mouse breast cancer cell line 4t1  (ATCC)

Journal: Scientific Reports

Article Title: Retarding breast tumor growth with nanoparticle-facilitated intravenous delivery of BRCA1 and BRCA2 tumor suppressor genes

doi: 10.1038/s41598-022-25511-9

Figure Lengend Snippet: Cell viability assessment of BRCA1 and BRCA2 plasmid delivery in (a) MCF-7 (b) MDA-MB-231 and (c) 4T1 cells treated with BRCA1 + NP, and BRCA2 + NP formulations for 48 h. The experiments were performed three times in each of the cell lines and values were presented as mean ± SD of triplicates in MTT assay. * indicated p < 0.05 compared to NPs control.

Article Snippet: Two human breast cancer cell lines MCF-7, and MDA-MB-231, one mouse breast cancer cell line 4T1 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Plasmid Preparation, MTT Assay

Journal: Scientific Reports

Article Title: Retarding breast tumor growth with nanoparticle-facilitated intravenous delivery of BRCA1 and BRCA2 tumor suppressor genes

doi: 10.1038/s41598-022-25511-9

Figure Lengend Snippet: Cytotoxicity imposed by BRCA1 + NP and BRCA2 + NP formulations in three breast cancer cell lines.

Article Snippet: Two human breast cancer cell lines MCF-7, and MDA-MB-231, one mouse breast cancer cell line 4T1 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Retarding breast tumor growth with nanoparticle-facilitated intravenous delivery of BRCA1 and BRCA2 tumor suppressor genes

doi: 10.1038/s41598-022-25511-9

Figure Lengend Snippet: Light microscopic images of two different cell lines following treatment with NP, BRCA1 + NP and BRCA2 + NP. Cells treated with BRCA1 + NP and BRCA2 + NP, revealed decrease in cellular density in MCF-7 (upper panel) and 4T1 (lower panel) cell line, compared to the untreated control cells (concentrated).

Article Snippet: Two human breast cancer cell lines MCF-7, and MDA-MB-231, one mouse breast cancer cell line 4T1 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Retarding breast tumor growth with nanoparticle-facilitated intravenous delivery of BRCA1 and BRCA2 tumor suppressor genes

doi: 10.1038/s41598-022-25511-9

Figure Lengend Snippet: Tumor regression study following intravenous delivery of NP, BRCA1 + NP, and BRCA2 + NP in 4T1 cells-induced tumor mouse model. 4T1 cells were inoculated subcutaneously on the mammary pad of mice. On day 8, as the tumor reached an approximate volume of ~ 25 mm 3 , mice were treated intravenously through tail-vein injection with 100 μL of NP (8 M Ca 2+ ), BRCA1 + NP (40 µg of plasmid) and BRCA2 + NP (40 µg of plasmid); followed by the 2nd dose at day 10. The tumor outgrowth was monitored until day 22. Five mice were used per group and data were represented as mean ± SD. Compared to NP control group, statistical analysis was significant, * when p < 0.05. (* p < 0.05 (NP control and BRCA1 + NP), * p < 0.05 (NP control and BRCA2 + NP).

Article Snippet: Two human breast cancer cell lines MCF-7, and MDA-MB-231, one mouse breast cancer cell line 4T1 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Injection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Retarding breast tumor growth with nanoparticle-facilitated intravenous delivery of BRCA1 and BRCA2 tumor suppressor genes

doi: 10.1038/s41598-022-25511-9

Figure Lengend Snippet: Transgene delivery of BRCA1 + NP and BRCA2 + NP in 4T1-induced tumor mouse model inhibited tumor growth. ( a ) Tumor images captured following sacrifice at day 22. ( b ) Quantitation of tumor volumes in NP control, BRCA1 plasmid-treated mice and BRCA2 plasmid-treated mice. ( c ) Quantitation of tumor weights. Statistical analysis was found significant compared to NP control group, * when p < 0.05.

Article Snippet: Two human breast cancer cell lines MCF-7, and MDA-MB-231, one mouse breast cancer cell line 4T1 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA).

