Journal: Cancer cell international

Article Title: Machine learning-based prognostic model of lactylation-related genes for predicting prognosis and immune infiltration in patients with lung adenocarcinoma.

doi: 10.1186/s12935-024-03592-y

Figure Lengend Snippet: Fig. 12 KRT81 promotes EMT and PI3K/AKT pathway activation. A WB detection of EMT-associated proteins (E-cadherin, N-cadherin, and Vimentin) after KRT81 knockdown. B, C KEGG and GSEA enrichment analysis of up-regulated DEGs. D WB detection of p-PI3K, PI3K, p-AKT, and AKT proteins levels in A549 cells after siNC, siRNA2 transfections.*p < 0.05, **p < 0.01

Article Snippet: The membranes were closed with 5% skimmed milk and incubated with primary antibodies KRT81 (Proteintech,11342– 1-AP), GAPDH (Proteintech, 10494–1-AP), MMP2 (Proteintech,10373–2-AP), MMP9 (Proteintech, 10375–2-AP), E-cadherin (Proteintech,20874–1-AP), N-cadherin (Proteintech, 22018–1-AP), Vimentin (Proteintech,10366–1-AP), p-PI3K (Cell Signaling, 17366), t-PI3K (Cell Signaling, 9655), p-AKT (Cell Signaling,13038S), t-AKT (Cell Signaling, 4685S), P53 (Proteintech, 10442–1-AP), Caspase3 (Cell Signaling, 9664 T) and Bcl2 (Cell Signaling, 4223 T) were combined overnight at 4 °C.

Techniques: Activation Assay, Knockdown, Transfection

Journal: British Journal of Nutrition

Article Title: Maslinic acid inhibits the metastatic capacity of DU145 human prostate cancer cells: possible mediation via hypoxia-inducible factor-1α signalling

doi: 10.1017/s0007114512000967

Figure Lengend Snippet: Fig. 7. Maslinic acid reduces the activation of Akt and extracellular signal-related kinase (ERK) in DU145 cells. (A) Serum-deprived cells were incubated with 0– 25 mM-maslinic acid for 6 h, and cell lysates were prepared with or without a 15 min epidermal growth factor (EGF) stimulation. Serum-deprived cells were incu- bated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 for 6 h and lysed without stimulation or after 15 min of EGF stimulation to determine phospho (p)-Akt or p-ERK1/2 levels. To identify hypoxia-inducible factor-1a (HIF-1a), serum-deprived cells were incubated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 in the absence or presence of EGF for 6 h. Total cell lysates were subjected to Western blotting. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each band was estimated by densitometric scanning of the exposed films, and the expression levels were normalised to those of b-actin. To determine vascular endothelial growth factor (VEGF) concentrations, serum-deprived cells were incubated with (B) 0–20 mM-LY294002 or (C) 0–40 mM-PD98059 in serum-free media with or without 10 mg/l EGF for 18 h. The 18 h conditioned media were concentrated and subjected to Western blotting with VEGF antibody. The volumes of media loaded onto the gel were adjusted for equivalent protein concen- trations. Photographs of chemiluminescent detection of the blots, which are representative of three independent experiments, are shown. The relative abundance of each band was quantified by densitometric scanning of the exposed films. The adjusted means (n 3) of each band with their standard errors are shown above each blot. a,b,c,d Mean values with unlike letters were significantly different (P , 0·05). Mr, molecular weight.

Article Snippet: The reagents used were as follows: maslinic acid (Cayman Chemical); antibodies against uPAR, TIMP-1, TIMP-2, intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM) and VEGF (Santa Cruz Biotechnology); antibodies against Akt, phospho-Akt, ERK1/2 and phosphoERK1/2 (Cell Signaling Technology); anti-uPA antibody (Calbiochem); anti-HIF-1a, anti-E-cadherin and Matrigele Matrix (BD Biosciences); epidermal growth factor (EGF; R&D Systems); an adhesion assay kit (Chemicon International); transwell filters (Costar).

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Molecular Weight

Journal: Cell adhesion & migration

Article Title: Ras guanyl nucleotide releasing protein 2 affects cell viability and cell-matrix adhesion in ECV304 endothelial cells.

doi: 10.4161/cam.24082

Figure Lengend Snippet: Figure 3. Effect of RasGRP2 overexpression on Rap1 activation and cell adhesion. (A) Rap1 activation assessed by Rap activity assay. Upper panel, active form of Rap1; middle panel, total Rap1. (B) Rap1 activation assessed by Rap activity assay with or without 10 μM BAPTA-AM (BA, intracellular calcium chelator) for 30 min. (C) Cell adhesion assessed by adhesion assay. Data are shown as the mean ± SD (n = 3) *P < 0.05, **P < 0.01 vs. ECV304mock.

Article Snippet: After washing with PBS containing 0.05% Tween 20 (PBS-T), the membranes were incubated with rabbit anti-RasGRP2 antibody (GeneTex), mouse anti-β-actin antibody, rabbit anti-Rap1 antibody (Santa Cruz), mouse antipan-Ras antibody (Cell Biolabs), rabbit anti-phospho-p44/42 MAPK antibody, or rabbit anti-p44/42 MAPK antibody (Cell Signaling) in Can Get Signal® Solution 1 (Toyobo) for 1 h. Subsequently, the membranes were washed thrice with PBS-T and incubated with anti-rabbit IgG antibody (GeneTex) or antimouse IgG antibody (DakoCytomation) in Can Get Signal® Solution 2 (Toyobo) for 1 h. After five additional washes with PBS-T, immunoreactive proteins were detected using ECL Prime Western Blotting Detection Reagents and Amersham hyperfilmTM ECL (GE Healthcare).

Techniques: Over Expression, Activation Assay, Activity Assay, Cell Adhesion Assay

Journal: Nature communications

Article Title: microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition.

doi: 10.1038/ncomms3977

Figure Lengend Snippet: Figure 3 | miR-181a regulates EMT and increases tumour burden and incidence of metastasis in vivo. (a) Total tumour burden (g) in pBABE (n ¼ 5) versus p181a-injected mice (n ¼ 7) at 3 weeks post i.p. injection (2 106 cells; top). Fisher’s exact test correlating the incidence of intraperitoneal nodules with miR-181a expression (bottom); **P ¼ 0.01, OR: 46.76, 95% CI: 1.6–1424. (b) Confirmation of miR-181a expression in pBABE (n ¼ 5) versus p181a (n ¼ 7) cells isolated from harvested tissues as determined by qRT–PCR. Student’s t-test, **Pr0.01. Error bars, s.d. (c) Fold change of epithelial and mesenchymal marker expression as determined by qRT–PCR in miR-181a- (n ¼ 7) versus pBABE-injected (n ¼ 5) tumour cells. Student’s t-test, *Pr0.05 and **Pr0.01. Error bars, s.d. (d) Quantification of primary ovarian and intraperitoneal tumour burden in mice 3 weeks after intrabursal injection of both pBABE (n ¼ 5) versus p181a (n ¼ 5) tumour cells (1 106 cells). Student’s t-test, ***Pr0.001. Error bars, s.d (e) Gross images of metastatic nodules (top; white arrows). Ovarian tumours have been removed from the p181a 1 mouse to observe the extensive dissemination of tumour nodules throughout the abdomen. Haematoxylin and eosin and IHC staining(s) for pan-cytokeratin and N-cadherin in representative pBABE and p181a 1 tumours. Original magnification, 200, scale bars, 20 mM (black). (f) Quantification by immunoscoring as shown by percentage of positive staining for pan-cytokeratin and N-cadherin in pBABE (n ¼ 5) and p181a 1 (n ¼ 5) intraperitoneal tumours. Student’s t-test, *Pr0.05. Error bars, s.d.

Article Snippet: After blocking with 5% non-fat milk (LabScientific, Inc.) in TBS-Tween buffer, the membranes were probed with antibodies against E-cadherin (1:250, Santa Cruz), N-cadherin (1:250, Santa Cruz), Smad7 (1:200, Santa Cruz), ACTR-IIA (1:500, Santa Cruz), ACTR-IIB (1:500, Santa Cruz), actin (1:1,000, Santa Cruz), keratin 8 (1:250, Epitomics), Col3A1 (1:200, Abcam), Smad2 (1:500, Cell Signaling), P-Smad2 (1:250, Cell Signaling), Smad3 (1:200, Cell Signaling), P-Smad3 (1:100, Cell Signaling) and fibronectin (1:1,000, BD Transduction Lab).

Techniques: In Vivo, Injection, Expressing, Isolation, Quantitative RT-PCR, Marker, Immunohistochemistry, Staining

Journal: Nature communications

Article Title: microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition.

doi: 10.1038/ncomms3977

Figure Lengend Snippet: Figure 4 | Stable inhibition of miR-181a results in reduced cellular motility and survival. (a) Fold change of miR-181a expression levels in SKOV3 cell line stably transduced with miR-181a TuD vector and a control decoy vector as assessed by qRT–PCR. Student’s t-test, ***Pr0.001 (n ¼ 3). Error bars, s.d. (b) Monitoring functional activity of miR-181a in SKOV3 miR-181a decoy transduced cells utilizing sensor vectors as measured via mean fluorescent intensity measurements using flow cytometry. (c) Morphological changes (top panels; original magnification, 700, scale bars, 10 mM (black)) upon functional inhibition of miR-181a. Western blotting (bottom) for epithelial (E-cadherin) and mesenchymal markers (Col3A1 and N-cadherin) to confirm mesenchymal-to-epithelial transition. (d) Effects of miR-181a decoy on E-cadherin and F-actin organization (both shown in green (FITC, fluorescein isothiocyanate); blue (DAPI (40,6-diamidino-2-phenylindole)) for nuclear staining). Original magnification, 1,000, scale bars, 5 mM (white). (e) Clonogenic assay (top) to assess cellular survival in miR-181a decoy cells shown as fold change in number of colonies in control versus miR-181a decoy cells (bottom). Colonies are shown in purple post staining with crystal violet. Student’s t-test, ***Pr0.001 are shown (n ¼ 3). Error bars, s.d. (f) Wound-healing assay (left) to evaluate cell motility in miR-181a decoy cells as quantified by per cent wound closure over time points shown (right). Original magnification, 100, scale bars, 20 mM (black). Student’s t-test, **Pr0.01 (n ¼ 3). Error bars, s.d. (all results are shown for SKOV3 cells).

Article Snippet: After blocking with 5% non-fat milk (LabScientific, Inc.) in TBS-Tween buffer, the membranes were probed with antibodies against E-cadherin (1:250, Santa Cruz), N-cadherin (1:250, Santa Cruz), Smad7 (1:200, Santa Cruz), ACTR-IIA (1:500, Santa Cruz), ACTR-IIB (1:500, Santa Cruz), actin (1:1,000, Santa Cruz), keratin 8 (1:250, Epitomics), Col3A1 (1:200, Abcam), Smad2 (1:500, Cell Signaling), P-Smad2 (1:250, Cell Signaling), Smad3 (1:200, Cell Signaling), P-Smad3 (1:100, Cell Signaling) and fibronectin (1:1,000, BD Transduction Lab).

Techniques: Inhibition, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Control, Quantitative RT-PCR, Functional Assay, Activity Assay, Cytometry, Western Blot, Staining, Clonogenic Assay, Wound Healing Assay

Journal: Cancer science

Article Title: Critical role of aquaporin 3 on growth of human esophageal and oral squamous cell carcinoma.

