bt 549 brca cell line  (ATCC)

Journal: Scientific Reports

Article Title: KLF15 suppresses tumor growth and metastasis in Triple-Negative Breast Cancer by downregulating CCL2 and CCL7

doi: 10.1038/s41598-022-23750-4

Figure Lengend Snippet: Methylation status of KLF15 promoter in breast carcinoma. ( A ) methylation status of KLF15 promoter in BRCA patients with data from TCGA samples. ( B ) Representative methylation of KLF15 in breast tumor and normal tissues as examined by MSP. ( C ) MSP analysis of KLF15 promoter methylation in breast cancer cell lines. ( D ) RT-qPCR results of KLF15 expression after treatment of Aza or Aza + TSA (A + T) in BT549 and MDA-MB231 cell lines. ( E ) Western blotting indicating KLF15 protein expression after Aza or A + T treatment in BT549 and MDA-MB231 cells.

Article Snippet: Nine BrCa cell lines (BT549, MDA-MB231, MCF7, T-47D, YCC-B1, ZR-75-1, MB468, SK-BR-3, SUM159) were obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Methylation, Quantitative RT-PCR, Expressing, Western Blot

Journal: Theranostics

Article Title: Tumor suppressor DRD2 facilitates M1 macrophages and restricts NF-κB signaling to trigger pyroptosis in breast cancer

doi: 10.7150/thno.58322

Figure Lengend Snippet: DRD2 is transcriptionally downregulated through promoter methylation in BrCa. ( A ) Heatmap of top 30 differentially expressed genes based on RNA-seq. Tissues derived from nor mal breast tissues and BrCa tissues were applied for RNA-seq analysis. Log2FC transformed and normalized values were used. ( B ) IHC staining of DRD2 in normal breast tissues and BrCa tissues. Bars, 60 μm. ( C ) Expression and methylation of DRD2 based on TCGA database. Data are presented as mean ± SD; p -value was calculated using two-tailed Student's t test. p < 0.0001. ( D ) Online database Kmplot was used to analyze the effects of DRD2 on the overall prognosis (left) of BrCa patients, the survival times of BrCa patients featured HER2-positive (middle), and the survival times of BrCa patients featured Luminal A (right). ( E ) mRNA expression (RT-PCR) and promoter methylation (MSP) analysis of DRD2 in BrCa cells lines and mammary epithelial cell lines were all detected. ( F ) mRNA expression of DRD2 after Aza treatment was determined by qRT-PCR. MDA-MB231 and BT549 were treated with Aza for 3 d. BrCa cells without Aza treatment were used as controls. Data are presented as mean ± SD; p -value was calculated using two-tailed Student's t test. ***, p < 0.001. BN, normal breast; BrCa: Breast cancer; Ctrl, Control; MSP, methylation-specific PCR; M, methylated; U, unmethylated.

Article Snippet: BrCa cell lines (MDA-MB231, BT549, YCCB1, 4T1, etc.), immortalized human mammary epithelial cell lines (MCF-10A, HMEC) and HEK293T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or collaborators and cultured in PRMI 1640 (Gibco) or DEME medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) according to standard protocols.

Techniques: Methylation, RNA Sequencing, Derivative Assay, Transformation Assay, Immunohistochemistry, Expressing, Two Tailed Test, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Control

Journal: Theranostics

Article Title: Tumor suppressor DRD2 facilitates M1 macrophages and restricts NF-κB signaling to trigger pyroptosis in breast cancer

doi: 10.7150/thno.58322

Figure Lengend Snippet: DRD2 expression inhibits BrCa cells tumorigenesis in vitro and in vivo . ( A and B ) Confirming ectopic DRD2 mRNA expression by RT-PCR (A) and protein expression by WB (B). ( C ) Measurement of proliferation in Vector- and DRD2-transfected BrCa cells by CCK8 assay. ( D and E ) Histogram statistics of colony formation (D) and soft agar formation assay (E) to determine proliferation rates. ( F ) Histogram statistics showing analysis of apoptosis determined by AO/EB assay. ( G ) Histogram statistics of cell cycle distribution by FC. ( H ) Histogram statistics of proliferation rates in BrCa cells. CCK8 was performed to analyze effect of 891 DRD2 expression on chemosensitivity of BrCa cells to PTX. DMSO was used as controls. ( I and J ) Histogram showing effects of DRD2 on metastatic abilities in wound-healing (I) and Transwell® assay (J). Transwell® coated without (left) or with (right) Matrigel were applied to detecting migrative or invasive abilities of BrCa cells. ( K ) The volume and weight measurements of subcutaneous tumor model in BALB/c mice (8 mice per group). Volume = length × width2 × 0.5. DRD2/4T1 cells were used, and vector-transfected 4T1 cells were used as controls. Data are presented as mean ± SD; P-value was calculated using two-tailed Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001. PTX, Paclitaxel. AO/EB, acridine orange/ethidium bromide.

Article Snippet: BrCa cell lines (MDA-MB231, BT549, YCCB1, 4T1, etc.), immortalized human mammary epithelial cell lines (MCF-10A, HMEC) and HEK293T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or collaborators and cultured in PRMI 1640 (Gibco) or DEME medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) according to standard protocols.

Techniques: Expressing, In Vitro, In Vivo, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Transfection, CCK-8 Assay, Tube Formation Assay, AO/EB Assay, Transwell Assay, Two Tailed Test

Journal: Theranostics

Article Title: Tumor suppressor DRD2 facilitates M1 macrophages and restricts NF-κB signaling to trigger pyroptosis in breast cancer

doi: 10.7150/thno.58322

Figure Lengend Snippet: DRD2 triggers pyroptosis during crosstalk with Mφ. ( A and B ) Murinebreast cancer cell 4T1 used to construct subcutaneous tumor model. HE (A, left), IHC (A, middle and right) and TUNEL (B) assays were performed in samples derived from subcutaneous tumor model. Bars, 80 μm in (A); Bars, 75 μm in (B). ( C and D ) Pyroptosis markers were examined by qRT-PCR (C), and necroptosis, apoptosis, as well as pyroptosis markers were detected by WB (D) in vector- and DRD2-transfected BrCa cells co-cultivated with Mφ. BrCa cells cultured alone were used as controls. Data are presented as mean ± SD from biological replicates. P-value was calculated using two-tailed Student's t test. ***, p < 0.001. ns: not significant. ( E ) Markers of inflammasome (NLRP3) and pyroptosis (GSDME) were detected by WB. M1 Mφ was induced by LPS (200 ng/ml, 3 d). Medium derived from M1 Mφ was filtrated and applied to incubate Vector- and DRD2-MB231 for 3 d. Tumor cells cultured alone were used as control.

Article Snippet: BrCa cell lines (MDA-MB231, BT549, YCCB1, 4T1, etc.), immortalized human mammary epithelial cell lines (MCF-10A, HMEC) and HEK293T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or collaborators and cultured in PRMI 1640 (Gibco) or DEME medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) according to standard protocols.

Techniques: Construct, TUNEL Assay, Derivative Assay, Quantitative RT-PCR, Plasmid Preparation, Transfection, Cell Culture, Two Tailed Test, Control

Journal: Theranostics

Article Title: Tumor suppressor DRD2 facilitates M1 macrophages and restricts NF-κB signaling to trigger pyroptosis in breast cancer

