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: PLoS ONE

Article Title: Genistein Affects Histone Modifications on Dickkopf-Related Protein 1 (DKK1) Gene in SW480 Human Colon Cancer Cell Line

doi: 10.1371/journal.pone.0040955

Figure Lengend Snippet: pCMV vector containing human DKK1 gene was used to transfect cells before sample collection for analysis. Empty pCMV vector was used as the control for the overexpression experiments. Three independent cell samples were analyzed and presented as the mean ± SEM. A ) mRNA expression of DKK1 and Cyclin D1 in regular and DKK1 -transfected SW480 cells. The mRNA expression was analyzed by RT-PCR. Data were normalized to internal control L7a. B ) DKK1 protein expression in regular cells and transfected SW480 cells. Whole cell protein extracts were collected from transfected SW480 cells and western blot analysis of DKK1 was performed as described in materials and methods. A representative blot is shown and the quantification represents the mean± SEM from 3 independent dishes. Actin was used as the loading control. C ) Cell cycle analysis of regular and DKK1 -transfected SW480 cells. Result was obtained by flow cytometry. Y-axis represents % of gated cells and x-axis shows different cell cycle stages, including G1, S and G2/M. D ) WST-1 proliferation assay in vector- and DKK1 -transfected SW480 cells. WST-1 signals were converted to actual cell numbers using a standard generated by serial dilutions of a known number of cells. Data were normalized to control for genistein treatment and vector for DKK1 transfection, respectively. Asterisks (*) indicate statistical significance compared with the respective control group (genistein to control; DKK1 to vector; p <0.05). For the knockdown of DKK1, siRNA duplexes against human DKK1 gene were transfected into SW480 cells. Scrambled sequences were used as the control siRNA. E ) mRNA expression of DKK1 and Cyclin D1 in control siRNA (N/S si) and DKK1 siRNA (DKK1 si)-treated SW480 cells by control and genistein treatments. The mRNA expression was analyzed by RT-PCR. Data were normalized to internal control L7a. F ) WST-1 proliferation assay in control siRNA and DKK1 siRNA SW480 cells. WST-1 signals were converted to actual cell numbers using a standard generated by serial dilutions of a known number of cells. Asterisks (*) indicate statistical significance compared to Control 0 µmol/L genistein ( p <0.05). The bracket indicates statistical difference between the N/S si and DKK1 si groups treated with genistein (p = 0.002).

Article Snippet: At the time of transfection, 300 nmol/L of siRNA against human scramble sequence (N/S si, QIAGEN, Valencia, CA) or 300 nmol/L of siRNA against human DKK1 ( DKK1 si, Integrated DNA Technologies) was prepared in 200 μL serum-free media (SFM).

Techniques: Plasmid Preparation, Control, Over Expression, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Cycle Assay, Flow Cytometry, Proliferation Assay, Generated, Knockdown

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of the levels of ncRNAs in different models of HD.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Transfection

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of protein and miRNA interactions of ncRNAs Meg3, Neat1, and Xist.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Expression of genes in HD associated with Huntington’s disease pathway (KEGG: 05016 and PANTHER: {"type":"entrez-protein","attrs":{"text":"P00029","term_id":"124076962","term_text":"P00029"}} P00029 ) and coded for protein interacting partners of NEAT1 or MEG3.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Expressing

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of co-expressed genes of MEG3, NEAT1, and XIST.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: MEG3, NEAT1 and XIST interacting protein enriched with HD pathway.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Transcription factors that bind within 5 Kb upstream sequences of NEAT1, MEG3, and XIST.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Sequencing

Journal: Nature Communications

Article Title: Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1

doi: 10.1038/ncomms5589

Figure Lengend Snippet: ( a ) HEK293 cells expressing SEP-CB1Rs were exposed to 10μM CP55940, ORG27569, WIN 55212-2 and 2-AG for 5, 10 and 15 min. Cell lysates were analysed using western blots with phospho-ERK1/2 (p-ERK1/2) or total ERK1/2. ( b ) Quantified time course in a showing ERK1/2 phosphorylation levels induced by 10 μM of each compound. ( c ) HEK293 cells expressing SEP-CB1Rs were exposed to 10μM CP55940, ORG27569, WIN 55212-2 and 2-AG for 5, 10 and 15 min with PTX pre-treatment, respectively. ( d ) Quantified time course in c showing ERK1/2 phosphorylation levels from cells pretreated with PTX. ( e , g , i , k ) HEK293 cells co-expressing SEP-CB1R and either control ( e ), β-arrestin-1 ( g ), β-arrestin-2 ( i ) siRNAs or β-arrestin-2 siRNA with PTX pretreatment ( k ) were exposed to 10 μM of CP55940, ORG27569, WIN 55212-2 and 2-AG, respectively, as indicated. ( f , h , j , l ). Graphs in f , h , j , l provide the quantified ERK1/2 phosphorylation levels induced by 10 μM of each compound as shown in e , g , i , k . Data represent the mean±s.e.m. of at least three independent experiments.

Article Snippet: Briefly, HEK293 cells that were 40–50% confluent in a six-well plate were transfected with the plasmid encoding rat CB1 and 2.6 mg of siRNA (Qiagen) sequences targeting β-arrestin 1, β-arrestin 2 or non-silencing RNA duplex for control.

Techniques: Expressing, Western Blot, Phospho-proteomics, Control

Journal: Nature Communications

Article Title: Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1

doi: 10.1038/ncomms5589

Figure Lengend Snippet: ( a ) HEK293 cells expressing SEP-CB1R were preincubated with 30μM dyngo-4a and imaged under TIRF before and after bath application of 10μM WIN55212-2. Representative kymograph shows prolonged SEP-CB1R dwell times in the presence of dyngo-4a. ( b ) HEK293 cells expressing SEP-CB1R were pre-incubated with 30μM dyngo-4a before exposure to agonists and compared with no dyngo-4a treatment. ( c ) Graph provides the quantified ERK1/2 phosphorylation induced by 10 μM of each compound shown in b . Data are expressed as a percentage of the level of phosphorylation at 5 min for each compound without dyngo-4a pre-treatment. ( d ) HEK293 cells expressing SEP-CB1Rs with and without dyngo-4a pretreatment were exposed to 10μM 2-AG as indicated with PTX pre-treatment. ( e ) HEK293 cells co-expressing SEP-CB1R and β-arrestin-1 siRNAs with and without dyngo-4a pretreatment were exposed to 10μM 2-AG as indicated. ( f ) Quantified time course in d , e showing ERK1/2 phosphorylation levels induced by 10μM 2-AG for 5, 15, 30, 45 and 60 min for cells either pre-treated with PTX or co-transfected with β-arrestin-1 siRNA. ( g ) HEK293 cells expressing SEP-CB1R were treated with PTX (or no PTX as a control), and then incubated with 30μM dyngo-4a before subsequent exposure to WIN 55,212-2. ( h ) Graph provides the quantified ERK1/2 phosphorylation induced by 10μM WIN 55,212-2 in the absence of PTX treatment shown in g . ( i ) HEK293 cells expressing SEP-CB1R with or without dominant-negative dynamin 2 K44A were exposed to agonists. ( j ) Graph provides the quantified ERK1/2 phosphorylation induced by 10 μM of each compound shown in i . All data are expressed as a percentage of the level of phosphorylation at 5 min for 2-AG without dyngo-4a pre-treatment and represent the mean±s.e.m. of at least three independent experiments.

Article Snippet: Briefly, HEK293 cells that were 40–50% confluent in a six-well plate were transfected with the plasmid encoding rat CB1 and 2.6 mg of siRNA (Qiagen) sequences targeting β-arrestin 1, β-arrestin 2 or non-silencing RNA duplex for control.

Techniques: Expressing, Incubation, Phospho-proteomics, Transfection, Control, Dominant Negative Mutation