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: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Boxshade alignment of LDLa module sequences from RXFP1 receptors from various mammalian species in comparison with the sequence of LB2 of the human LDLr. The conserved cysteine residues are highlighted in red, conserved amino acids are highlighted in black, and conservative amino acid substitutions are shaded.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: RXFP1 variants and their corresponding mutation as well as the template DNA and primers used in QuikChange site-directed mutagenesis

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Mutagenesis, Construct, Sequencing, Residue

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: H2 relaxin-induced cAMP response of RXFP1-LB2 (A) and G12del-RXFP1-LB2 and S34A-RXFP1-LB2 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars (not visible) represent S.E. of triplicate determinations from at least three independent experiments.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Pooled activity data for RXFP1 mutants compared with wild type receptor Competition binding data showing the pIC 50 values, receptor expression data showing the cell surface expression, and cAMP activity data showing the pEC 50 values as well as percent forskolin-stimulated maximum responses of RXFP1 and all its variants used in this study. NA, no activity.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Binding Assay, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: H2 relaxin-induced cAMP response of SLGYFP-RXFP1-LB2, NITK-RXFP1-LB2, LLH-RXFP1-LB2, and NGVD-RXFP1-LB2 (A) and SLGYFP-RXFP1-LB2, NITK-RXFP1-LB2, SLGYFP NITK-RXFP1-LB2, and SLGYFP NITK C-term-RXFP1-LB2 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars (not visible) represent S.E. of triplicate determinations from at least three independent experiments.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: 1H,15N HSQC of 1 mm13C,15N-labeled SLGYFP NITK-LB2 in 50 mm imidazole, 10 mm CaCl2, 10% D2O, pH 6. 0 and 25 °C acquired at 800 MHz. Assignments of observed peptide and side chain NH resonances are indicated. Numbering is based on the chimeric LB2 module in Protein Data Bank code 2M7P.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Labeling

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Input for the structure calculation and characterization of the bundle of 20 CYANA conformers of SLYFP NITK-LB2 (residues 1–42) ali, aliphatic; aro, aromatic.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: A, ensemble of the 20 lowest energy structures of SLYFP NITK-LB2. Structures are represented as an overlay of the backbone structures in MOLMOL that overlay with an r.m.s.d. of 0.35 Å between residues 6 and 42. The side chains from the two “add-back” regions SLYFP and NITK have been represented as lines. B, overlay of SLYFP NITK-LB2 (blue) onto the structure of RXFP1 LDLa (orange) (Protein Data Bank code 2JM4). The side chains of Leu-7, Tyr-9, and Lys-17 overlay with good agreement. The overall r.m.s.d. of the two structures between residues 6 and 42 is 1.68 Å.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Overlay of the mean structure of SLYFP NITK-LB2 (blue), RXFP1 LDLa (orange), and LB2 (pink) represented as a cartoon model. The side chains of Lys-17 and equivalent Arg in LB2 are represented as sticks in addition to Asn-33 (RXFP1) and the equivalent Asn-32 from LB2. The overlay demonstrates that the chimera is more “RXFP1”-like despite the LB2 scaffold.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: