u118 atcc  (ATCC)

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA675-5p inhibitor’s dual role as novel therapeutic alternative or sensitizing treatment in resistant glioma models

doi: 10.1016/j.omtn.2025.102647

Figure Lengend Snippet: miRNA657-5p inhibitor has an anti-proliferative effect mediated by HIF-1α down-regulation (A) Viability of TMZ-resistant glioma cell lines (T98, LN18, and U118) cells, assessed by means of Trypan blue exclusion test, and expressed as the percentage of viable cells compared with untreated cells after treatment with miRNA675-5p inhibitor for 18 h. ∗∗ p < 0.01 vs. scramble-treated cells. (B) ATP level in T98, LN18, and U118 cells analyzed by Cell Tox Green kit and expressed as relative fluorescence units compared with scramble-treated cells. ∗∗∗ p < 0.001 vs. control cells. (C) Gene expression analysis for BAX , BAD , and BCL-2 analyzed by real-time PCR in T98, LN18, and U118 cells after treatment with miRNA675-5p inhibitor. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as fold of induction (FOI) of the ratio between treated and scramble-treated cells. ∗ p < 0.05; ∗∗ p < 0.01 inhibitor- vs. scramble-treated cells. (D) Analysis of caspase 3/7 activity evaluated by Caspase-Glo 3/7 Assay and expressed as relative luminescent units. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 inhibitor- vs. scramble-treated cells. (E) In silico analysis of an integrative map reconstructed after the public database (The Cancer Genome Atlas Program) interrogation based on protein-protein interaction (called interactome). (F) Gene expression analysis for HIF-1α analyzed with real-time PCR in T98, LN18, and U118 cells after treatment with miRNA675-5p inhibitor. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as the FOI of the ratio between treated and scramble-treated cells. ∗∗ p < 0.01 inhibitor- vs. scramble-treated cells. (G) ELISA-based HIF-1α nuclear (black columns) and cytoplasm (gray columns) quantification after miRNA675-5p inhibitor treatment. The data are expressed as absorbance at 450 nm. ∗∗ p < 0.01 and ∗∗∗ p < 0.001 vs. control cells in nuclear vs. cytoplasmatic localization. Graphs represent mean values ± SD of three independent experiments.

Article Snippet: U251 (ICLC, Ospedale San Martino, Genova, Italy), T98, LN18 and U118 ATCC (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI1640 (U251 and T98) or DMEM High Glucose (LN18 and U118), both supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, and 2 mM glutamine (all Euroclone, Milan, Italy) in a humidified atmosphere of 5% of CO 2 at 37°C.

Techniques: CellTox Assay, Fluorescence, Control, Gene Expression, Real-time Polymerase Chain Reaction, Activity Assay, Caspase-Glo Assay, In Silico, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA675-5p inhibitor’s dual role as novel therapeutic alternative or sensitizing treatment in resistant glioma models

doi: 10.1016/j.omtn.2025.102647

Figure Lengend Snippet: miRNA675-5p inhibitor effect is due to oxidative stress (A) Luminescent assay (ROS-Glo H2O2 Assay kit) was applied to measure H 2 O 2 levels in cell culture medium of different cell lines after treatment with scramble (as control)- or miRNA675-5p inhibitor. Data were expressed as relative luminescence units (RLUs) obtained by luciferase counts normalized for the amount of total proteins quantified by Bradford assay. ∗∗∗ p < 0.001 vs. control cells; # p < 0.05 U251 vs. average ROS level in T98, LN18, and U118 cells. (B) Gene expression analysis for detox-related genes ( SOD-1 and CAT-1 ) and (C) for HIF-1α-related genes ( NRF-2 , Nf-kb , and KEAP1 ) analyzed by means of Real-time PCR in all cells. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as the FOI of the ratio between treated and scramble-treated cells. ∗ p < 0.05, ∗∗ p < 0.01; ∗∗∗ p < 0.001 vs. scramble-treated cells. (D) ATP levels in U251, T98, LN18, and U118 cells after treatment with MitoTEMPO, a ROS scavenger, and inhibitor analyzed by Cell Tox Green expressed as relative fluorescence units compared with inhibitor-treated cells alone. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. inhibitor-treated cells; ### p < 0.001 inhibitor + MitoTEMPO vs. inhibitor alone. (E) Gene expression analysis of apoptosis-related genes ( BAX , BAD , and BCL-2 ) by real-time PCR in all cells after inhibitor of inhibitor + MitoTEMPO treatment. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as the FOI of the ratio between MitoTEMPO plus inhibitor and inhibitor-treated cells. (F–H) Relative GSH (F) and GSSG (G) abundance and GSH/GSSG ratio (H) in U251 and T98 cell line in control condition vs. inhibitor-treated cells obtained by LC-MS analysis ( n = 9). ∗∗∗ p < 0.001 vs. scramble-treated cells; # p < 0.05 and ### p < 0.001 U251 scramble-treated (control) cells vs. T98 scramble-treated (control) cells. Graphs represent mean values ± SD of three independent experiments.

Article Snippet: U251 (ICLC, Ospedale San Martino, Genova, Italy), T98, LN18 and U118 ATCC (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI1640 (U251 and T98) or DMEM High Glucose (LN18 and U118), both supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, and 2 mM glutamine (all Euroclone, Milan, Italy) in a humidified atmosphere of 5% of CO 2 at 37°C.

Techniques: Luminescence Assay, H2O2 Assay, Cell Culture, Control, Luciferase, Bradford Assay, Gene Expression, Real-time Polymerase Chain Reaction, CellTox Assay, Fluorescence, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular Therapy. Nucleic Acids

Article Title: miRNA675-5p inhibitor’s dual role as novel therapeutic alternative or sensitizing treatment in resistant glioma models

doi: 10.1016/j.omtn.2025.102647

Figure Lengend Snippet: miRNA675-5p sensitizes resistant cells to subsequent TMZ treatment (A and B) Representative images of immunoblots (A) and quantification histograms (B) of regulatory associated protein of mtor complex 1 (RPTOR) expression. RPTOR level was assessed in U251 (left) and T98 (right) scramble (black bars) cell lines, treated with TMZ (light gray bars) miRNA675-5p inhibitor (gray bars), or inhibitor followed by TMZ treatment (dark gray bars). Mean ± SD. ∗Significant difference, ANOVA and Tukey’s test, n = 3, p < 0.05. (C) Viability of resistant glioma cell lines (T98) cells, assessed by means of Trypan blue exclusion test, and expressed as live or dead cells after miRNA675-5p inhibition and subsequent TMZ challenge, in absence or presence of the refresh period. ∗∗ p < 0.01 dead vs. live cells; ## p < 0.01 dead cells in control vs. TMZ-treated cells. (D) Cytotoxicity evaluation of the average of T98, LN18, and U118 cells analyzed by Cell Tox Green expressed as relative fluorescence units (RFUs) compared with scramble-treated cells, in absence or presence of the refresh period. (E) Gene expression analysis of apoptosis related genes ( BAX , BAD , and BCL-2 ) by real-time PCR in all cells after TMZ treatment subsequent to miRNA675-5p inhibition. Data were normalized to β-ACTIN , and the ΔΔCt values were expressed as fold of induction (FOI) of the ratio between inhibitor + TMZ- and scramble + TMZ-treated cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 of inhibitor + TMZ- vs. scramble + TMZ-treated cells. (F) Comparison of HIF-1α expression level between U251 basal level and T98 basal and treated cells. Data were normalized to β-ACTIN , and the ΔCt values were depicted in the graph. ∗∗ p < 0.01 inhibitor alone vs. inhibitor + TMZ treatment. (G) Gene expression analysis of HIF-1α and miRNA675-5p levels by real-time PCR in all cells after TMZ treatment subsequent to miRNA675-5p inhibition. Data were normalized to β-ACTIN or U6 , respectively housekeeping reference for gene and miRNA expression, and the ΔΔCt values were expressed as FOI of the ratio between inhibitor + TMZ- and scramble + TMZ-treated cells. ∗ p < 0.05, ∗∗∗ p < 0.001 inhibitor + TMZ-vs. scramble + TMZ-treated cells.

Article Snippet: U251 (ICLC, Ospedale San Martino, Genova, Italy), T98, LN18 and U118 ATCC (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI1640 (U251 and T98) or DMEM High Glucose (LN18 and U118), both supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin, and 2 mM glutamine (all Euroclone, Milan, Italy) in a humidified atmosphere of 5% of CO 2 at 37°C.

Techniques: Western Blot, Expressing, Inhibition, Control, CellTox Assay, Fluorescence, Gene Expression, Real-time Polymerase Chain Reaction, Comparison

Journal: International Journal of Cancer. Journal International du Cancer

Article Title: Human BLCAP transcript: new editing events in normal and cancerous tissues

doi: 10.1002/ijc.25022

Figure Lengend Snippet: Increased editing activity observed on BLCAP mRNA. A172 and U118 astrocytoma cell lines were transfected with EGFP empty vector or a vector expressing EGFP-ADAR2. The editing events present in BLCAP mRNA are expressed as a percentage ( y -axis). On the x -axis are the untransfected astrocytoma U118 and A172 cell lines, the cell lines transfected with EGFP vector and with EGFP-ADAR2. ADAR2 increased the number of BLCAP transcripts undergoing editing in both cell lines.

Article Snippet: Tissue and cell-line samples used in this study were identified by progressive identification numbers, ID 1–21: (ID1) human heart from Clonthech, BD Biosciences; (ID2) human bladder from Clonthech, BD Biosciences; (ID3) normal blood lymphocytes, patient 2630; (ID4) normal epithelial tissue from colonic normal mucosa, patient 2630; (ID5) skin fibroblast from patient 2630; (ID6) white matter, patient n.22; (ID7) human bladder carcinoma grade II, (ID8) human bladder carcinoma grade III; (ID9) human bladder carcinoma grade III 727-1; (ID10) tumorigenic human bladder cell line (TCC) HU 609 (kind gift from Dr. Toben Ørntoft, Denmark); (ID11) human bladder cell line (TCC) HCV 29 (kind gift from Dr. Toben Ørntoft, Denmark); (ID12) human bladder epithelial invasive carcinoma cell line (TCC) T24 (ATCC); (ID13) colorectal cancerous epithelial tissue from a polyp developed by patient 2630; (ID14) colorectal cancerous epithelial tissue from patient 425; (ID15) epithelial colorectal cancer cell line from patient 425 (MYH deficient), VACO425; (ID16) epithelial colorectal cancer cell line CACO2; (ID17) epithelial colorectal cancer cell line HRT18; (ID18) pediatric astrocytoma grade IV patient n.22; (ID19) astrocytoma cell line U118 MG (ATCC); (ID20) astrocytoma cell line A172 (ATCC); (ID21) HeLa cell line (ATCC).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing