Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: EPHA2 receptor consists of the ligand-binding domain, a cysteine-rich domain, two fibronectin-III domains, a tyrosine kinase (TK) domain, a sterile alpha motif (SAM), and a PDZ-binding motif. Novel mutations (yellow) and known SNPs were found in ligand-binding domain and TK domain by us recently

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Ligand Binding Assay, Sterility, Binding Assay

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Thirty five SCC, 39 MPM tumor samples, and six cell lines have been used to determine EPHA2 gene amplification. Fold change relative to reference gene LINE-1

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Amplification

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a Lysates of six MPM cell lines and the Met-5A, a mesothelial control cell line, were immunoblotted with EPHA2 antibody. b Immunohistochemistry representative pictures of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. c Protein expression quantity of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. d Protein expression quantity of 48 NSCLC SQ and 24 adjacent normal samples were used in SSC TMA. H&E, EPHA2, phospho-(p-)EPHA2, and ephrin A1 were stained and scored. N: normal, T: Tumor

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Control, Immunohistochemistry, Expressing, Staining

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: BEAS2B EPHA2 isogenic cells were used to treat a Taxol, b cisplatin, c SU11274, and d rapamycin. Mutation G391R cells showed resistant to cisplatin inhibition but sensitive to MET inhibitor SU11274 and mTOR inhibitor Rapamycin. EV: empty vector

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Mutagenesis, Inhibition, Plasmid Preparation

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a MPM cell lines H28, H513, H2052, H2373, H2461, and H2596 were treated with cisplatin with 1, 5, and 10 μM for 48 h. b HEK293 EPHA2 isogenic cells treatment with doxazosin for 48 h

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques:

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a The crystal structure of EphA2 in the auto-inhibited conformation. The distance between Y772 and K702 shown by dotted line is 20.3 Å, b The EphA2 conformation after MD simulations showing the minimum distance between Y772 and K702 which is 3.4 Å, c The distribution of the distance between Y772 and K702 in the wild type and mutants A859D and T647M, d The surface showing the ATP binding site in EphA2, and e The distribution of the volume in (Å 3 ) of the ATP-binding site for the wild type and the two mutants calculated from the snapshots of the MD simulations

Article Snippet: The wild-type EPHA2 construct (pCMV6-AC-GFP–EPHA2) was purchased from OriGene (Rockville, MD).

Techniques: Binding Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Early Lymphocyte Loss and Increased Granulocyte/Lymphocyte Ratio Predict Systemic Spread of Streptococcus pyogenes in a Mouse Model of Acute Skin Infection

doi: 10.3389/fcimb.2018.00101

Figure Lengend Snippet: Bacterial loads in blood (A) , liver (B) , spleen (C) , and lung (D) of mice infected with S. pyogenes strains AP1 or 5448. Female C57BL/6J mice were infected subcutaneously on the back with 5 × 10 7 CFU S. pyogenes . After the indicated time points, mice were sacrificed, blood was drawn by cardiac puncture, and livers, spleens and lungs were dissected and homogenized in PBS. Blood samples and organ homogenates were plated onto blood agar plates in 10-fold serial dilutions. Bacterial loads were determined by counting colonies after 18 h of incubation at 37°C. For simplicity sake, the value of 10 0 (corresponding to one hypothetical colony) was inserted for all samples with sterile culture. The graphs show mean ± SD of 7–10 mice per group, combined from three independent complete time course experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Unpaired T -test.

Article Snippet: Female C57BL/6J mice (8 to 12 weeks old) were purchased from Harlan (Venray, The Netherlands) and housed in a pathogen-free animal facility at the Helmholtz Centre for Infection Research (Braunschweig, Germany) under standard conditions according to institutional guidelines.

