Journal: Viruses

Article Title: Upregulation of GLS1 Isoforms KGA and GAC Facilitates Mitochondrial Metabolism and Cell Proliferation in Epstein–Barr Virus Infected Cells

doi: 10.3390/v12080811

Figure Lengend Snippet: c-Myc regulates KGA and GAC protein expression in EBV-infected cells. ( a – c ) Raji ( a ), Namalwa ( b ), and C666-1 ( c ) were treated with the vehicle control DMSO or 25 µM or 50µM of c-Myc inhibitor 10074-G5 for 24 h. Cell lysates collected from the treated cells were Western blotted for c-Myc and GLS1 expression with anti-c-Myc and anti-GLS1 antibodies, respectively. β-actin was used as loading control. Percentages of c-Myc inhibition in the inhibitor treated cells were compared with the vehicle control treated cells. ( d , e ) Namalwa ( d ) and C666-1 ( e ) were transduced with lentiviral constructs of control shRNA or c-Myc shRNA for 72 h. Whole cell lysates collected after 72 h of transduction from the control shRNA and c-Myc shRNA cells were used for determining the protein levels of c-Myc, KGA, and GAC by Western blot analysis with anti-c-Myc and anti-GLS1 antibodies, respectively. β-actin was used as loading control. Percentage inhibition of c-Myc was calculated and compared with the control shRNA cells.

Article Snippet: Lentiviruses encoding c-Myc shRNA and control shRNA were produced by transiently transfecting HEK 293T cells with lentiviral packaging plasmids (Gag-Pol, Rev and VSV-G) and plasmid encoding c-Myc shRNA (Cat #29435, Addgene, Cambridge, MA, USA) or control shRNA, as described previously [ ].

Techniques: Expressing, Infection, Control, Western Blot, Inhibition, Transduction, Construct, shRNA

Journal: Journal of Translational Medicine

Article Title: HBx-induced MiR-1269b in NF-κB dependent manner upregulates cell division cycle 40 homolog (CDC40) to promote proliferation and migration in hepatoma cells

doi: 10.1186/s12967-016-0949-y

Figure Lengend Snippet: Primers used in plasmid construction

Article Snippet: CDC40 knockdown primers were synthesized from GenePharma (Shanghai, China).

Techniques: Plasmid Preparation, Sequencing

Journal: Journal of Translational Medicine

Article Title: HBx-induced MiR-1269b in NF-κB dependent manner upregulates cell division cycle 40 homolog (CDC40) to promote proliferation and migration in hepatoma cells

doi: 10.1186/s12967-016-0949-y

Figure Lengend Snippet: Primers used in qRT-PCR

Article Snippet: CDC40 knockdown primers were synthesized from GenePharma (Shanghai, China).

Techniques: Sequencing

Journal: Journal of Translational Medicine

Article Title: HBx-induced MiR-1269b in NF-κB dependent manner upregulates cell division cycle 40 homolog (CDC40) to promote proliferation and migration in hepatoma cells

doi: 10.1186/s12967-016-0949-y

Figure Lengend Snippet: miR-1269b directly targets the 3′UTR of CDC40. a The predicted binding sites for miR-1269b in the 3′UTR of CDC40 and the induced mutations in the binding sites are shown. b qRT–PCR ( left ) and representative Western blot analysis ( right ) of CDC40 levels in HepG2 and SMMC-7721 cells transfected with the control vector or ASO-nc vs miR-1269b or ASO-miR1269b-transduced cells. c Luciferase reporter assays were performed in HepG2 and SMMC-7721 cell lines that were co-transfected with miR-1269b in the presence of CDC40 3′UTR ( top ). In the controls, the mutated 3′UTR sequences were included ( bottom ). Columns, mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: CDC40 knockdown primers were synthesized from GenePharma (Shanghai, China).

Techniques: Binding Assay, Quantitative RT-PCR, Western Blot, Transfection, Control, Plasmid Preparation, Luciferase

Journal: Journal of Translational Medicine

Article Title: HBx-induced MiR-1269b in NF-κB dependent manner upregulates cell division cycle 40 homolog (CDC40) to promote proliferation and migration in hepatoma cells

doi: 10.1186/s12967-016-0949-y

Figure Lengend Snippet: CDC40 promotes cell proliferation and migration, which is regulated by miR-1269b. a Colony formation assays were performed to test the influence of CDC40 on proliferation in HepG2 and SMMC-7721 cells. b Cell cycle progression in the transfected cells was analyzed using flow cytometry. The chart shows the populations of cells in the different phases of the cell cycle. c Transwell migration assays were performed to detect the effect of CDC40 on the migration of HepG2 and SMMC-7721 cells. d The influence of CDC40 on the protein levels of the EMT-associated molecules E-cadherin and vimentin was determined using western blot analysis. Cells were transfected 48 h before proteins were harvested. Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: CDC40 knockdown primers were synthesized from GenePharma (Shanghai, China).

Techniques: Migration, Transfection, Flow Cytometry, Western Blot

Journal: Journal of Translational Medicine

Article Title: HBx-induced MiR-1269b in NF-κB dependent manner upregulates cell division cycle 40 homolog (CDC40) to promote proliferation and migration in hepatoma cells

doi: 10.1186/s12967-016-0949-y

Figure Lengend Snippet: Overexpression of CDC40 abrogated the repression of the phenotypes induced by ASO-miR-1269b in HepG2 and SMMC-7721 cells. a Colony formation rates. b Cell cycle progression. c Migratory ability. d Corresponding levels of the EMT protein markers E-cadherin and vimentin. Error bars indicate the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: CDC40 knockdown primers were synthesized from GenePharma (Shanghai, China).

Techniques: Over Expression

Journal: BMC Veterinary Research

Article Title: Canine mammary cancer cells direct macrophages toward an intermediate activation state between M1/M2

doi: 10.1186/s12917-015-0473-y

Figure Lengend Snippet: CSF-1 and Twist-1 are key mediators of macrophage activation. Twist-1 protein levels were depleted by treating with siRNA against Twist-1 for 24 h. Immunofluorescence was carried out using anti-Twist-1 antibody ( a ). The effects of the reduction of Twist-1 levels on LPS-induced activation of macrophages were assayed by expression of MHC II ( b (i)); granularity ( b (ii)); and phagocytosis ( c ). All experiments are replicates of 3. Horizontal bars indicate statistically significant differences on a Student t-test, where P < 0.05, unless where indicated; “ns” indicates non-significant differences

Article Snippet: For transient transfection of small interfering RNA (siRNA) knockdown experiments, RAW264.7 cells were transfected with siRNA using the Amaxa Cell Line Nucleofector Kit V (Lonza,VCA-1003) according to the manufacturer's instructions.

Techniques: Activation Assay, Immunofluorescence, 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: 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: Stem Cells International

Article Title: Cancer Stem Cell Marker Endoglin (CD105) Induces Epithelial Mesenchymal Transition (EMT) but Not Metastasis in Clear Cell Renal Cell Carcinoma

doi: 10.1155/2019/9060152

Figure Lengend Snippet: CD105(+) ccRCC tumor cells are mesenchymal cells with enhanced motility and invasion capability. (a) Western blot and (b) qRT-PCR of EMT markers of CD105(+) cells and parental cells. (c) Migration assay and (d) modified 3D transwell assay of CD105(+) cells and parental cells ( ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The shRNA plasmids for CD105 knockdown were constructed from pSicoR (Addgene, #11579) with target sequences of shENG1: 5′-GAAAGAGCTTGTTGCGCAT-3′ and shENG2: 5′-AACAGTCCATTGTGACCTTCA-3′, as previously reported [ ].

Techniques: Western Blot, Quantitative RT-PCR, Migration, Modification, Transwell Assay

Journal: Stem Cells International

Article Title: Cancer Stem Cell Marker Endoglin (CD105) Induces Epithelial Mesenchymal Transition (EMT) but Not Metastasis in Clear Cell Renal Cell Carcinoma

doi: 10.1155/2019/9060152

Figure Lengend Snippet: CD105 knockdown induces loss of mesenchymal markers and inhibition of motility and invasion. (a) Western blot and (b) qRT-PCR of EMT markers of CD105(+) cells and shRNA-mediated CD105 knockdown CD105(+) cells. (c) Migration assay and (d) modified 3D transwell assay of CD105(+) cells and shRNA-mediated CD105 knockdown CD105(+) cells ( ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The shRNA plasmids for CD105 knockdown were constructed from pSicoR (Addgene, #11579) with target sequences of shENG1: 5′-GAAAGAGCTTGTTGCGCAT-3′ and shENG2: 5′-AACAGTCCATTGTGACCTTCA-3′, as previously reported [ ].

Techniques: Knockdown, Inhibition, Western Blot, Quantitative RT-PCR, shRNA, Migration, Modification, Transwell Assay

Journal: Stem Cells International

Article Title: Cancer Stem Cell Marker Endoglin (CD105) Induces Epithelial Mesenchymal Transition (EMT) but Not Metastasis in Clear Cell Renal Cell Carcinoma

doi: 10.1155/2019/9060152

Figure Lengend Snippet: MYC overexpression can reverse this process. (a) Western blot of MYC in CD105(+) cells, shRNA-mediated CD105 knockdown CD105(+) cells, and MYC overexpressed cells. (b) qRT-PCR of EMT markers of shRNA-mediated CD105 knockdown CD105(+) cells before and after MYC overexpression. (c) Migration assay of shRNA-mediated CD105 knockdown CD105(+) cells and MYC overexpressed cells. (d) Migration assay of shRNA-mediated CD105 knockdown CD105(+) cells and NANOG overexpressed cells. (e) qRT-PCR analysis of EMT markers of CD105(+) cells before and after the treatment of TGF- β type I receptor kinase inhibitor LY-364947 (50 nM) ( ∗∗ p < 0.01).

