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: Identification of Tomato microRNAs in Late Response to Trichoderma atroviride

doi: 10.3390/ijms25031617

Figure Lengend Snippet: General analysis in Trichoderma atroviride T11-treated (T11) Solanum lycopersicum or untreated (C) plants libraries. ( A ) Length distribution and abundance of the miRNAs that showed at least 10 read counts against their miRNA* sequence in S. lycopersicum plants treated (T11) or not (C). The percentage is calculated over the total number of reads that aligned to the miRNA candidates identified in plants from both conditions (including those with less than 10 read counts against their miRNA* sequence and/or those that were solely transcribed in one sample per condition). The length of the miRNAs identified ranged from 20–24 nt; ( B ) Venn diagram showing the number of miRNAs (that displayed at least 10 read counts against their miRNA* sequence) identified in both conditions, solely in T11 (green) or in untreated control plants (blue).

Article Snippet: Quality control of the sequencing data and the identification of known and novel miRNA candidates were performed by Sequentia Biotech S.L. (Barcelona, Spain) as follows.

Techniques: Sequencing, Control

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Basic characterization of the G-quadruplex motifs under investigation. ( A ) and ( B ) CD spectra and melting curves of the G-quadruplex motifs and their mutated control oligonucleotides (A: MMP16; B: ARPC2). ( C ) Melting temperature at different concentrations of the ARPC2 G-quadruplex. All melting experiments were carried out at 100 mM KCl, except for the MMP16 G-quadruplex-forming sequence, since it did not unfold in the measured temperature range. D) RT-PCR experiments to confirm expression of MMP16 and ARPC2 in HEK293 cells. Additional experiments were carried out with HeLa cells. Control experiments without reverse transcription are shown (–).

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Circular Dichroism, Control, Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Reverse Transcription

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Inhibition of translation by the G-quadruplex motifs of MMP16 and ARPC2, as determined by dual luciferase assays. ( A ) Dual luciferase assays for the isolated G-quadruplex motifs and ( B ) for the full-length 5′-UTR of ARPC2. The ratio of Renilla and firefly luciferase activity is normalized to the value of the empty psiCHECK-2 vector. The G-quadruplex motif in the 5′-UTR of Renilla luciferase reduces enzyme synthesis. Values given are averages ± standard deviations of three experiments.

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Inhibition, Luciferase, Isolation, Activity Assay, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Pull-down assays. ( A ) and ( B ) G-quadruplex motifs of the MMP16 (A) and ARPC2 (B) mRNAs were coupled to streptavidin agarose beads. Whole-cell extract of HEK293 cells was added to the beads. After several washing steps, proteins were eluted with buffers containing the indicated K + -concentrations. Proteins were analyzed by SDS-PAGE. Proteins that bind exclusively to the G-quadruplex-sequence (Q) are indicated. GQ-M represents an independent experiment, in which the HEK293 extract was first incubated with the mutated oligonucleotide and then with the G-quadruplex-forming RNA. The indicated proteins were identified by MS-based peptide mass fingerprinting. ( C ) Pull-down assay with the additional ARPC2 control oligonucleotide with two G-stretches (ARPC2–2xG). 1. ME2 (66 kDa); 2. YB-1 (36 kDa); 3. U2AF65 (54 kDa); 4. hnRNPH (50 kDa); 5. YB-1 (36 kDa) hnRNPF (46 kDa); 6. RPS2 (32 kDa); 7. Nucl (76 kDa); 8. RBM14 (70 kDa); 9. SRSF1 (28 kDa); 10. SRSF1 (28 kDa) RPS6 (29 kDa); 11. RPL7 (29 kDa); 12. SRSF9 (26 kDa), RPS6 (29 kDa); 13. SRSF9 (26 kDa), RPL14 (23,5 kDa); 14. RPL10 (25 kDa) RPS9 (23 kDa); 15. RPL26 (17 kDa); 16. RPL27a (17 kDa); 17. RPS9 (23 kDa); a. Actin (42 kDa); b. hnRNPA3 (39 kDa); c. hnRNPA2B1 (37 kDa) and hnRNPA3 (39 kDa); d. hnRNPA2B1 (37 kDa); e. hnRNPA1 (39 kDa); f. YB-1 (36 kDA); g. YB-1/Actin (36 kDa, 42 kDa).

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: SDS Page, Sequencing, Incubation, Peptide Mass Fingerprinting, Pull Down Assay, Control

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Proteins identified to bind to the G-quadruplex motifs of the ARPC2 and MMP16 mRNA

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Protein Binding, RNA Binding Assay

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Association ( k a ) and dissociation ( k d ) rates and dissociation constants (K D ) determined by SPR spectroscopy; n. d.: not determined due to too weak interactions

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Spectroscopy

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Analysis of protein-RNA interactions by SPR spectroscopy. ( A ) Binding parameters for Δnucleolin to the ARPC2 and MMP16 G-quadruplex and the mutated sequence were analyzed in the concentration range of 1–20 nM and 10–1000 nM, respectively. Data were fitted to 1:1 binding with mass transfer. Sensorgrams are shown in gray and the respective fits in black. ( B ) Binding of U2AF65 to 100 nM of the ARPC2 and MMP16 G-quadruplex oligonucleotides and experiments with the respective controls. ( C ) Additional experiments with the G-rich control oligonucleotides ARPC2 2xG in comparison to the G-quadruplex and the exhaustively mutated oligonucleotide.

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Spectroscopy, Binding Assay, Sequencing, Concentration Assay, Control, Comparison

Journal: Nucleic Acids Research

Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

doi: 10.1093/nar/gku290

Figure Lengend Snippet: Analysis of protein-RNA interactions by SPR spectroscopy. Binding of SRSF1 ( A ) and EFHD2 ( B ) to 100 nM of the G-quadruplex and mutated control sequences of ARPC2 and MMP16.

Article Snippet: The G-quadruplex-forming sequences of the MMP16 and ARPC2 RNAs and their mutated controls were purchased from Purimex GmbH, Grebenstein, Germany: MMP16 GQ, AACGA GGGAGGGAGGGAGAGGG AGAGA-biotin MMP16 mut, AACGAGAGAGAGAGAGAGAGAGAGAGA-biotin ARPC2 GQ, AGCC GGGGGCUGGGCGGGGACCG GG CUUGU-biotin ARPC2 mut, AGCCGUAGACUGAGCGAAGACCGAGCUUGU-biotin ARPC2–2xG, AGCCGUAGACUGGGCGAAGACCGGGCUUGU-biotin.

Techniques: Spectroscopy, Binding Assay, Control