doi: 10.1111/j.1349-7006.2011.01927.x

Figure Lengend Snippet: Fig. 4. Aquaporin 3 (AQP3)-siRNA inhibits the expression of integrins and the activation of the FAK-MAPK pathway in squamous cell carcinoma (SCC). (a) Typical photographs of KYSE30 treated with AQP3-siRNA. Cells was treated with AQP3-siRNA for 24 h, and then the floating cells and adhesive cells were separated. The upper columns are composed of adherent cells and the lower ones are floating cells. (b) Time-dependent inhibition of cell adhesion of SCC by treatment with AQP3-siRNA. The KYSE30 cells were pretreated with AQP3-siRNA, and then the cells were placed onto fibronectin-coated plates. The time-dependent changes of the adherent cells were counted and expressed as a percentage. The data are the average of three independent experiments. (c) Percentage of dead cells within the floating cells after treatment with AQP3-siRNA. The dead cells within the total floating cells at 12, 24 and 48 h after treatment with AQP3-siRNA were counted and expressed as a percentage. The data are the average of three independent experiments. (d) Western blot analysis for the expression of integrin a5 and b1 by treatment with AQP3-siRNA on KYSE30 (left) and SAS (right). Cells were treated with AQP3-siRNA (20 and 40 nM), negative control siRNA and vehicle (Veh). Samples were collected for 24 h after the treatment. GAPDH was used to evaluate equivalent loading. (e) Western blot analysis for the expression of integrin a5 and b1 by treatment with AQP3-siRNA on adenocarcinoma cell lines such as HT-29 and Caco2. Cells were treated with AQP3-siRNA (40 nM), negative control siRNA and vehicle (Veh), and then the samples were collected. GAPDH was used to evaluate equivalent loading. (f) Effects of AQP3-siRNA on phosphorylation of FAK, MAPK and MEK. Western blot analysis for phosphorylated FAK (Tyr 925), MAPK (p44 ⁄ 42 MAP kinase, Thr202 ⁄ Tyr204) and MEK1 ⁄ 2 (Ser217 ⁄ 221). Cells were treated with AQP3-siRNA (20 and 40 nM), negative control siRNA and vehicle (Veh). Samples were collected for 24 h after the treatment. GAPDH was used to evaluate equivalent loading.

Article Snippet: Antibodies against focal adhesion kinase (FAK), phosphorylated FAK, phosphorylated MEK1 ⁄ 2, p44 ⁄ 42MAPK and phosphorylated p44 ⁄ 42MAPK were from Cell Signalling Technologies (Beverly, MA, USA).

Techniques: Expressing, Activation Assay, Adhesive, Inhibition, Western Blot, Negative Control, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

doi: 10.1371/journal.ppat.1003974

Figure Lengend Snippet: (A) Predicted duplex formation between miR-BART9 and human E-cadherin 3′UTR (Wt). The seed sequence region is highlighted in bold. The putative target sequence of E-cadherin 3′UTR at nt 1795–1801. Mut indicates the mutated E-cadherin 3′UTR sequence used as a control in the reporter assay. Mutated bases are specified by underlining. (B) Luciferase activity of the wild type (Wt) or mutant (Mut) E-cadherin 3′UTR reporter in BM1, TW04 and HK1 cells expressing miR-BART9 or LacZ. (C) Luciferase activity of the wild type (Wt) or mutant (Mut) E-cadherin 3′UTR reporter in HK1-EBV and C666-1 cells treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl). (D) Top panel: Immunoblotting analysis of E-cadherin in BM1, TW04 and HK1 cells expressing miR-BART9 or LacZ. (Left) or HK1-EBV cells treated with an LNA-modified miR-BART9 antisense oligo (anti-BART9) or scramble control (anti-Ctrl) (Right). GAPDH was used as a loading control. Bottom panel: E-cadherin protein levels were normalized to GAPDH levels, and then compared with the LacZ or anti-Ctrl cells whose normalized levels were expressed as 1.0. Bar graphs provide the means ± SEM of independent experiments and two-tailed Student's t-test were performed (*, P<0.05; **, P<0.01). (E) Representative immunofluorescence staining of E-cadherin and DAPI staining to detect the nucleus in BM1 and TW04 cells expressing miR-BART9 or LacZ. Arrows indicate cell-cell junctions. Scale bar = 20 µm. (F) Representative IHC staining of GFP, human Mac2BP and E-cadherin in sections of primary tumors formed by BM1 cells expressing miR-BART9 or LacZ. Scale bar = 500 µm.

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-CDH1 (E-cadherin) antibody (1∶5000; Cell signaling #4065), anti-LMP1 monoclonal antibody (S12) (1∶1000; a gift from Dr. Yu-Sun Chang, Chang Gung University), rat monoclonal anti-LMP2A antibody (1∶1000; Bio-Rad MCA2466), anti-EBNA1 antibody (1∶1000; a gift from Dr. Mei-Ru Chen, National Taiwan University) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1∶10000; GeneTex #213110-01, #213111-01) and the resultant bands were detected using ECL reagents (Millipore).