Techniques: Infection, Incubation, Sterility

Journal: Cell death & disease

Article Title: PLOD3 promotes lung metastasis via regulation of STAT3.

doi: 10.1038/s41419-018-1186-5

Figure Lengend Snippet: Fig. 1 Endogenous PLOD3 plays an important role in lung cancer. a Representative microscopic images of lung cancers and their normal tissue counterparts stained with an anti-PLOD3 antibody (left panel, scale bar, 100 μm). Staining intensity was scored as follows in all patient samples (n = 59). The scores are calculated by multiplying the staining intensity with the percentage of stained cells in primary lung cancer relative to the paired normal tissue. Statistical significance was determined by Student’s t-test. ***P < 0.001. b PLOD3 upregulation is positively correlated with the stage of lung cancer in all patient samples (n = 59). c Box plot analysis of the PLOD3 mRNA levels obtained from ONCOMINE from lung cancer patients. Gene: PLOD3; Analysis type: cancer vs normal analysis; Data type: mRNA; Sample type: clinical specimen; Lung. d The effects of PLOD3 on the overall survival of lung cancer patients, using Kaplan–Meier plotter analysis. e R-H460 and A549 cells were cultured up to 80% confluence, followed by changing of the medium to fresh serum-free RPMI1640. After 6 h, collected supernatants were analyzed via immunoblotting with anti-PLOD3 antibody (left panel) and enzyme-linked immunosorbent assay (ELISA) at 450 nm (right panel). Statistical significance was determined by Student’s t-test. ***P < 0.001. f R-H460 and A549 cells (1 × 106 cells) were inoculated subcutaneously into the backs of nude mice. After tumorigenesis, mice were killed. Serum collected from each group was analyzed via ELISA. Statistical significance was determined by Student’s t-test. **P < 0.01

Article Snippet: Human PLOD3 cDNA (wild type) purchased from OriGene (Cat No. SC324563) was amplified via polymerase chain reaction (PCR) and cloned into pcDNA3.1-HA vectors.

Techniques: Staining, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell death & disease

Article Title: PLOD3 promotes lung metastasis via regulation of STAT3.

doi: 10.1038/s41419-018-1186-5

Figure Lengend Snippet: Fig. 2 PLOD3 possesses greater potential to promote lung cancer malignancy. a PLOD3 protein expression was assessed via western blot analysis, following transfection of HA-PLOD3 in H460 cells for 48 h. b A transwell assay was performed to evaluate the motility of PLOD3- overexpressing H460 cells. PLOD3 displayed superior potential to promote cell migration. The relative numbers of migratory cells were determined and presented as the mean ± SD values from three independent experiments. Statistical significance was determined by Student’s t-test. ***P < 0.001. c The invasion assay revealed different cell motilities in PLOD3-overexpressing lung cancer cells. PLOD3 expression promoted the invasion of H460 cells. The relative numbers of invasive cells were determined and presented as the mean ± SD values from three independent experiments. Statistical significance was determined by Student’s t-test. ***P < 0.001. d PLOD3 protein and mRNA levels were determined via western blotting (lower) and quantitative reverse transcription polymerase chain reaction (qRT-PCR; upper), respectively, after the indicated concentration of siPLOD3 was transfected in R-H460 cells for 24 h. The qRT-PCR data are expressed as the mean ± SD values *P < 0.05, **P < 0.01. e The cell viability assay for siPLOD3-treated cells for 24 h; x-axis: siCON, siPLOD3 (concentration). f A transwell assay was conducted to evaluate the motility of PLOD3 knockdown R-H460 cells at 12 h. Statistical significance was determined by Student’s t-test. **P < 0.01. g Cell invasion assay revealed that siPLOD3 had a limited effect on pro-metastasis at 24 h in R-H460 cells. Statistical significance was determined by Student’s t-test. **P < 0.01. h R-H460 cells were treated with siPLOD3 and then incubated for 24 and 48 h. The cells were then scraped with a sterile 200-μl pipette tip for the scratch assay

Article Snippet: Human PLOD3 cDNA (wild type) purchased from OriGene (Cat No. SC324563) was amplified via polymerase chain reaction (PCR) and cloned into pcDNA3.1-HA vectors.

