Journal: Frontiers in Immunology

Article Title: Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

doi: 10.3389/fimmu.2020.01171

Figure Lengend Snippet: Flow cytometric analysis of apoptosis induction in breast cancer cells treated with 20 μg/ml of immobilized and solution-phase rfhSP-D for 24 h.

Article Snippet: Human breast cancer cell lines, triple-negative BT20 (ER − /PR − /HER2 − ) (ATCC-HTB19), triple-positive BT474 (ER + /PR + /HER2 + ) (ATCC-HTB20), and HER2-positive SKBR3 (ER − /PR − /HER2 + ) (ATCC-HTB30), were cultured in complete RPMI medium (Sigma-Aldrich), supplemented with 10% v/v fetal bovine serum (FBS), 2 mM l -glutamine, 100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) and left to grow at 37°C under 5% v/v CO 2 .

Techniques:

Journal: Frontiers in Immunology

Article Title: Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

doi: 10.3389/fimmu.2020.01171

Figure Lengend Snippet: Apoptosis induction in BT20, BT474, and SKBR3 cell lines following HA challenge in the presence and absence of rfhSP-D (A) . The data were expressed as the mean of three independent experiments ( n = 3). A significant difference was seen among treated and untreated samples, as made evident by the shift in the fluorescence intensity. Staurosporine (1 μM/ml) was used as a positive control. Proliferative effects of rfhSP-D treatment on BT474 and SKBR3 breast cancer cell lines (B) . BT474 and SKBR3 cells were seeded in wells pre-coated with HA, HA + rfhSP-D, and rfhSP-D alone. The percentage of proliferative cells was evaluated by staining with mouse anti-human KI-67 antibody, and KI-67-stained cells were measured via flow cytometry. The data were generated from at least three independent experiments ( n = 3) and presented as mean ± SD (* p < 0.1, ** p < 0.01, and **** p < 0.0001). The statistical analysis was performed between rfhSP-D and HA + rfhSP-D-treated breast cancer cells. The secretion levels of FL-SP-D were confirmed and analyzed via western blotting (C) . Culture medium collected from BT20, BT474, and SKBR3 cell lines was passed through a maltose Agarose column, and the eluted fractions were validated via western blotting; FL-SP-D was detected at ~43 kDa only for BT474. Both BT20 and SKBR3 cell lines did not secrete any FL-SP-D. Secreted FL-SP-D by BT474 was tested for its ability to induce apoptosis (D) . No effect of secreted FL-SP-D was seen in terms of cell viability and apoptosis induction.

Article Snippet: Human breast cancer cell lines, triple-negative BT20 (ER − /PR − /HER2 − ) (ATCC-HTB19), triple-positive BT474 (ER + /PR + /HER2 + ) (ATCC-HTB20), and HER2-positive SKBR3 (ER − /PR − /HER2 + ) (ATCC-HTB30), were cultured in complete RPMI medium (Sigma-Aldrich), supplemented with 10% v/v fetal bovine serum (FBS), 2 mM l -glutamine, 100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) and left to grow at 37°C under 5% v/v CO 2 .

Techniques: Fluorescence, Positive Control, Staining, Flow Cytometry, Generated, Western Blot

Journal: Frontiers in Immunology

Article Title: Hyaluronic Acid Present in the Tumor Microenvironment Can Negate the Pro-apototic Effect of a Recombinant Fragment of Human Surfactant Protein D on Breast Cancer Cells

doi: 10.3389/fimmu.2020.01171

Figure Lengend Snippet: Intracellular signaling to show phosphorylation of p53 in rfhSP-D-treated BT20 and SKBR3 cell lines (A) . Breast cancer cell lines were allowed to adhere to HA or HA-bound rfhSP-D, and the phosphorylation status of p53 was evaluated using total cell lysates with a PathScan Antibody Array Kit (Cell Signaling). Data were generated from at least three independent experiments and presented as mean ± SD. rfhSP-D treatment resulted in upregulation of p21 and p27 cell cycle inhibitors (B) . BT20, BT474, and SKBR3 (0.4 × 10 6 ) cells were seeded in a six-well-plate pre-coated with rfhSP-D and HA + rfhSP-D. The treated cells were lysed and pelleted down. The pelleted cells were subjected to RNA isolation, followed by cDNA synthesis and qPCR. The comparative quantification method was performed to calculate the efficiencies of each gene for each individual PCR and is based on the second differential maximum method or takeoff analysis. The takeoff results obtained with p21/p27 primers were normalized with the housekeeping gene TBP. qPCR assay was conducted in triplicates, and error bars represent ± SEM. Unpaired one-way ANOVA test was used to determine the significance; * p < 0.05, ** p < 0.01, and *** p < 0.001 ( n = 3). The statistical analysis was performed between rfhSP-D and HA + rfhSP-D-treated breast cancer cells.

Article Snippet: Human breast cancer cell lines, triple-negative BT20 (ER − /PR − /HER2 − ) (ATCC-HTB19), triple-positive BT474 (ER + /PR + /HER2 + ) (ATCC-HTB20), and HER2-positive SKBR3 (ER − /PR − /HER2 + ) (ATCC-HTB30), were cultured in complete RPMI medium (Sigma-Aldrich), supplemented with 10% v/v fetal bovine serum (FBS), 2 mM l -glutamine, 100 U/ml penicillin (Sigma-Aldrich), 100 μg/ml streptomycin (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich) and left to grow at 37°C under 5% v/v CO 2 .

Techniques: Phospho-proteomics, Ab Array, Generated, Isolation, cDNA Synthesis

Journal: The EMBO Journal

Article Title: Reciprocal priming between receptor tyrosine kinases at recycling endosomes orchestrates cellular signalling outputs

doi: 10.15252/embj.2020107182

Figure Lengend Snippet:

Article Snippet: BT549 , LGC ATCC® , HTB‐122.

Techniques: Virus, Recombinant, Extraction, Multiplex sample analysis, Transfection, Molecular Weight, Marker, Reverse Transcription, Protease Inhibitor, Membrane, Cell Culture, Clinical Proteomics, Spectroscopy, Imaging, cDNA Synthesis, Plasmid Preparation, Negative Control, Mutagenesis, Clone Assay, Generated, Software, Microscopy

Journal: PLOS ONE

Article Title: Ribitol alters multiple metabolic pathways of central carbon metabolism with enhanced glycolysis: A metabolomics and transcriptomics profiling of breast cancer

doi: 10.1371/journal.pone.0278711

Figure Lengend Snippet: A) KRAS expression in MCF-7 cell line by Real-Time qRT-PCR B) Relative KRAS expression in MDA MB-231 and T-47D breast cancer cell lines by Real-Time qRT-PCR C) KRAS protein expression by western blot of control, and ribitol-treated breast cancer cell lines MDA MB-231, MCF-7, and T-47D (D) Relative KRAS expression in xenograft MCF-7 tumors after ribitol treatment by Real-Time qRT-PCR.

Article Snippet: The human breast cancer cell lines MCF7 (ATCC- HTB-22), T47D (ATCC-CRL2865), and MDA-MB-231 (MDA-231) (ATCC-HTB-26) were used.

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