Techniques: Quantitation Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: Reprogramming the tumor immune microenvironment using engineered dual-drug loaded Salmonella

doi: 10.1038/s41467-024-50950-5

Figure Lengend Snippet: a Experimental scheme. Briefly, 4T1 cells were implanted into the mammary fat pad of BALB/c mice. When tumors reached approximately 140 mm 3 , mice received bacteria through i.v injection (day 0). Doxy (+) and CSF-1R antibody (αCSF-1R) were administered as shown. Pimary tumors and metastatic lungs were collected on day 15 and 35, respectively ( b , c ) and flow cytometry was performed on day 9 ( d , e ). b Average growth curves of 4T1 primary tumors ( n = 6 mice examined from two independent experimental replicates; * P = 0.0144, **** P < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test). c Individual images (left) and metastatic nodule count (right) of lungs ( n = 6 exami n ed from two independent experimental replicates; **** P < 0.0001; ns not significant; unpaired two-tailed t -test). d Flow cytome t ry gating of M1 (F4/80 + CD86 + ) and M2 (F4/80 + CD206 + ) macrophages . e Ratio of M1/M2 macrophages in lungs (left) and tumors (right) ( n = 6 mice examined from two independent experimental replicates; **** P < 0.0001; ns, not significant; unpaired two-tailed t -test). f In vivo and ex vivo bioluminescent imaging of MDA-MB-231-FLuc-GFP lung metastasis. Left, images of the mice on day 0 (pre-treatment) and on day 14 (post-treatment) and the excised lungs on day 14; right, quantitation of bioluminescence levels in the lungs. g Frequency of macrophages phagocytosing tumor cells (F4/80 + GFP + ) in the metastatic lungs of ( f ) ( n = 3 mice/group, from one experiment, unpaired two-tailed t -test). h Experimental scheme. i Average growth curves of subcutaneous tumors ( n = 7 mice examined from two independent experimental replicates; **** P < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test). j Kaplan-Meier survival curves ( n = 7 mice examined from two independent experimental replicates; **** P < 0.0001; Log-rank (Mantel-Cox) test). k Metastatic scores of livers. Metastatic nodules on the liver surface were counted at the experimental end point. The scores were classified as follows: 0, 0%; 1, <20%; 2, 20%–50%; 3, >50% ( n = 7 mice examined from two independent experimental replicates; **** P < 0.0001; unpaired two-tailed t -test).

Article Snippet: CT26 (mouse colon carcinoma, CRL-2638), 4T1 (mouse breast cancer, CRL-2539), and HEK293T (human epithelial cell, CRL-3216) cell lines were purchased from the American Type Culture Collection (ATCC, USA).

Techniques: Bacteria, Injection, Flow Cytometry, Two Tailed Test, In Vivo, Ex Vivo, Imaging, Quantitation Assay

Journal: Cancers

Article Title: Evaluation of the Mammalian Aquaporin Inhibitors Auphen and Z433927330 in Treating Breast Cancer

doi: 10.3390/cancers16152714

Figure Lengend Snippet: Auphen treatment of metastatic breast cancer cells increases overall survival in vivo. ( A ) Schematic of orthotopic injection of 4T1 cells in NOD SCID mice treated with either vehicle ( n = 15) or Auphen ( n = 15). ( B ) Growth of tumors in ( A ). ( C ) Kaplan–Meier plot showing overall survival (OS). p -values for OS were determined using the log-rank (Mantel–Cox) test. ( D ) Quantification of median lung metastasis using Welch’s t -test. ( E ) Schematic of orthotopic injection of 4T1 cells in BALB/cJ mice treated with either vehicle ( n = 14) or Auphen ( n = 14). ( F ) Tumor burden of ( E ). ( G ) Kaplan–Meier plot showing overall survival (OS) of ( E ). p -values for OS were determined using the log-rank (Mantel–Cox) test. ( H ) Quantification of median lung metastasis was analyzed using Goodman and Kruskal’s gamma test.