Techniques: Expressing, Western Blot, Transfection, Transwell Assay, Migration, Invasion Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Viability Assay, Knockdown, Incubation, Sterility, Transferring, Wound Healing Assay

Journal: Cell death & disease

Article Title: PLOD3 promotes lung metastasis via regulation of STAT3.

doi: 10.1038/s41419-018-1186-5

Figure Lengend Snippet: Fig. 3 Loss of PLOD3 suppressed R-H460 tumor metastasis in vivo. a Representative micrographs with metastatic nodules are shown via Bouin’s staining. b Hematoxylin and eosin staining were performed using xenografts. c, d The number and volume of metastatic nodules were determined microscopically in PLOD3 xenograft tumors. Statistical significance was determined by Student’s t-test. *P < 0.05, **P < 0.01. e, f Estimation of hPLOD3 mRNA and PLOD3 protein levels in the lung nodules. Statistical significance was determined by ANOVA analysis. ***P < 0.001

Article Snippet: Human PLOD3 cDNA (wild type) purchased from OriGene (Cat No. SC324563) was amplified via polymerase chain reaction (PCR) and cloned into pcDNA3.1-HA vectors.

Techniques: In Vivo, Staining

Journal: Cell death & disease

Article Title: PLOD3 promotes lung metastasis via regulation of STAT3.

doi: 10.1038/s41419-018-1186-5

Figure Lengend Snippet: Fig. 4 MMP-2/9 and uPA expression in siPLOD3- or HA-PLOD3-treated lung cancer cells. a hMMP-2/9 mRNA levels were determined after siPLOD3 treatment in vivo. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01. b In vivo immunofluorescence of siCON- and siPLOD3-treated groups. c hMMP-2/9 and hu-PA mRNA levels after the indicated concentration of HA-PLOD3 was transfected were determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). Statistical significance was determined by Student’s t-test. *P < 0.05. d H460 cells were transfected with siCON or the indicated concentration of siPLOD3 for 24 h. hMMP-2/9 and hu-PA mRNA levels were determined via RT-qPCR. Statistical significance was determined by ANOVA analysis. **P < 0.01, ***P < 0.001. e–g Correlation of hPLOD3 with hMMP-2 or hMMP-9 or hu-PA transcripts in the cbioportal datasets of lung cancer patients

Article Snippet: Human PLOD3 cDNA (wild type) purchased from OriGene (Cat No. SC324563) was amplified via polymerase chain reaction (PCR) and cloned into pcDNA3.1-HA vectors.

Techniques: Expressing, In Vivo, Concentration Assay, Transfection, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cell death & disease

Article Title: PLOD3 promotes lung metastasis via regulation of STAT3.

doi: 10.1038/s41419-018-1186-5

Figure Lengend Snippet: Fig. 5 PLOD3 regulated the malignant phenotype via STAT3 signaling. a Immunostaining of PLOD3, pS-STAT3, and STAT3 in PLOD3 knockdown xenograft tumors. b Protein levels of PLOD3, pS-STAT3, pY-STAT3, and STAT3 were determined via western blotting in PLOD3 xenograft tumors. Statistical significance was determined by Student’s t-test. *P < 0.05, **P < 0.01. c Levels of the indicated proteins were determined via western blotting after siPLOD3 treatment in R-H460 cells. d Levels of the indicated proteins were determined via western blotting in HA-PLOD3- overexpressing H460 cells. e, f The HA-PLOD3 and STAT3 inhibitor S3I-201-treated H460 cells were analyzed via a transwell assay to evaluate cell motility and invasiveness. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01

Article Snippet: Human PLOD3 cDNA (wild type) purchased from OriGene (Cat No. SC324563) was amplified via polymerase chain reaction (PCR) and cloned into pcDNA3.1-HA vectors.

Techniques: Immunostaining, Knockdown, Western Blot, Transwell Assay

Journal: Cell death & disease

Article Title: PLOD3 promotes lung metastasis via regulation of STAT3.

doi: 10.1038/s41419-018-1186-5

Figure Lengend Snippet: Fig. 6 Correlation between the PLOD3-STAT3 signaling pathway and the survival and prognosis of lung cancer patients. a The in situ proximity ligation assay was performed for R-H460 cells stained with STAT3, pS-STAT3, and pY-STAT3. Statistical significance was determined by Student’s t-test. **P < 0.01; ***P < 0.001. b Immunoblotting analysis of lysates after immunoprecipitation from R-H460 cells with expressing PLOD3 and STAT3. c Representative microscopic images of specimens of lung cancer patients stained with an anti-PLOD3, anti-STAT3, anti-pS-STAT3, and anti-pY-STAT3 antibodies. d Correlation between PLOD3 and STAT3 transcripts in the cbioportal datasets of lung cancer patients

Article Snippet: Human PLOD3 cDNA (wild type) purchased from OriGene (Cat No. SC324563) was amplified via polymerase chain reaction (PCR) and cloned into pcDNA3.1-HA vectors.

Techniques: In Situ, Proximity Ligation Assay, Staining, Western Blot, Immunoprecipitation, Expressing

Journal: Cell death & disease

Article Title: PLOD3 promotes lung metastasis via regulation of STAT3.

doi: 10.1038/s41419-018-1186-5

Figure Lengend Snippet: Fig. 7 PLOD3-induced metastasis is independent of the RAS-MAPK pathway. a Ras expression assessed via western blot analysis following transfection with siPLOD3 in R-H460 cells (upper). Ras activity was determined via an enzyme-linked immunosorbent assay-based activity assay after siPLOD3 treatment in R-H460 cells (lower, x-axis: siCON, siPLOD3). b Protein levels of MAP kinases were determined via western blotting after siPLOD3 treatment in R-H460 cells. c A cell invasion assay showing that SB and SP have a limited effect on pro-metastatic PLOD3 in R-H460 cells. (upper). Protein levels of indicated proteins determined via western blotting after treatment with siPLOD3 and MAP kinase inhibitor (p38 inhibitor, SB203580 (SB); JNK inhibitor, SP600125 (SP)) in R-H460 cells (lower). Statistical significance was determined by Student’s t-test. ***P < 0.001

Article Snippet: Human PLOD3 cDNA (wild type) purchased from OriGene (Cat No. SC324563) was amplified via polymerase chain reaction (PCR) and cloned into pcDNA3.1-HA vectors.

Techniques: Expressing, Western Blot, Transfection, Activity Assay, Enzyme-linked Immunosorbent Assay, Invasion Assay

Journal: bioRxiv

Article Title: Trained Immunity Affecting Dendritic Cell Differentiation and Function in Rheumatoid Arthritis

doi: 10.1101/2025.02.28.640673

Figure Lengend Snippet: (A) Representative flow cytometry plots of bone marrow and spleen from arthritis afflicted RA-SKG and control SKG mice, gated on live CD45 + Lin − cells and showing the CD11c + MHCII + DC cell population. Full gating hierarchy is presented in Figure S2A; average percentage of cells in DC gates for all mice in each group is indicated. (B) Absolute numbers of cDC1, cDC2a, cDC2b, and pDC cells in the bone marrow and spleen of arthritis afflicted RA-SKG and control SKG mice. DCs are defined as live CD45 + Lin − CD11c + MHCII + cells, where the lineage markers are CD3, CD19, NK1.1, Ly6G, F4/80, CD64 and TER119. DCs are subdivided into B220 + PDCA1 + pDCs and B220 − PDCA1 − cDCs, which are further subdivided into the cDC1, cDC2a, and cDC2b subsets based on the expression of XCR1, CD172a/SIRPα, and CLEC12A markers, as shown in Figure S2. Bars represent means ± SEM; data are from n=9 mice per group and consolidated from two independent experiments; statistical analyses used Student’s t -test; * p <0.05, ** p <0.01, ns – not significant. Marrow cell counts are per two tibias and femurs. (C-D) Expression of activation and checkpoint markers on DCs in the bone marrow and spleen of RA-SKG and control SKG mice, gating on DCs as live CD45 + Ly6G − F4/80 − CD11c + MHCII + cells. Bars represent means ± SEM; data from n=5-9 mice per group; MFI – mean or median fluorescence intensity; statistical analyses with Student’s t -test; * p <0.05, ** p <0.01, *** p <0.001; ns – not significant.