Techniques: Sequencing, Control, Reporter Assay, Luciferase, Activity Assay, Mutagenesis, Expressing, Concentration Assay, Modification, Western Blot, Two Tailed Test, Immunofluorescence, Staining, Immunohistochemistry

Journal: PLoS Pathogens

Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

doi: 10.1371/journal.ppat.1003974

Figure Lengend Snippet: (A) Protein level of E-cadherin was increased after introducing pcDNA6/His-CDH1, which contains CDH1 open reading frame without 3′-UTR. Transwell migration assay (B) and Matrigel invasion assay (C) for miR-BART9- or LacZ-expressing BM1 cells with or without ectopic expression of E-cadherin. (D) HK1-EBV cells were transfected with 10 nM siRNA negative control (si-Neg) or CDH1 siRNA (si-CDH1). Expression of E-cadherin was examined by Western blotting. GAPDH was used as a loading control. (E) Transwell migration assay (Upper) and Matrigel invasion assay (Middle) of HK1-EBV cells treated with an LNA-modified miR-BART9 antisense oligo (anti-BART9), scramble control (anti-Ctrl), or anti-BART9 plus E-cadherin siRNA (si-CHD1). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and are expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P<0.05; ***, P<0.001). Scale bar = 200 µm.

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-CDH1 (E-cadherin) antibody (1∶5000; Cell signaling #4065), anti-LMP1 monoclonal antibody (S12) (1∶1000; a gift from Dr. Yu-Sun Chang, Chang Gung University), rat monoclonal anti-LMP2A antibody (1∶1000; Bio-Rad MCA2466), anti-EBNA1 antibody (1∶1000; a gift from Dr. Mei-Ru Chen, National Taiwan University) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1∶10000; GeneTex #213110-01, #213111-01) and the resultant bands were detected using ECL reagents (Millipore).

Techniques: Transwell Migration Assay, Invasion Assay, Expressing, Transfection, Negative Control, Western Blot, Control, Modification, Two Tailed Test

Journal: PLoS Pathogens

Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

doi: 10.1371/journal.ppat.1003974

Figure Lengend Snippet: Expression levels of matrix metalloproteases MMP1, MMP2, MMP9, MMP10 and MMP12 (A) and E-cadherin (CDH1), α-catenin (CTNNA1) and vimentin (B) in BM1 and TW04 cells infected with lentivirus expressing the miR-BART9 or control (LacZ) vector. mRNA levels were determined via qPCR. The data were normalized to cellular EEF1A1 levels and expressed as the fold change relative to the appropriate cell line. Bar graphs provide the means ± SEM of three independent experiments and two-tailed Student's t-test were performed (*, P<0.05; **, P<0.01; ***, P<0.001). (C) Western blot analysis for the expression of indicated EMT markers. GAPDH protein was used as a protein loading control. (D) Representative immunofluorescence staining of vimentin and DAPI staining to detect the nucleus in BM1 and TW04 cells expressing miR-BART9 or LacZ. Scale bar = 20 µm. (E) Representative immunofluorescence staining of E-cadherin (CDH1) and vimentin and DAPI staining to detect the nucleus in sections of primary tumors formed by BM1 cells expressing miR-BART9 or LacZ. Scale bar = 20 µm.

Article Snippet: The membranes were incubated with a rabbit monoclonal anti-CDH1 (E-cadherin) antibody (1∶5000; Cell signaling #4065), anti-LMP1 monoclonal antibody (S12) (1∶1000; a gift from Dr. Yu-Sun Chang, Chang Gung University), rat monoclonal anti-LMP2A antibody (1∶1000; Bio-Rad MCA2466), anti-EBNA1 antibody (1∶1000; a gift from Dr. Mei-Ru Chen, National Taiwan University) followed by horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1∶10000; GeneTex #213110-01, #213111-01) and the resultant bands were detected using ECL reagents (Millipore).

Techniques: Expressing, Infection, Control, Plasmid Preparation, Two Tailed Test, Western Blot, Immunofluorescence, Staining