4t1  (ATCC)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer

doi: 10.1007/s00018-023-05046-6

Figure Lengend Snippet: MAPK12-G4 binding selectivity and anticancer properties of naphthalene diimide compound. A Chemical formula of synthetic naphthalene diimide derivatives, TGS24 and another isogenic compound TGS25. B ITC profiles showing intermolecular interactions between wild-type MAPK12-GQ and TGS24, TGS25 in potassium phosphate buffer (pH 7.0) and 100 mM KCl at 25 °C temperature. Top panels: enthalpic heat released versus time at 25 °C during titrations. Bottom panels: thermogram of the integrated peak intensities plotted against the molar ratio of the complex. Thermodynamic parameters of intermolecular interactions (change in binding enthalpy (ΔH), entropy (ΔS), binding stoichiometry (n) and binding free energy (ΔG) are estimated from best-fit curves. C Flow cytometric analysis of apoptosis in MDAMB-231 cells by Annexin V-FITC-PI dual staining assay. 2 × 105 cells treated with TGS24 (50 nM and 100 nM) for 24 h. FITC: Fluorescein isothiocyanate; PI: Propidium iodide. In the typical flow cytometric quadrants, lower left quadrant (Annexin V-FITC negative/PI negative) denotes live cells, lower right quadrant (Annexin V-FITC positive/PI negative) denotes early apoptotic cells, upper quadrants (Annexin V-FITC positive/PI positive and Annexin V-FITC negative/PI positive) define late apoptotic and dead cells. D Percentage of live (Annexin V-FITC negative/PI negative), early apoptotic (Annexin V-FITC positive/PI negative), and late apoptotic/dead (Annexin V-FITC positive/PI positive and Annexin V-FITC negative/PI positive) cells calculated from flow cytometric analyses in MDAMB-231 cells treated with TGS24 (50 nM and 100 nM) for 24 h. Error bars represent mean ± SE (N = 3). E pGL4.72[hRlucCP] luciferase constructs having the inserts containing MAPK12 promoter sequences having both of wild-type G4-elements (wt), single-deletion of GQ-1 motif (GQ(d1)) or GQ-2 motif (GQ(d2)), and double-deletions of GQ-1 and GQ-2 (GQ-null) ahead of the hRluc coding region. The promoter sequences are cloned into KpnI and HindIII restriction sites. hRluc, Renilla luciferase gene; P1, promoter sequences. F Dual-luciferase assays. Evaluation of MAPK12 promoter activities using the luciferase constructs with or without the wild-type G4-forming sequences in MDAMB-231 cells. Promoter activities are determined by Renilla/Firefly luminescence values in control cells and cells treated with TGS24 (50 nM and 100 nM) and TGS25 (1 and 2 μM) for 24 h. Error bars represent mean ± SE (N = 3). Statistical differences in the luciferase activities compared to that of the control cells use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001). G Tumor regression by TGS24 (800 pg per kg body weight) treatment and without treatment (H) in BALB/c mice (average body weight 25 g) bearing palpable 4T1-breast tumors. Tumor volume measured up to 21 days at a 5 days interval after tumor implantation. Statistical analyses of tumor regression compared to that of the 0th day post-TGS24 treatment use two-tailed Student's t test (*P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: 4T1 (murine mammary carcinoma) tumor cells were acquired from the National Centre for Cell Science (NCCS) central cell repository, Pune and grown in RPMI 1640 media, containing 1 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine, 100 units/l penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamycin sulfate, 50 µg/ml plasmocure, and 10% FBS at 37 °C with 5% CO 2 .

Techniques: Binding Assay, Staining, Luciferase, Construct, Clone Assay, Two Tailed Test, Tumor Implantation