Article Snippet: For competitive bone marrow transplantations, cohorts of wild type CByJ.SJL(B6)-Ptprca/J (JAX 006584, congenic for CD45.1) mice were irradiated with 2 doses of 4.5Gy, delivered 3 hours apart, in a RS-2000 irradiator (Rad Source), and engrafted with a 1:1 mix of bone marrow cells from CD45.2 + SKG mice with active arthritis (RA-SKG) and from control CD45.1 + allotype-marked mice, delivered as intravenous injection in sterile PBS.

Techniques: Flow Cytometry, Control, Expressing, Activation Assay, Fluorescence

Journal: Cell Death Discovery

Article Title: Bisphenol-A disrupts mitochondrial functionality leading to senescence and apoptosis in human amniotic mesenchymal stromal cells

doi: 10.1038/s41420-025-02620-8

Figure Lengend Snippet: Expression of p53, p21 and p27 cell-cycle regulating genes was analysed by RT-PCR 24 h after exposure to increasing BPA concentrations (0.05, 0.1, 0.2, 0.3, 0.35 and 0.4 μM). Results are presented as fold-change relative to control conditions (MetOH) ( A ). Furthermore, p21 protein expression and its nuclear translocation in hAMSC were assessed 24 h after exposure to increasing BPA concentrations (0.1, 0.2, 0.3, and 0.4 μM) using immunofluorescence analysis. Pictures were acquired at ×20 magnification (scale bar, 50 μm) ( B ). p21-positive cells were identified by a rosy-red signal, while nuclei were stained with DAPI (blue). The total number of p21 positive cells was quantified and is reported in ( C ). Fluorescence intensity of p21, measured as Normalized Integrated Density, is presented in ( D ). Histograms represent the mean values ± standard deviation from n = 4 ( A ) and n = 3 ( B ) independent experiments. Statistical analysis was performed versus the control condition represented by the MetOH: p < 0.01(**), p < 0.001(***), p < 0.0001(****).

Article Snippet: Subsequently, cells were washed three times with Tris Buffered Saline (TBS) for 5 min each. hAMSC were incubated with p21 primary antibody (Waf1/Cip1/CDKN1A p21, sc-6246, Santa Cruz Biotechnology, Texas, USA), diluted 1:100 in normal goat serum (NGS, Invitrogen, Waltham USA, #10000C) and incubated overnight at 4 °C in the dark.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Translocation Assay, Immunofluorescence, Staining, Fluorescence, Standard Deviation

Journal: Cell Death Discovery

Article Title: Bisphenol-A disrupts mitochondrial functionality leading to senescence and apoptosis in human amniotic mesenchymal stromal cells

doi: 10.1038/s41420-025-02620-8

Figure Lengend Snippet: Alteration in mitochondrial function, evidenced by enhanced production of ROS in hAMSC after BPA exposure, trigger the downstream activation of several signalling pathways. ROS accumulation activates an antioxidant response, which is marked by an elevated production of Nrf2 and HO-1. Concurrently, the high ROS level induce sterile inflammation, resulting in increased transcription of factors involved in inflammasome complex formation and activation. However, this increased transcription does not translate to higher production of IL-1β, the downstream effector of the inflammasome pathway. Instead, oxidative stress promotes p53 stabilization and upregulates p21 and p27 genes, as well as components of the senescence-associated secretory phenotype (SASP). Ultimately, the senescent state serves as a prelude to apoptosis, which occurs when hAMSC are exposed to the highest BPA concentrations.

Article Snippet: Subsequently, cells were washed three times with Tris Buffered Saline (TBS) for 5 min each. hAMSC were incubated with p21 primary antibody (Waf1/Cip1/CDKN1A p21, sc-6246, Santa Cruz Biotechnology, Texas, USA), diluted 1:100 in normal goat serum (NGS, Invitrogen, Waltham USA, #10000C) and incubated overnight at 4 °C in the dark.

Techniques: Activation Assay, Sterility

Journal: International Journal of Molecular Sciences

Article Title: Telomerase Inhibitor TMPyP4 Alters Adhesion and Migration of Breast-Cancer Cells MCF7 and MDA-MB-231

doi: 10.3390/ijms20112670

Figure Lengend Snippet: Cancer cell survival after exposure to telomerase inhibitor, TMPyP4. MCF7, MDA-MB-231, and MCF-12A cells were exposed to different concentrations of TMPyP4 (10–100 µM; open bars), doxorubicin (DOX, 0.1 µM) or a combination of both compounds (grey bars) for 24, 48, or 72 h. The viability was assessed using MTT assay. Cell survival was assessed relative to the control sample (C). * p < 0.05, TMPyP4 relative to TMPyP4+DOX; # p < 0.05, relative to control sample. Tests were performed in biological triplicates (each replicate consisted of 8 technical replicates/wells).

Article Snippet: Both human breast adenocarcinoma cell lines MCF7 (ER+, p53 wild type) and MDA-MB-231 (tripel-negative, p53 mutant) and non-tumorigenic epithelial cell line MCF12A were obtained from the American Type Culture Collection (HTB-22, HTB-26, CRL-10782).

Techniques: MTT Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Telomerase Inhibitor TMPyP4 Alters Adhesion and Migration of Breast-Cancer Cells MCF7 and MDA-MB-231

doi: 10.3390/ijms20112670

Figure Lengend Snippet: TMPyP4 alters telomerase expression and activity. The contribution of TMPyP4 to telomerase expression in MCF7 ( A ) and MDA-MB-231 cells ( B ) was assessed using qPCR. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was used as a housekeeping/relative gene. Cells were treated for 72 h with TMPyP4 (10, 20 or 50 μM), DOX (0.1 μM) or a combination of those two compounds, total RNA was isolated, poly(A+)mRNA was reversely transcribed, and hTERT gene expression was assessed. Similarly, the influence of studied compounds on telomerase activity was analyzed using telomeric repeat amplification protocol (TRAP) assay in MCF7 ( C ) and MDA-MB-231 cells ( D ); cell treatment, as mentioned in the expression study. The basal level of hTERT ( E ) in studied cell lines was assessed using western blot after 24 h from seeding and densitometry analysis, relative to loading control GAPDH, was performed to reveal the difference ( F ). The same amount of total protein from both cell lines was used, i.e., 40 µg. * p < 0.05, relative to control sample.

Article Snippet: Both human breast adenocarcinoma cell lines MCF7 (ER+, p53 wild type) and MDA-MB-231 (tripel-negative, p53 mutant) and non-tumorigenic epithelial cell line MCF12A were obtained from the American Type Culture Collection (HTB-22, HTB-26, CRL-10782).