Article Snippet: The shRNA plasmids for CD105 knockdown were constructed from pSicoR (Addgene, #11579) with target sequences of shENG1: 5′-GAAAGAGCTTGTTGCGCAT-3′ and shENG2: 5′-AACAGTCCATTGTGACCTTCA-3′, as previously reported [ ].

Techniques: Over Expression, Western Blot, shRNA, Knockdown, Quantitative RT-PCR, Migration

Journal: Stem Cells International

Article Title: Cancer Stem Cell Marker Endoglin (CD105) Induces Epithelial Mesenchymal Transition (EMT) but Not Metastasis in Clear Cell Renal Cell Carcinoma

doi: 10.1155/2019/9060152

Figure Lengend Snippet: CD105 knockdown does not change the metastasis of ccRCC in the tail vein injection mouse model. (a) The lung weight analysis of mice received the CD105(+) cells and shRNA-mediated CD105 knockdown CD105(+) cells. Representative images of lung metastasis in both gross view (b) and H&E staining (c) established by tail vein injection of cancer cells.

Article Snippet: The shRNA plasmids for CD105 knockdown were constructed from pSicoR (Addgene, #11579) with target sequences of shENG1: 5′-GAAAGAGCTTGTTGCGCAT-3′ and shENG2: 5′-AACAGTCCATTGTGACCTTCA-3′, as previously reported [ ].

Techniques: Knockdown, Injection, shRNA, Staining

Journal: Aging Cell

Article Title: Liver osteopontin is required to prevent the progression of age‐related nonalcoholic fatty liver disease

doi: 10.1111/acel.13183

Figure Lengend Snippet: Liver OPN is p53 regulated. (a) Protein levels of p53 were evaluated by immunohistochemistry in liver sections of 3‐, 10‐, and 20‐month‐old (m) wild‐type (WT) mice ( n = 3–5). (b) OPN protein levels from liver homogenates were measured in WT and p53‐KO male and female mice fed a chow diet (CD) and a high‐fat diet (HFD) by immunoblotting using glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as loading control ( n = 4–6). (c) Liver p53 and OPN protein levels were evaluated in WT mice fed a HFD injected with p53‐dominant positive adenovirus (adp53) and GFP (adGFP), using transferrin or GAPDH as a loading control ( n = 4–6). (d) Protein levels of p53 and OPN in siCtrl and sip53 HepG2 cells were measured by immunoblotting using GAPDH as loading control. Extracellular OPN was measured using an ELISA ( n = 4–5). (e) OPN protein levels from HepG2 cells silenced for p53 and Hep3B cells treated with palbociclib (Palbo) were measured by immunoblotting using GAPDH as loading control. OPN media levels from Hep3B cells treated either with vehicle (Veh) or with palbociclib (Palbo) were measured by ELISA ( n = 4–8). Values are means ± SEM . Significant differences are denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001 (Student's t test)

Article Snippet: Data obtained from the Cell Line Encyclopedia (© 2019 The Broad Institute of MIT & Harvard) showed that the cell line with the least p53 expression, the Hep3B, also had the lowest OPN expression.

Techniques: Immunohistochemistry, Western Blot, Control, Injection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Genetics

Article Title: Long Noncoding Ribonucleic Acid MSTRG.59589 Promotes Porcine Skeletal Muscle Satellite Cells Differentiation by Enhancing the Function of PALLD

doi: 10.3389/fgene.2019.01220

Figure Lengend Snippet: MSTRG.59589 promoted muscle differentiation by enhancing the expression of PALLD . (A) Quantitative reverse transcription PCR (QRT-PCR) results showing that the expression level of the PALLD increases continuously from satellite cell proliferation to differentiation. (B) QRT-PCR results showing that PALLD is more highly expressed in the heart, skeletal muscle, and spleen than in other tissues. RNA sequencing and QRT-PCR results showing that the expression of PALLD is down-regulated (C, D) or up-regulated (E) after knockdown or overexpression of MSTRG.59589. (F) QRT-PCR results showing that PALLD is successfully significantly inhibited by SI-2. (G) QRT-PCR results showing that PALLD knockdown significantly decreases the messenger RNA expression levels of MYOD, MYOG, and MYHC.

Article Snippet: The small interfering RNAs (siRNAs) for knockdown of PALLD were designed and synthesized by GenePharma (Suzhou, China).

Techniques: Expressing, Reverse Transcription, Quantitative RT-PCR, RNA Sequencing, Knockdown, Over Expression, RNA Expression