Article Snippet: Mouse 4T1 (CRL-2539) and human AU565 (CRL-2351), BT474 (HTB-20), MCF-7, and MDA-MB-231 cells were purchased from ATCC.

Techniques: In Vivo, Injection

Journal: Cancers

Article Title: Evaluation of the Mammalian Aquaporin Inhibitors Auphen and Z433927330 in Treating Breast Cancer

doi: 10.3390/cancers16152714

Figure Lengend Snippet: Z433927330 treatment of metastatic mammary tumors in vivo. ( A ) Schematic of orthotopic injection of 4T1 cells in syngeneic BALB/cJ mice treated with either vehicle ( n = 4, weekly and biweekly) or Z433927330 ( n = 4, weekly and biweekly). ( B ) Tumor growth curves for biweekly treatment. ( C ) Quantification of lung metastasis using Fisher’s exact test. ( D ) Kaplan–Meier plot showing overall survival for biweekly treatment. p -values for OS were determined using the log-rank (Mantel–Cox) test. ( E ) Tumor growth curves for weekly treatment. ( F ) Quantification of lung metastasis using Goodman and Kruskal’s gamma. ( G ) Kaplan–Meier plot showing overall survival for weekly treatment. p -values for OS were determined using the log-rank (Mantel–Cox) test.

Article Snippet: Mouse 4T1 (CRL-2539) and human AU565 (CRL-2351), BT474 (HTB-20), MCF-7, and MDA-MB-231 cells were purchased from ATCC.

Techniques: In Vivo, Injection

Journal: Cancer & Metabolism

Article Title: Disrupting Na + ion homeostasis and Na + /K + ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo

doi: 10.1186/s40170-024-00343-5

Figure Lengend Snippet: Metabolic characterization and response to ouabain treatment on [Na + ] i . a Principal component analysis of intracellular metabolites (mean values given in Supplementary Table S2). Intracellular concentrations of b lactate (left panel) and c phosphocholine (right panel) were significantly higher in all cancer cells with respect to control epithelial cells ( n = 5). Extracellular metabolite concentrations of d glucose, e glutamine and f lactate after 24 h cell culture. Respective metabolite concentration from fresh media were subtracted such that negative concentrations refer to metabolite consumption while positive concentrations refer to production ( n = 5). g MTT cytotoxicity assay dose response curves following 24 h treatment with ouabain. Measured EC 50 values were: 4T1: 40 µM, MDA-MB-231: 0.4 µM, HCC1954: 0.2 µM, MCF-7: 0.04 µM. h Cell viability in response to 1 µM ouabain for 1 h measured by trypan blue exclusion assay measured no change in cell viability. i Representative TQF 23 Na NMR spectra showing proportionality with cell number. The Tm-DOTP reference peak is from the internal standard. j Quantification of TQF 23 Na NMR relative to cell number and cell volume. Baseline [Na + ] i was higher in all cancer cells with respect to control epithelial cells ( n = 5). Treatment with 1 µM ouabain for 1 h led to a significant increase in [Na + ] i in all human cancer cell lines compared to vehicle control ( n = 5). [Na + ] i was unchanged in the murine 4T1 cell line following 1 h treatment with 1 µM ouabain, ( p = 0.7, n = 5). Significance was assessed using a two-tailed unpaired t-test, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data plotted as mean ± SD

Article Snippet: Human cell lines MDA-MB-231 (CRM-HTB-26); MCF-7 (HTB-22); HCC1954 (CRL-2338) and 4T1 (CRL-2539) mouse cell line were obtained from American Type Culture Collection (ATCC).

Techniques: Cell Culture, Concentration Assay, Cytotoxicity Assay, Trypan Blue Exclusion Assay, Two Tailed Test

Journal: Cancer & Metabolism

Article Title: Disrupting Na + ion homeostasis and Na + /K + ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo

doi: 10.1186/s40170-024-00343-5

Figure Lengend Snippet: Extracellular acidification rate with NKA inhibition . a Seahorse XFe24 glycolytic stress test of extracellular change in pH for the MDA-MB-231 cancer cells (± SEM representing biological reproducibility of n = 3 biological repeats where each n = 3 technical repeats were first averaged). Complete time courses for the other cell lines are given in Supplementary Fig. S8. The stress test comprised 10 mM glucose, 1 μM oligomycin, and 100 mM 2-deoxy-D-glucose indicated by arrows. b Oxygen consumption rate (OCR) measured simultaneously with the ECAR data in panel a . c Plot of the measured ECAR glycolytic rate vs OCR during the 10 mM glucose time window defined in Supplementary Fig. S6 ( n = 3 biological repeats each with n = 3 technical repeats) showing a reduction in glycolytic rate and no change in OCR. Quantified extracellular acidification rate corresponding to glycolytic rates during the 10 mM glucose time window (left panels) and their corresponding OCR (right panels), in control and ouabain treated cells: d 4T1: ECAR ( p = 0.24), OCR ( p = 0.39); e MDA-MB-231: ECAR decreased by 52% ( p = 0.006), OCR ( p = 0.40); f HCC1954: ECAR decreased by 21% ( p = 0.08), OCR ( p = 0.66); g MCF-7: ECAR decreased by 38% ( p = 0.015), OCR ( p = 0.16). n = 3 biological repeats each with n = 3 technical repeats, significance was assessed using a nested unpaired t-test. ns p > 0.05, * p < 0.05, ** p < 0.01

Article Snippet: Human cell lines MDA-MB-231 (CRM-HTB-26); MCF-7 (HTB-22); HCC1954 (CRL-2338) and 4T1 (CRL-2539) mouse cell line were obtained from American Type Culture Collection (ATCC).

Techniques: Inhibition

Journal: Cancer & Metabolism

Article Title: Disrupting Na + ion homeostasis and Na + /K + ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo

doi: 10.1186/s40170-024-00343-5

Figure Lengend Snippet: Glycolytic flux measured with 2 H-NMR. a Time-series of 2 H-NMR spectra showing metabolism of [6,6- 2 H 2 ] d -glucose to [3,3- 2 H 2 ] l -lactate by 4T1 cells in suspension. No 2 H label was lost to HOD, serving as an internal chemical shift and intensity standard at 16.7 mM. Time course and empirical fits performed in Matlab of the normalized peak integrals of the [6,6- 2 H 2 ] d -glucose and [3,3- 2 H 2 ] l -lactate spectral peaks: b 4T1 cells and c MDA-MB-231 cells for vehicle control (filled symbols) and ouabain treated (open symbols). d Quantified glycolytic flux in MCF-10A cells was 0.020 ± 0.003 nmol (pL cells) −1 s −1 ( n = 9). 4T1 cells had a higher baseline glycolytic rate of 0.043 ± 0.007 nmol (pL cells) −1 s −1 ( n = 8) and unchanged rate after ouabain treatment, 0.045 ± 0.010 nmol (pL cells) −1 s −1 ( n = 9, p = 0.6949). Human breast cancer cells all showed higher baseline glycolytic rates than control epithelial cells, MDA-MB-231: 0.054 ± 0.003 nmol (pL cells) −1 s −1 ( n = 7); HCC1954: 0.034 ± 0.006 nmol (pL cells) −1 s −1 ( n = 7); MCF-7: 0.031 ± 0.006 nmol (pL cells) −1 s −1 ( n = 7). Human cells showed a decreased glycolytic rate following ouabain-treatment vs vehicle control, MDA-MB-231: 0.020 ± 0.004 nmol (pL cells) −1 s −1 ( n = 7; p < 0.0001); HCC1954: 0.019 ± 0.008 nmol (pL cells) −1 s −1 ( n = 6; p = 0.004); MCF-7: 0.023 ± 0.003 nmol (pL cells) −1 s −1 ( n = 5; p = 0.029). e Schematic of the proposed mechanism of the effect of ouabain inhibition of NKA on glycolytic flux (Figure created using BioRender.com). ns p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001. Data plotted as mean ± SD

Article Snippet: Human cell lines MDA-MB-231 (CRM-HTB-26); MCF-7 (HTB-22); HCC1954 (CRL-2338) and 4T1 (CRL-2539) mouse cell line were obtained from American Type Culture Collection (ATCC).

Techniques: Suspension, Inhibition

Journal: Cancer & Metabolism

Article Title: Disrupting Na + ion homeostasis and Na + /K + ATPase activity in breast cancer cells directly modulates glycolysis in vitro and in vivo

doi: 10.1186/s40170-024-00343-5

Figure Lengend Snippet: Glycolytic flux affected by intracellular [Na] i and NKA function. a Schematic of the proposed mechanism of the effect of gramicidin-A on [Na + ] i and glycolysis. Gramicidin introduces an artificial Na + leak, increasing [Na + ] i and glycolytic metabolism (Figure created using BioRender.com). b 23 Na TQF spectra showing the intracellular [Na + ] i peak relative to a reference capillary in MDA-MB-231 cells following membrane permeabilization with gramicidin and varying concentrations of titrated extracellular [Na + ] e . c Quantification of TQF 23 Na NMR spectra (proportional to [Na + ] i ) following exposure to isotonic solutions of titrated [Na + ] e ( n = 4), for 4T1 cells and MDA-MB-231 cells. d Quantification of glycolytic fluxes measured by the rate of [6,6- 2 H 2 ] d -glucose to [3,3- 2 H 2 ] l -lactate conversion at different concentrations of titrated [Na + ] i in murine 4T1 and human MDA-MB-231 breast cancer cells ( n = 4). e Glycolytic fluxes in panel d replotted as a function of pump current derived from the analytical expression given by Silverman et al. f Glycolytic flux measured at the highest concentration of 70 mM [Na + ] e following treatment with 1 µM ouabain treatment was not significantly altered in murine 4T1 cells but was significantly decreased in human MDA-MB-231 cells. ns p > 0.05, **** p < 0.0001. Data plotted as mean ± SD

Article Snippet: Human cell lines MDA-MB-231 (CRM-HTB-26); MCF-7 (HTB-22); HCC1954 (CRL-2338) and 4T1 (CRL-2539) mouse cell line were obtained from American Type Culture Collection (ATCC).

Techniques: Membrane, Derivative Assay, Expressing, Concentration Assay

Journal: Pharmaceutics

Article Title: Study of Cytotoxicity and Internalization of Redox-Responsive Iron Oxide Nanoparticles on PC-3 and 4T1 Cancer Cell Lines

doi: 10.3390/pharmaceutics15010127

Figure Lengend Snippet: Evaluation of cytotoxicity of MNP-PEG and MNP-SS on PC-3 ( A ) and 4T1 ( B ) cells. Differences were considered statistically significant at: *— p < 0.05, **— p < 0.01, ***— p < 0.001.

Article Snippet: PC-3 human prostate cancer cells and 4T1 mouse breast cancer cells (American Type Culture Collection, Manassas, VA, USA) were cultured in a 5% CO 2 atmosphere at 37 °C in DMEM/F12 medium containing 10% fetal bovine serum (Sigma-Aldrich, Burlington, VT, USA), 1% L-glutamine (Gibco, Waltham, MA, USA), and 1% antibiotics (penicillin and streptomycin).

Techniques:

Journal: Pharmaceutics

Article Title: Study of Cytotoxicity and Internalization of Redox-Responsive Iron Oxide Nanoparticles on PC-3 and 4T1 Cancer Cell Lines

doi: 10.3390/pharmaceutics15010127

Figure Lengend Snippet: TEM images of the 4T1 cells after 2 h incubation with MNP-SS ( A – D ) and MNP-PEG ( E – H ): ( A )—cytoplasm of 4T1 cell with a vesicle filled with MNP-SS nanoparticles; the vesicle is enlarged in ( B , C )—outer membrane and cytoplasm of the 4T1 cell with attached clumps of MNP-SS nanoparticles and just formed vesicles; the vesicle is enlarged in ( D )—filled with nanoparticles; ( E )—4T1 cell plasmalemma with attached MNP-PEG nanoparticles, enlarged in ( F – H )—MNP-PEG nanoparticles in the vicinity of the 4T1 cell plasmalemma.

