAKD or 100 nm DAKD (4 nm [3H]DAKD+ 96 nm cold DAKD) for 90 min. Cells were washed, and specific binding was determined as described under “Experimental Procedures.” The data are expressed as % of control binding without CT-peptide or antibody. D and E, HLMVEC were transfected with specific CPM siRNA or nonspecific control (NC) siRNA using an Amaxa nucleofector. After 48 h cells were cytokine-treated as above, and CPM expression (D) and activity (E) were determined as well as the binding of B1R with its agonist, 4 nm [3H]DAKD or 100 nm DAKD (4 nm [3H]DAKD + 96 nm cold DAKD), for 90 min (F). The data in B, C, E, and F are expressed as the mean ± S.E. (n = 3). * = p < 0.05 versus control (Student's t test). The data in A and D are representative of three experiments.

Article Snippet: D and E , HLMVEC were transfected with specific CPM siRNA or nonspecific control ( NC ) siRNA using an Amaxa nucleofector.

Techniques: Binding Assay, Concentration Assay, Incubation, Expressing, Activity Assay, Inhibition, Positive Control, Transfection

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 is correlated with NSCLC metastasis and could promote NSCLC cell migration in vitro . (A) Representative IHC images of PFN1 expression on the NSCLC tissues. (B) The staining index of PFN1 on the tissue chip. ** p < 0.01. (C) Representative IHC images of PFN1 expression on the tissue chip. (D) The expression of PFN1 in TCGA LUAD data. ** p < 0.01. (E) The Kaplan–Meier survival analysis of PFN1 in NSCLC patients. (Data source: TCGA LUAD dataset) (F,G) Wound healing assays conducted to evaluate the migration ability of PFN1 -overexpressing (F) and PFN1 knockdown (KD) (G) H1299 cells. ** p < 0.01; scale bar, 500 μm. (H,I) Transwell migration assays conducted to evaluate the migration of PFN1 -overexpressing (H) and PFN1 KD (I) H1299 cells. ** p < 0.01; scale bar, 500 μm. EV, empty vector; OE, PFN1 overexpression; NC, negative control; si-1/ 2, PFN1 siRNA1 1/2.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Migration, In Vitro, Expressing, Staining, Knockdown, Plasmid Preparation, Over Expression, Negative Control

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 could promote MVs secretion in NSCLC. (A) Heatmap of differentially expressed proteins between EV and PFN1 OE cells. (B) GO enrichment analysis of differentially expressed proteins. (C) COG/KOG analysis of differentially expressed proteins. (D) MVs extracted from EV-expressing and PFN1 -overexpressing cells, using continuous differential centrifugation, identified using transmission electron microscopy. Scale bar, 100 nm. (E,F) Flow cytometry (E) and western blotting (F) were used to quantify MVs in PFN1 -overexpressing and EV-expressing cells. ARF6 and actin were used as MV markers. (G) Expression of PFN1 and annexin A1 in lung tumor tissues detected using immunofluorescence. (H) The staining index of p-MLC on the tissue chip. ** p < 0.01. (I) Representative IHC images of p-MLC expression. (J) Spearman rank correlation analysis was used to assess the relationship between PFN1 and p-MLC expression on the tissue chip; p and r values are shown in the plot.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Expressing, Centrifugation, Transmission Assay, Electron Microscopy, Flow Cytometry, Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: MVs derived from PFN1 OE cells promote migration in NSCLC cells. (A) MVs collected from sera of patients with NSCLC quantified using flow cytometry. ** p < 0.01. (B) Protein expression of ARF6 and β-actin in MVs collected from sera of patients with NSCLC detected using western blotting. (C) Effect of PFN1 -overexpressing cell supernatants on cell migration evaluated through wound healing assays. ** p < 0.01; scale bar, 500 μm. (D) PKH67-labeled MVs taken up by H1299 cells. DAPI was used to stain the nuclei of H1299 cells. Scale bar, 500 μm. (E,F) Wound healing (E) and Transwell migration (F) assays conducted to evaluate the migration of H1299 cells after treatment with MVs derived from EV-expressing and PFN1 -overexpressing cells; ** p < 0.01; scale bar, 500 μm.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Derivative Assay, Migration, Flow Cytometry, Expressing, Western Blot, Labeling, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: PFN1 promotes in vivo NSCLC metastasis by elevating MV secretion. (A) Schematic illustration of the mouse model of metastatic tumor established to determine the role of PFN1 in tumor metastasis. (B) Body weight changes in mice after intracardiac injection of PFN1 -overexpressing and EV-expressing cell lines. (C,D) Representative images of lung (C) and liver (D) metastases of the mouse model. The number of metastases is displayed in the right-hand side graph. * p < 0.05, ** p < 0.01. (E) Representative images of HE-stained lung tissues of the mouse model. (F) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (G) Representative images of HE-stained liver tissues of the mouse model. (H) Representative IHC images of PFN1 and p-MLC expression in liver tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (I) Body weight changes in mice after intracardiac injection of H1299 cells and MVs. (J) Representative images of lung metastases of the mouse model. The number of metastases is shown in the bottom graph. * p < 0.05. (K) Representative images of HE-stained lung tissues of the mouse model. (L) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph; * p < 0.05.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: In Vivo, Injection, Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: Mechanisms underlying the promotion of MLC phosphorylation by PFN1. (A,B) Protein expression after PFN1 overexpression (A) and knockdown (B) measured using western blotting. (C) Protein expression in PFN1 mutants measured using western blotting. (D) PFN1 interactions with ROCK1/2 confirmed using co-IP. (E) Protein expression after treatment with Y27632 (10 µM) measured using western blotting. (F) Effect of PFN1 on ROCK1 activity. ** p < 0.01. (G) Effect of PFN1 on ROCK2 activity. (H) Flow cytometry measuring changes in the amount of MVs after treatment with Y27632; * p < 0.05.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Phospho-proteomics, Expressing, Over Expression, Knockdown, Western Blot, Co-Immunoprecipitation Assay, Activity Assay, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: ROCK1 inhibitor Y27632 partially reversed the promotion of lung cancer metastasis by PFN1 in vitro and in vivo . (A,B) Wound healing assays conducted to evaluate the effect of Y27632 (A) and Y27632 combined with MVs (B) on cell migration. ** p < 0.01; scale bar, 500 μm. (C) Transwell migration assays conducted to evaluate the effect of Y27632 and Y27632 combined with MVs on cell migration. ** p < 0.01; scale bar, 500 μm. (D) Schematic diagram of the mouse model of metastatic tumor established to determine the effect of Y27632 on PFN1-induced lung cancer metastasis. (E) Body weight changes in mice after intracardiac injection of PFN1 -overexpressing H1299 cells and intraperitoneal injection of Y27632 (10 mg/kg). (F) Representative images of lung and liver metastatic tissue in mice. The number of metastatic nodules is shown in the right-hand side graph. * p < 0.05. (G,H) Representative images of HE-stained lung (G) and liver (H) metastases. (I) Representative IHC images of PFN1 and p-MLC expression in lung tissues. The staining index is shown in the right-hand side graph. ** p < 0.01. (J) Representative IHC images of PFN1 and p-MLC expression in liver tissues. The staining index is shown in the right-hand side graph; ** p < 0.01.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: In Vitro, In Vivo, Migration, Injection, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer

doi: 10.3389/fphar.2022.890891

Figure Lengend Snippet: Schematic diagram of the role of PFN1 in NSCLC metastasis. In the initiation stage of NSCLC, cells with upregulated PFN1 secret more MVs through PFN1 interactions with the ROCK/p-MLC pathway. These MVs contain numerous oncogenenic moleculars, which could enhance migration abilities of PFN1 normal expressed NSCLC cells, and untimately promote progression and metastasis of NSCLC.

Article Snippet: PFN1 siRNA and control scramble siRNA were synthesized by Guangzhou RiboBio Co. (Guangzhou, China), and the sequences are listed in .

Techniques: Migration

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Macrophage Activation and Polarization by HCC-Derived Exosomal lncRNA TUC339

doi: 10.3390/ijms19102958

Figure Lengend Snippet: Knockdown of TUC339 in THP-1 cells leads to increased pro-inflammatory cytokine production, increased co-stimulatory molecule expression, enhanced phagocytosis, and reduced viability. THP-1 cells were transfected with siRNAs against TUC339 or unrelated siRNA control. Both ( a ) qRT-PCR and ( b ) Northern blot analysis showed effective knockdown of TUC339 by siRNAs. Upon TUC339 knockdown, IL-1β ( c ) and TNF-α ( d ) mRNAs were elevated in THP-1 cells, which is shown by qRT-PCR. IL-1β ( e ) and TNF-α ( f ) secretion were elevated, which is shown by ELISA. ( g ) CD86 mRNA was elevated as shown by qRT-PCR. ( h ) Phagocytosis was enhanced in LPS challenged THP-1 cells upon TUC339 knockdown. ( i ) Cell viability was reduced in THP-1 cells upon TUC339 knockdown, which is shown by the CCK-8 assay. Data represent mean ± SEM of three independent experiments. * p < 0.05.

Article Snippet: Three independent siRNAs against TUC339 and lincRNA-VLDLR along with unrelated control siRNA (si-ctrl) were purchased from Ribobio (Guangzhou, China).

Techniques: Knockdown, Expressing, Transfection, Control, Quantitative RT-PCR, Northern Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Macrophage Activation and Polarization by HCC-Derived Exosomal lncRNA TUC339

doi: 10.3390/ijms19102958

Figure Lengend Snippet: TUC339 promotes the THP-1 macrophages transition to the M(IL-4) phenotype. THP-1 macrophage cells were cultured in the presence of IFN-γ (20 ng/mL) plus LPS (100 ng/mL) or in IL-4 (20 ng/mL). Polarization-specific biomarkers were analyzed by qRT-PCR assays using RNA collected from macrophages at 18 h post-treatment. CXCL10 ( a ) and CXCL11 ( b ) were elevated in M(IFN-γ + LPS), CCL17 ( c ) and CCL22 ( d ) were elevated in M(IL-4). ( e ) Expression of TUC339 was elevated in M(IL-4) macrophages. ( f ) Differential expression of TUC339 during macrophage re-polarization. ( g – j ) Macrophages were transfected with siRNA-TUC339 or unrelated siRNAs control and then stimulated with IL-4 for M(IL-4) polarization. Knockdown of TUC339 increased the expression of M1 maker CXCL10 ( g ) and CXCL11 ( h ) and decreased expression of the M2 maker CCL17 ( i ) and CCL22 ( j ). Data are representative of three separate experiments and show the means ± SEM. * p < 0.05.

Article Snippet: Three independent siRNAs against TUC339 and lincRNA-VLDLR along with unrelated control siRNA (si-ctrl) were purchased from Ribobio (Guangzhou, China).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Quantitative Proteomics, Transfection, Control, Knockdown