human ews cell line a 673  (ATCC)

Journal: Journal of Cell Communication and Signaling

Article Title: Circulating miR34a levels as a potential biomarker in the follow-up of Ewing sarcoma

doi: 10.1007/s12079-020-00567-2

Figure Lengend Snippet: miR34a reduces tumor growth and improves EWS cell chemosensitivity. The effects of miR34a on EWS cells were evaluated and the controls (parental cell line A673 and the empty vector pMIF-EV) and the miR34a-overexpressing variants (pMIF-miR34a #11, pMIF-miR34a #28, pMIF-miR34a #31, and pMIF-miR34a #39) were compared. (a) Evaluation of miR34a expression by RT-qPCR in all transduced selected clones (pMIF-miR34a #11, #28, #31, and #39) compared to controls (A673 and pMIF-EV). The relative miRNA levels were normalized to the expression of RNU6b. The data are shown as the mean ± SE (**p < 0.01; ***p < 0.001, one-way ANOVA). (b) Cell growth in monolayer conditions is displayed. Data are represented as the mean value ± SE (*p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA). (c) The ability to grow in anchorage-independent conditions is shown by the pictures; the number above each picture represents the mean number of colonies observed in soft-agar ± SE (scale bar: 200 μm) (***p < 0.001, one-way ANOVA). (d) Cell sensitivity to DXR and VCR is represented. The columns show the mean values ± SE (*p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA)

Article Snippet: The human EWS cell line A673 was provided by American Type Culture Collection (Rockville, MD, USA).

Techniques: Plasmid Preparation, Expressing, Quantitative RT-PCR, Clone Assay

Journal: Journal of Cell Communication and Signaling

Article Title: Circulating miR34a levels as a potential biomarker in the follow-up of Ewing sarcoma

doi: 10.1007/s12079-020-00567-2

Figure Lengend Snippet: Stable expression of miR34a impairs EWS tumor growth in mice. (a) Tumor growth in mice after s.c. injection of A673, pMIF-EV, pMIF-miR34a #11, and pMIF-miR34a #39 cells. Volume values are shown as the mean ± SE (*p < 0.05, ***p < 0.001; one-way ANOVA, compared to pMIF-EV). (b) Single tumor growth curves of mice injected with pMIF-miR34a #39. Animals became positive starting from 39 days after cell injection, when animals of the other groups had already been sacrificed but then tumor growth was very rapid

Article Snippet: The human EWS cell line A673 was provided by American Type Culture Collection (Rockville, MD, USA).

Techniques: Expressing, Injection

Journal: Journal of Cell Communication and Signaling

Article Title: Circulating miR34a levels as a potential biomarker in the follow-up of Ewing sarcoma

doi: 10.1007/s12079-020-00567-2

Figure Lengend Snippet: In situ expression of miR34a and evaluation of proliferation markers in tumors developed from injection of EWS cells expressing or not expressing miR34a. (a) The tissue sections were hybridized with miR34a or RNU6b (used as positive control) probes to perform ISH. Positivity to miR34a or RNU6b is shown as blue precipitates. All slides were counterstained with nuclear fast red. The pink sections are negative. Positive sections or those with positive zones are in blue, and the intensity depends on the amount of expression. Representative images are shown for tumors developed from injection of control (A673, pMIF-EV) or miR34a transduced cells (pMIF-miR34a #11, pMIF-miR34a #39) (magnification 20x. Scale bar: 50 μm). (b) Expression of Ki-67 and Cyclin D1 in the same tumors. Representative images of cell positivity for Ki-67 and Cyclin D1 are displayed (magnification 40x. Scale bar: 50 μm). For pMIF-miR34a #39 tumors, details are presented to show their differential expression in the area with or without expression of miR34a. The % of positive cells is shown ± SE (**p < 0.01; one-way ANOVA). All the pictures were taken from tumor samples isolated at the time of mouse sacrifice.

Article Snippet: The human EWS cell line A673 was provided by American Type Culture Collection (Rockville, MD, USA).

Techniques: In Situ, Expressing, Injection, Positive Control, Control, Quantitative Proteomics, Isolation

Journal: Journal of Cell Communication and Signaling

Article Title: Circulating miR34a levels as a potential biomarker in the follow-up of Ewing sarcoma

doi: 10.1007/s12079-020-00567-2

Figure Lengend Snippet: Detection of released miR34a in vitro and in the plasma of mice bearing EWS cell-induced tumors expressing or not expressing miR34a. (a) miR34a expression levels were analyzed by RT-qPCR in controls (A673 and pMIF-EV) and miR34a transduced cells (pMiF-miR34a #11 and pMIF-miR34a #39), as well as in their supernatants. pMIF-EV was used as the calibrator. The data are presented as the mean ± SE (*p < 0.05; **p < 0.01; ***p < 0.001, one-way ANOVA). (b) Circulating levels of miR34a in the plasma of mice bearing xenografts (pMIF-EV and pMIF-miR34a #11 cells) are shown at days 12, 19, and 26 after cell injection. miR34a amounts were measured by RT-qPCR absolute quantification and expressed as # copies/μl plasma. The results are represented as the mean ± SE (*p < 0.05; Student’s t test). (c) Tumor volumes from the same mice bearing xenografts are plotted at days 5, 12, 19, and 26 from cell injection and are presented as the mean ± SE (**p < 0.01; Student’s t test)

Article Snippet: The human EWS cell line A673 was provided by American Type Culture Collection (Rockville, MD, USA).