Article Snippet: PC-3 human prostate cancer cells and 4T1 mouse breast cancer cells (American Type Culture Collection, Manassas, VA, USA) were cultured in a 5% CO 2 atmosphere at 37 °C in DMEM/F12 medium containing 10% fetal bovine serum (Sigma-Aldrich, Burlington, VT, USA), 1% L-glutamine (Gibco, Waltham, MA, USA), and 1% antibiotics (penicillin and streptomycin).

Techniques: Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

doi: 10.1186/s13046-022-02565-1

Figure Lengend Snippet: SUSD4 expression suppresses tumor growth and affect EGFR dynamics. Tumor growth monitored in a syngeneic mouse model. 4 T1-Luc2 cells stably expressing mouse SUSD4, or mock control cells were injected into the mammary fat pad of BALB/c mice ( n = 10 mice per group). In first experiment ( A ) 2 × 10 6 cells were injected per mouse and in the second ( B ) 5 × 10 6 cells were injected per mouse (Error bars represents standard error of the mean). ( C ) Migration and invasion assay ( D ) of 5 × 10 4 4 T1-Luc2 cells incubated for 48 h under serum. (E) Immunodetection of SUSD4 from lysates of BT-20 cells stably expressing human SUSD4 or mock control cells treated with deglycosylation enzymes to assess SUSD4 N-glycosylation status. (F) Protein profile comparison between SUSD4-expressing BT-20 cells and mock control cells using the Human XL Oncology Array ( n = 2 technical repeats). (G) Expression of SUSD4 and EGFR, independent of each other, in individual cell types present in breast cancer tumors. Representative immunoblots depicting total EGFR levels and phospho-EGFR at Tyr1045 (H) or Tyr1086 (I) in SUSD4-expressing BT-20 or MDA-MB-468 cells and corresponding mock control cells. Densitometric analysis of total EGFR and phospho-EGFR levels together with the ratio of phosphorylated EGFR to total EGFR in BT-20 cells (J) and MDA-MB-468 cells (K). (L) EGFR ubiquitination assessed by immunoprecipitation of EGFR in serum-starved EGF-treated BT-20 cell expressing SUSD4 or mock control cells followed by immunoblot detection of ubiquitin. All results were repeated in at least three independent biological experiments unless otherwise specified. The p -value was estimated with t test when two groups were compared and with Two-way ANOVA test when two or more groups were compared for two independent parameters * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001

Article Snippet: The 4 T1-Luc2 mouse breast cancer cells (ATCC) were harvested with Versene (Thermo Fisher Scientific), counted, washed two times in PBS, and resuspended in PBS.

Techniques: Expressing, Stable Transfection, Injection, Migration, Invasion Assay, Incubation, Immunodetection, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: Representative results of leptin-dose effects on increase in protein levels of Notch (ligands and receptors) and molecular targets as determined by immunocytochemistry in 4T1 cells (A) and western blot (WB) in 4T1 (B and C), EMT6 (D and E) and MMT (F and G) cells. Cells were cultured for 24 h and leptin dose-induced (0, 0.6, 1.2 and 6.2 nM) effects were determined as described (See M &M). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software. (a) P<0.05 when comparing levels of protein to control (basal). Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments. Bar = 200 µm.

Article Snippet: The mouse mammary tumor (MT) cell line 4T1 (CRL-2539; ATCC), EMT6 (CRL-2755) and MMT060562 (MMT; CCL-51) were cultured with complete RPMI-1640 containing 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin on uncoated flat-bottomed plastic plates (cell densities of 1.0, 2.0 or 4.0×10 5 cells/well for 24, 12 or 6 well plates).