Techniques: Expressing, Activity Assay, Isolation, Gene Expression, Amplification, TRAP Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Telomerase Inhibitor TMPyP4 Alters Adhesion and Migration of Breast-Cancer Cells MCF7 and MDA-MB-231

doi: 10.3390/ijms20112670

Figure Lengend Snippet: TMPyP4 efficiently decreases hTERT accumulation in a time-dependent manner. MCF7 ( A ) and MDA-MB-231 ( B ) cells were subjected to treatment with TMPyP4 (20 µM), BIBR 1532 (20 µM) or DOX (0.1 µM) in a time-course experiment. After individual time intervals (24, 48, or 72 h) cells were lysed, and immunoidentification of hTERT was performed. The concentration of compounds was adjusted according to MTT assay. GAPDH was used as a loading control. Densitometry analysis of the western blot results was performed relative to the loading control, GAPDH ( C , D ). * p < 0.05, relative to control sample.

Article Snippet: Both human breast adenocarcinoma cell lines MCF7 (ER+, p53 wild type) and MDA-MB-231 (tripel-negative, p53 mutant) and non-tumorigenic epithelial cell line MCF12A were obtained from the American Type Culture Collection (HTB-22, HTB-26, CRL-10782).

Techniques: Concentration Assay, MTT Assay, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Telomerase Inhibitor TMPyP4 Alters Adhesion and Migration of Breast-Cancer Cells MCF7 and MDA-MB-231

doi: 10.3390/ijms20112670

Figure Lengend Snippet: The contribution of TMPyP4 to cell-cycle modification. MCF7 (5.1) and MDA-MB-231 (5.2) were subjected to flow cytometry assessment ( A ), followed by immunodetection of cell cycle associated proteins ( B ) and densitometry analysis of the blots i.e. Cyclin B1 ( C ), Cyclin D1 ( D ), Cyclin E ( E ), respectively. Both studied cell lines were subjected to the analysis of cell cycle after treatment with 10, 20, 50 μM TMPyP4, 0.1 μM DOX or a combination of both compounds for 72 h since no significant effects were revealed in shorter time interval neither in MTT nor in a clonogenic assay. Detection of cell-cycle phase was performed in MCF7 (5.1) and MDA-MB-231 cells (5.2) using propidium iodide staining and flow cytometry. Immunodetection of cyclins B1, D1, and E after 72 h treatment with TMPyP4 (0, 10, 20 or 50 μM), DOX (0.1 μM) or a combination of these compounds was performed using western blot in MCF7 (5 B1 ) as well as MDA-MB-231 cells (5 B2 ) followed by densitometry analysis (5 C1 , D1 , E1 ,5 C2 , D2 , E2 , respectively). * p < 0.05, relative to control sample. Densitometry analysis was performed out of 3 scanned membranes from 3 independent experiments relative to the loading control, GAPDH.

Article Snippet: Both human breast adenocarcinoma cell lines MCF7 (ER+, p53 wild type) and MDA-MB-231 (tripel-negative, p53 mutant) and non-tumorigenic epithelial cell line MCF12A were obtained from the American Type Culture Collection (HTB-22, HTB-26, CRL-10782).

Techniques: Modification, Flow Cytometry, Immunodetection, Clonogenic Assay, Staining, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Telomerase Inhibitor TMPyP4 Alters Adhesion and Migration of Breast-Cancer Cells MCF7 and MDA-MB-231

doi: 10.3390/ijms20112670

Figure Lengend Snippet: TMPyP4 does not induce DNA damage/repair-associated pathways. The influence of TMPyP4 (0.5, 5, 10 or 20 µM) on individual proteins was assessed in both MCF7 ( A ) and MDA-MB-231 cells ( B ). Cells were treated for 72 h, followed by lysis and immunodetection. Typical result out of 3 replicates was demonstrated. In experiments that were assessing proteins with a low basal level, the additional positive control was included (2 μM DOX, 2 h treatment). Densitometry analysis was performed out of 3 scanned membranes from 3 independent experiments, relative to the loading control, GAPDH (hTERT, γ-H2A.X, P53) or tubulin (pATM, DNA-PKCs, PARP, p21, PCNA). * p < 0.05, relative to control sample.