doi: 10.7150/thno.58322

Figure Lengend Snippet: DRD2 restricts NF-κB signaling activation by interrupting phosphorylation of TAK1. ( A and B ) Representative IF staining of DRD2 (green) and p-p65 (red) in BrCa cells treated by LPS (A) or THP1-derived Mφ (B). And BrCa cells were seeded in glass coverslips for 24h before LPS (5 μg/ml, serum-free, 24 h) or Mφ treatment. Images were taken by confocal microscopy. Vector-transfected BrCa cells were used as the control. Nuclei were stained with DAPI. Bars, 10 μm in (A); 5 μm in (B). ( C ) WB was applied to detect cytoplasmic and nuclear expression of p-p65. PCNA and Actin were used to prove the protein integrity of nucleus and cytoplasm respectively. ( D and E ) WB was used to analyze the activation status of NF-κB signaling and its upstream regulator TAK1 after being treated by LPS (5 μg/ml, serum-free, 24 h) (D) and THP1-derived Mφ (3 d) (E). ( F and G ) IB was used to confirm the binding of proteins obtained by Co-IP in MDA-MB231. Cells were treated with LPS (5 μg/ml, serum-free, 24 h) or CM (3 d) before Co-IP. The binding of DRD2, β-arrestin2 and p-p65 were analyzed by IB in DRD2-expressed MDA-MB231 with or without LPS treatment (F). The binding of DRD2 and β-arrestin2 were analyzed by IB in in Vector- and DRD2-expressed MDA-MB231 with or without CM treatment (G). ( H ) IB was sued to determine the binding of TAB1, TAK1, and β-arrestin2 in Vector- and DRD2-expressed MDA-MB231. Co-IP was used to obtain possible binding proteins of TAB1 in samples with or without LPS treatment. IgG was used as negative control, and the input protein was used as positive control. And GAPDH was used for protein integrity. CM, Conditioned Medium.

Article Snippet: BrCa cell lines (MDA-MB231, BT549, YCCB1, 4T1, etc.), immortalized human mammary epithelial cell lines (MCF-10A, HMEC) and HEK293T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or collaborators and cultured in PRMI 1640 (Gibco) or DEME medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) according to standard protocols.

Techniques: Activation Assay, Phospho-proteomics, Staining, Derivative Assay, Confocal Microscopy, Plasmid Preparation, Transfection, Control, Expressing, Binding Assay, Co-Immunoprecipitation Assay, Negative Control, Positive Control

Journal: Theranostics

Article Title: Tumor suppressor DRD2 facilitates M1 macrophages and restricts NF-κB signaling to trigger pyroptosis in breast cancer

doi: 10.7150/thno.58322

Figure Lengend Snippet: DRD2 antagonizes NF-κB signaling through interacting and downregulating DDX5 and eEF1A2. ( A ) qRT-PCR was used to detect mRNA expression of p65 and IL-10 in Vector- and DRD2- transfected BrCa cells. ( B ) Venn diagram showed potential binding proteins of DRD2 identified by mass spectrum analysis. And mass spectrum was performed in Co-IP isolated proteins in both MDA-MB231 and BT549. ( C ) mRNA expression of DDX5 , eEF1A2 , and ICAM-1 were analyzed by qRT-PCR in Vector- and DRD2- transfected BrCa cells. ( D ) Protein expression of DDX5 and eEF1A2 were determined by WB in Vector- and DRD2- transfected BrCa cells. The expression was analyzed in both MDA-MB231 and BT549. ( E ) The binding of DRD2, DDX5 and eEF1A2 was examined by Co-IP and IB in 293T and MDA-MB231. ( F ) ERBB1 (EGFR) and ERBB2 (HER2) mRNA expression were determined by qRT-PCR in Vector- and DRD2- transfected BrCa cells. ( G ) WB was used to detect the effect of DDX5 and eEF1A2 on NF-κB activation was analyzed by WB in MDA-MB231 by stimulation of LPS. GAPDH was used for protein integrity. Data are presented as mean ± SD from biological replicates. P-value was calculated using two-tailed Student's t test. *, p < 0.05 **, p < 0.01; ***, p < 0.001.

Article Snippet: BrCa cell lines (MDA-MB231, BT549, YCCB1, 4T1, etc.), immortalized human mammary epithelial cell lines (MCF-10A, HMEC) and HEK293T were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) or collaborators and cultured in PRMI 1640 (Gibco) or DEME medium supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) according to standard protocols.

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, Binding Assay, Co-Immunoprecipitation Assay, Isolation, Activation Assay, Two Tailed Test