Techniques: Immunocytochemistry, Western Blot, Cell Culture, Software, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: Leptin-induced transcriptional expression of Notch receptors and targeted genes was abrogated by DAPT in 4T1 cells. Levels of Notch receptors mRNA (A, Notch1; B, Notch2 and C, Notch3) and targeted genes (D, Hey2 and E, survivin) as determined by real-time RT-PCR. GAPDH was used as internal control. (F–M) Representative results of leptin upregulation of protein levels of Notch1–3 and activated NICD1 as well as Notch targeted genes, Hey2 and survivin in 4T1 cells as determined by western blot (WB). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for (G) NICD1; (H) Notch1; (I) Notch 2; (J) Notch 3; (K) Hey2 and (M) survivin. 4T1 cells were cultured in a medium containing 0 or 1.2 nM leptin for 24 h. (a) P<0.05 when comparing levels of mRNA or protein to control (basal). Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: The mouse mammary tumor (MT) cell line 4T1 (CRL-2539; ATCC), EMT6 (CRL-2755) and MMT060562 (MMT; CCL-51) were cultured with complete RPMI-1640 containing 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin on uncoated flat-bottomed plastic plates (cell densities of 1.0, 2.0 or 4.0×10 5 cells/well for 24, 12 or 6 well plates).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software, Cell Culture, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: (A) Representative results of CSL-siRNA inhibition on leptin upregulation of protein levels of Notch 1-3, activated NICD1 and Notch targeted genes, CSL, survivin and Hey2 in 4T1 cells as determined by western blot (WB). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for (B) CSL; (C) Notch1; (D) NICD1; (E) Notch 2; (F) Notch 3; (G) Hey2 and (H) survivin. Cells cotransfected with siRNA oligonucleotides (CSL-siRNA or control-siRNA) were treated with leptin (0 and 1.2 nM) for 24 h. (a) P<0.05 when comparing protein levels to cells treated with control-siRNA (basal) or CSL-siRNA. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: The mouse mammary tumor (MT) cell line 4T1 (CRL-2539; ATCC), EMT6 (CRL-2755) and MMT060562 (MMT; CCL-51) were cultured with complete RPMI-1640 containing 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin on uncoated flat-bottomed plastic plates (cell densities of 1.0, 2.0 or 4.0×10 5 cells/well for 24, 12 or 6 well plates).

Techniques: Inhibition, Western Blot, Software, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: Leptin transcriptional activation of CBF (or CSL) promoter is linked to several leptin-induced signaling pathways. (A) 4T1 cells were transiently transfected with a CBF-Luc reporter construct and incubated with leptin (0 and 1.2 nM) alone or plus siRNA oligonucleotides (SignalSilence control-siRNA and STAT3, MEK1, AKT1, mTOR, JNK, p38-siRNA). (B) Protein levels of kinases after siRNA treatment were determined by WB analysis using β-actin as loading control. (C) 4T1 cells transfected with CBF-Luc reporter were incubated with leptin alone or plus pharmacological inhibitors of JAK2/STAT3 (AG490, 30 µM), MEK/MAPK/ERK1/2 (PD98059, 30 µΜ), PI-3K/AKT1 (Wortmannin, 50 nM), PKC-Ca dependant (Gö6976, 30 µΜ), p38 kinase (SB203580, 2 µΜ), JNK (SP600125, 30 µM) and mTOR (Rapamycin, 20 nM) signaling pathways for 24 h. Luciferase activity was determined as described (see M &M) and expressed as a percent of basal or leptin-treated cells. (a) P<0.05 when comparing levels of luciferase activity to control (basal) or leptin-treated cells. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: The mouse mammary tumor (MT) cell line 4T1 (CRL-2539; ATCC), EMT6 (CRL-2755) and MMT060562 (MMT; CCL-51) were cultured with complete RPMI-1640 containing 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin on uncoated flat-bottomed plastic plates (cell densities of 1.0, 2.0 or 4.0×10 5 cells/well for 24, 12 or 6 well plates).