Techniques: In Vitro, Clinical Proteomics, Expressing, Quantitative RT-PCR, Injection, Quantitative Proteomics

Journal: American Journal of Cancer Research

Article Title: Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway

doi:

Figure Lengend Snippet: Pharmacological inhibition of the proton pump V-ATPase causes tumor necrosis in a Ewing’s sarcoma xenograft model. A. A673 cells were subcutaneously injected in NOD/SCID mice. Mice were then treated with different concentration of omeprazole, a V-ATPase inhibitor (0 mg/kg n = 14, 2.5 mg/kg n = 7, 10 mg/kg n = 14), to reduce intratumoral acidosis. H&E staining of tumor sections, representative images of the necrotic area, scale bar, 100 µm; B. Box plot of the tumor volume. Mean ± SE; C. Box plot of the tumor necrosis. Mean ± SE (*P < 0.05, **P < 0.01).

Article Snippet: HOS, Saos2, MG-63 (osteosarcoma), and A673 (Ewing sarcoma) human cell lines were purchased from the American Type Culture Collection, cultured in IMDM plus 20 unit/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at pH 7.4, and incubated at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Inhibition, Injection, Concentration Assay, Staining

Journal: American Journal of Cancer Research

Article Title: Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway

doi:

Figure Lengend Snippet: Extracellular acidosis promotes cell survival in bone sarcoma via the NF-κB pathway. (A) Heat map representation of the fold increase of the expression of apoptosis and stress related-genes of osteosarcoma cells (MG63, HOS, Saos-2) after short-term acidosis. mRNA were analyzed by deep-sequencing after that cells were cultured at pH 6.5 compared to physiological medium (pH 7.4) for 24 h. Colors on the heat map indicate the log2 ratios of expression (representing normalized read counts). Red, upregulation; green, downregulation. (B) Cells were cultured under different pH (6.5 and 7.4) for 24 h as described in (A), and NF-κB mRNA expression and activation were quantified in cell lysates. (C) NF-κB1 (p50) and RelB nuclear protein concentration of osteosarcoma and Ewing sarcoma cells. Mean ± SE (n = 3 for HOS and MG63, and n = 5 for A673, *P < 0.05). (D) Signaling pathways of the tumor necrosis factor receptor (TNFR) family that mediates both survival and apoptotic pathways. The draw represents the pathways and adaptor molecules that are involved in the signaling that follows the acid stress: the NF-κB activation pathway via Akt (1), via the canonical pathway (2), via the non-canonical pathway (3), the activation of the AP1 complex transcriptional factor (4), and the TRAF-mediated death signaling with the activation of caspase cascade (5).

Article Snippet: HOS, Saos2, MG-63 (osteosarcoma), and A673 (Ewing sarcoma) human cell lines were purchased from the American Type Culture Collection, cultured in IMDM plus 20 unit/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at pH 7.4, and incubated at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Expressing, Sequencing, Cell Culture, Activation Assay, Protein Concentration, Protein-Protein interactions

Journal: American Journal of Cancer Research

Article Title: Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway

doi:

Figure Lengend Snippet: Targeting of cIAP protein or of TRAF1 inhibit cell viability of A673 cells when cultured in acid condition. A. Inhibition of cell viability as verified by cell counting after the treatment with LCL161 (25 mM). The inhibitor was more effective at pH 6.5 than at pH 7.4. Mean ± SE (n = 3, *P < 0.05); B. Induction of apoptosis as verified by the counting of apoptotic bodies after the treatment with LCL161 (25 mM) of cells cultured at low pH (6.5). Mean ± SE (n = 3, *P < 0.05); C. mRNA analysis of TRAF1 expression by Q-RT-PCR after the treatment with specific siRNA at low pH (pH 6.5); D. Inhibition of cell viability as verified by acid phosphatase indirect assay after the treatment with specific siRNA at low pH (pH 6.5). Mean ± SE (n = 19, ****P < 0.001); E. Correlation of mRNA level between different proteins of the TRAF-cIAP destruction complex in Ewing sarcoma xenografts with acid-related markers V-ATPase V1B2 or LAMP2 (n = 6, *P < 0.05, **P < 0.01).

Article Snippet: HOS, Saos2, MG-63 (osteosarcoma), and A673 (Ewing sarcoma) human cell lines were purchased from the American Type Culture Collection, cultured in IMDM plus 20 unit/mL penicillin, 100 μg/mL streptomycin, and 10% FBS at pH 7.4, and incubated at 37°C in a humidified 5% CO 2 atmosphere.

Techniques: Cell Culture, Inhibition, Cell Counting, Expressing, Reverse Transcription Polymerase Chain Reaction