Journal: Cancer Microenvironment

Article Title: Hypoxia Induces the Acquisition of Cancer Stem-like Phenotype Via Upregulation and Activation of Signal Transducer and Activator of Transcription-3 (STAT3) in MDA-MB-231, a Triple Negative Breast Cancer Cell Line

doi: 10.1007/s12307-018-0218-0

Figure Lengend Snippet: Hypoxia significantly increased tumorigenic and stem-like properties in RU cells. The effect of 48 h incubation under hypoxia, compared to RU and RR cells incubated under normoxia, in RU cells on: a GFP expression as illustrated by flow cytometry histogram plots, b SRR2 luciferase activity, and c Proportion of CD44high/CD24low cells as illustrated by flow cytometry dot plots. d Colony formation ability was assessed for RU cells after 24 h incubation under hypoxia. Cells were then re-plated at density of 1000 cells/well in duplicate in six-well plates under normoxia. The number of colonies formed from 1000 cells after 7 days was graphed. e Cellular viability was measured by MTT assay after 48 h exposure to increasing concentration of cisplatin (3.32–332 μM). (*) denotes a significant difference between hypoxic and native RU cells (one-way ANOVA followed by a post-hoc Tukey test, p < 0.05). Data are represented as mean ± SD (n = 3)

Article Snippet: SRR2 Luciferase Reporter Assay and Flow Cytometry Luciferase reporter assay was performed using luciferase assay system kit (#E4530, Promega, Corporation, Madison, USA) according to the manufacturer’s protocol, on costar white polystyrene opaque 96-well plates (#3912, Corning, NY, USA) and analyzed on the FLUOstar Omega multi-mode microplate reader (BMG Labtech, Ortenburg, Germany).

Techniques: Incubation, Expressing, Flow Cytometry, Luciferase, Activity Assay, MTT Assay, Concentration Assay

Journal: Cancer Microenvironment

Article Title: Hypoxia Induces the Acquisition of Cancer Stem-like Phenotype Via Upregulation and Activation of Signal Transducer and Activator of Transcription-3 (STAT3) in MDA-MB-231, a Triple Negative Breast Cancer Cell Line

doi: 10.1007/s12307-018-0218-0

Figure Lengend Snippet: STAT3 activation drives hypoxia-induced RU/RR conversion. a Western blot is depicting the effect of 24 and 48 h incubation under hypoxia on the protein expression profile of RU cells as compared to native RU and native RR cells. b Higher amount of STAT3 was pulled down with SRR2 probe, compared to native RU cells, in hypoxic RU cells after 48 h hypoxia. c Lower amount of STAT3 was pulled down with SRR2 probe in hypoxic RU cells in the presence of STAT3 inhibitor (20 μM Stattic)

Article Snippet: SRR2 Luciferase Reporter Assay and Flow Cytometry Luciferase reporter assay was performed using luciferase assay system kit (#E4530, Promega, Corporation, Madison, USA) according to the manufacturer’s protocol, on costar white polystyrene opaque 96-well plates (#3912, Corning, NY, USA) and analyzed on the FLUOstar Omega multi-mode microplate reader (BMG Labtech, Ortenburg, Germany).

Techniques: Activation Assay, Western Blot, Incubation, Expressing

Journal: Cancer Microenvironment

Article Title: Hypoxia Induces the Acquisition of Cancer Stem-like Phenotype Via Upregulation and Activation of Signal Transducer and Activator of Transcription-3 (STAT3) in MDA-MB-231, a Triple Negative Breast Cancer Cell Line

doi: 10.1007/s12307-018-0218-0

Figure Lengend Snippet: Suppression of STAT3 effectively inhibits RU/RR conversion. The effect of STAT3 silencing using siRNA in hypoxic RU cells (48 h hypoxia) on: a Protein expression profile as depicted by Western blot, b GFP expression as illustrated by flow cytometry histograms, and c SRR2 luciferase activity. d Cellular viability was measured after 48 h cisplatin (30 uM) exposure in hypoxic RU cells transfected with either scrambled or STAT3 siRNAs. e Clonogenic survival assay was conducted in hypoxic RU cells transfected with either scrambled or STAT3 siRNA. Cells were then re-plated at density of 1000 cells/well in duplicate in six-well plates under normoxia. The number of colonies formed from 1000 cells was graphed. f qRT-PCR results of CCL2, ADM and IGFBP7 genes expression in hypoxic RU cells (24 h hypoxia) normalized to GAPDH, and further normalized to native RU cells, and g qRT-PCR results of CCL2, ADM and IGFBP7 genes expression after STAT3 silencing using siRNA in hypoxic RU cells (24 h hypoxia) normalized to GAPDH, and further normalized to hypoxic RU cells subjected to scrambled siRNA

Article Snippet: SRR2 Luciferase Reporter Assay and Flow Cytometry Luciferase reporter assay was performed using luciferase assay system kit (#E4530, Promega, Corporation, Madison, USA) according to the manufacturer’s protocol, on costar white polystyrene opaque 96-well plates (#3912, Corning, NY, USA) and analyzed on the FLUOstar Omega multi-mode microplate reader (BMG Labtech, Ortenburg, Germany).

Techniques: Expressing, Western Blot, Flow Cytometry, Luciferase, Activity Assay, Transfection, Clonogenic Cell Survival Assay, Quantitative RT-PCR

Journal: Cancer Microenvironment

Article Title: Hypoxia Induces the Acquisition of Cancer Stem-like Phenotype Via Upregulation and Activation of Signal Transducer and Activator of Transcription-3 (STAT3) in MDA-MB-231, a Triple Negative Breast Cancer Cell Line

doi: 10.1007/s12307-018-0218-0

Figure Lengend Snippet: Hypoxia-induced conversion of RU/RR cells is independent of c-Myc. a Lower amount of c-Myc was pulled down with SRR2 probe, compared to native RU cells, in hypoxic RU cells after 48 h hypoxia. b Successful knockdown of c-Myc in hypoxic RU cells as depicted by Western blot after 48 h hypoxia. c siRNA knockdown of c-Myc did not reverse the chemoresistance in hypoxic RU cells after 48 h cisplatin exposure. d Hypoxia-mediated suppression of c-Myc, compared to RU cells carrying empty vector (RU/EV), was abrogated in c-Myc overexpressing RU cells (RU/Myc) as depicted by Western blot after 48 h hypoxia. e Abrogation of c-Myc suppression under hypoxia in overexpressing c-Myc RU cells (RU/Myc), compared to RU/EV cells, did not enhance their clonogenic potential. Colony formation ability was assessed for RU/Myc and RU/EV cells after 24 h incubation under hypoxia. Cells were then re-plated at density of 1000 cells/well in duplicate in six-well plates under normoxia. The number of colonies formed from 1000 cells after 7 days was graphed

Article Snippet: SRR2 Luciferase Reporter Assay and Flow Cytometry Luciferase reporter assay was performed using luciferase assay system kit (#E4530, Promega, Corporation, Madison, USA) according to the manufacturer’s protocol, on costar white polystyrene opaque 96-well plates (#3912, Corning, NY, USA) and analyzed on the FLUOstar Omega multi-mode microplate reader (BMG Labtech, Ortenburg, Germany).

Techniques: Knockdown, Western Blot, Plasmid Preparation, Incubation