Techniques: Activation Assay, Transfection, Construct, Incubation, Luciferase, Activity Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: A) Representative results from immunocytochemistry (ICC, hematoxylin staining) of leptin and DAPT effects on migration of 4T1 cells as compared to basal conditions. B) Quantitative assessment of cell migration under the effects of leptin and DAPT. C) Representative results from ICC after addition of control-siRNA (Ctr-siRNA), CLS-siRNA and leptin. D) Quantitative assessment of cell migration under the effects of leptin, CSL-siRNA and Crt-siRNA. E) Effects of leptin and DAPT on 4T1 cell proliferation. F) Effects of leptin, Ctr-siRNA and CSL-siRNA on 4T1 cell proliferation. Results from cell migration (Boyden chamber cell migration assay) and proliferation (MTT cell proliferation assay) were obtained after 24 h and normalized to basal conditions (see Material and Methods). Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: The mouse mammary tumor (MT) cell line 4T1 (CRL-2539; ATCC), EMT6 (CRL-2755) and MMT060562 (MMT; CCL-51) were cultured with complete RPMI-1640 containing 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin on uncoated flat-bottomed plastic plates (cell densities of 1.0, 2.0 or 4.0×10 5 cells/well for 24, 12 or 6 well plates).

Techniques: Immunocytochemistry, Staining, Migration, Cell Migration Assay, MTT Cell Proliferation, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: (A) DAPT inhibition of γ-secretase abrogated leptin transcriptional induction of VEGF (A); VEGFR-2 (B) and IL-1α mRNA (C) as determined by real-time RT-PCR and normalized to the GAPDH expression. CSL-siRNA also inhibited leptin-induced effects on protein levels of VEGF (D); VEGFR-2 (E) and IL-1α (F) mRNA. Representative results of DAPT (G) and CSL-siRNA (M) inhibition of leptin upregulation of protein levels of VEGF, VEGFR-2, IL-1α, ERα and OB-R in 4T1 cells as determined by western blot (WB). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for VEGF (H and N); VEGFR-2 (I and O); IL-1α (J and P); ERα (K and Q) and OB-R (L and R) in cells treated with leptin (0 and 1.2 nM) and DAPT for 24 h or cotransfected with CSL-siRNA and control (Ctr)-SiRNA, respectively. (a) P<0.05 when comparing levels of antigens to basal conditions and control-siRNA. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: The mouse mammary tumor (MT) cell line 4T1 (CRL-2539; ATCC), EMT6 (CRL-2755) and MMT060562 (MMT; CCL-51) were cultured with complete RPMI-1640 containing 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin on uncoated flat-bottomed plastic plates (cell densities of 1.0, 2.0 or 4.0×10 5 cells/well for 24, 12 or 6 well plates).

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Western Blot, Software, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: Anti-IL-1R tI antibodies abrogated leptin transcriptional induction of Notch1 (A); Notch2 (B); Notch3 (C) and targeted genes, Hey2 (D) and survivin (E) in 4T1 cells as determined by real-time RT-PCR and normalized to the GAPDH expression. Blockade of IL-1R tI with antibodies also inhibited leptin-induced effects on protein levels of Notch and targeted genes. Representative results of IL-1R tI blockade on leptin upregulation of protein levels of Notch1, NICD1, Notch2, Notch3, Hey2) and survivin (F) as determined by western blot (WB). The WB results were normalized to β\-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for Notch1 (G); NICD1 (H); Notch2 (I); Notch3 (J), Hey2 (K) and survivin (M). 4T1 cells treated were treated with leptin (0 and 1.2 nM) and IL-1R tI antibodies or nonspecific species-matched IgG (Control Ab) for 24 h. (a) P<0.05 when comparing Basal to Leptin+Control Ab and, (b) comparing Leptin+IL-1R tI Ab to Leptin+Control Ab. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: The mouse mammary tumor (MT) cell line 4T1 (CRL-2539; ATCC), EMT6 (CRL-2755) and MMT060562 (MMT; CCL-51) were cultured with complete RPMI-1640 containing 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 µg/ml streptomycin on uncoated flat-bottomed plastic plates (cell densities of 1.0, 2.0 or 4.0×10 5 cells/well for 24, 12 or 6 well plates).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Software, Derivative Assay