Article Snippet: Both human breast adenocarcinoma cell lines MCF7 (ER+, p53 wild type) and MDA-MB-231 (tripel-negative, p53 mutant) and non-tumorigenic epithelial cell line MCF12A were obtained from the American Type Culture Collection (HTB-22, HTB-26, CRL-10782).

Techniques: Lysis, Immunodetection, Positive Control, Control

Journal: International Journal of Molecular Sciences

Article Title: Telomerase Inhibitor TMPyP4 Alters Adhesion and Migration of Breast-Cancer Cells MCF7 and MDA-MB-231

doi: 10.3390/ijms20112670

Figure Lengend Snippet: TMPyP4 triggers migration impairment in breast-cancer cells. MCF7 and MDA-MB-231 cells were incubated until reaching ca 70% confluence followed by a scratch with a sterile pipette tip. Samples were treated for 48 h with TMPyP4 (0.5 or 5 μM), and the wound healing properties were documented using a Zeiss Axiovert microscope (magnification 100 ×). Typical result out of 3 replicates was demonstrated.

Article Snippet: Both human breast adenocarcinoma cell lines MCF7 (ER+, p53 wild type) and MDA-MB-231 (tripel-negative, p53 mutant) and non-tumorigenic epithelial cell line MCF12A were obtained from the American Type Culture Collection (HTB-22, HTB-26, CRL-10782).

Techniques: Migration, Incubation, Sterility, Transferring, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Telomerase Inhibitor TMPyP4 Alters Adhesion and Migration of Breast-Cancer Cells MCF7 and MDA-MB-231

doi: 10.3390/ijms20112670

Figure Lengend Snippet: TMPyP4-mediated inhibition of adhesion properties of cancer cells. MCF7 and MDA-MB-231 cells were grown till reaching 70% confluence and exposed to 10, 20 or 50 μM TMPyP4 for 24 h. Then cells were trypsinized, and 5 × 10 5 cells were transferred to fresh dishes. Cells were analyzed under the phase contrast microscope and photographed after 15, 30, 60 min, 3 h and 18 h (Zeiss Axiovert microscope; magnification 100×). The most significant difference was observed after 18 h. (All studied time intervals were shown in the ). Typical result out of 3 replicates was demonstrated.

Article Snippet: Both human breast adenocarcinoma cell lines MCF7 (ER+, p53 wild type) and MDA-MB-231 (tripel-negative, p53 mutant) and non-tumorigenic epithelial cell line MCF12A were obtained from the American Type Culture Collection (HTB-22, HTB-26, CRL-10782).

Techniques: Inhibition, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Telomerase Inhibitor TMPyP4 Alters Adhesion and Migration of Breast-Cancer Cells MCF7 and MDA-MB-231

doi: 10.3390/ijms20112670

Figure Lengend Snippet: TMPyP4 down-regulates proteins engaged in adhesion pathway without inducing apoptosis. MCF7 and MDA-MB-231 cells were subjected to treatment with TMPyP4 (20 µM), BIBR 1532 (20 µM) or DOX (0.1µM) in a time-course experiment (24, 48, or 72 h). After individual time intervals, cells were lysed, and immunoidentification of B1 integrin, FAK (including p-FAK Y397 and p-FAK Y576/577) and paxillin (including p-paxillin) were performed ( A ). GAPDH was used as a loading control. Additionally, caspase-3 detection was performed, but due to a mutated caspase-3 coding gene in MCF7 cells, immunodetection did not reveal any signal. Densitometry analysis ( B ) was performed out of 3 scanned membranes from 3 independent experiments relative to the loading control, GAPDH. * p < 0.05, relative to control sample.

Article Snippet: Both human breast adenocarcinoma cell lines MCF7 (ER+, p53 wild type) and MDA-MB-231 (tripel-negative, p53 mutant) and non-tumorigenic epithelial cell line MCF12A were obtained from the American Type Culture Collection (HTB-22, HTB-26, CRL-10782).

Techniques: Control, Immunodetection