primary human mammary epithelial cells hmec  (Cell Applications Inc)

Journal: Frontiers in Oncology

Article Title: Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway

doi: 10.3389/fonc.2021.654940

Figure Lengend Snippet: ER stress genes are abundantly expressed in IBC tissue sections and IBC cell lines. (A) RNA isolated from healthy and IBC tumor tissue was prepared, converted to cDNA, and gene expression was quantified by real-time RT-PCR using specific primers for ER stress markers as indicated. Each data point represents the average gene expression from six IBC and six healthy control samples. Each point represents the average ± the standard deviation of three experiments. (***) p<0.005, (****) p<0.001 indicates a statistically significant difference compared with healthy tissue. Each reaction was done in triplicate. (B) RNA isolated from HMEC, SUM149PT, and SUM190PT cells was converted to cDNA, and gene expression was quantified by real-time RT-PCR using specific primers for ATF4, CHOP, GADD34, GRP78, IRE1α, and XBP-1. Each point represents the average ± the standard deviation of three experiments. (**) p<0.01, (***) p<0.005, (****) p<0.001 indicate a statistically significant difference compared with HMEC cells. Each reaction was done in triplicate. (C) ER stress genes proteins are abundantly expressed in IBC cell lines. Lysates prepared from HMEC, SUM149PT, and SUM190PT cells, were tested for protein levels of PERK, IRE1α, calnexin, ERO1α, and PDI. Blots were reprobed with anti-β-actin antibody as a loading control for normalization. Fold expression of each protein was calculated by considering the expression of the protein in HMEC as 1. (D) Immunostaining of HMEC and SUM149PT cells seeded in eight-well chamber slides. Cells were fixed, permeabilized, and then stained with primary monoclonal antibodies against ER stress markers, including calnexin, ERO1α, and IRE1α. Cells were developed with Alexa-488 coupled secondary antibody (green). Nuclei were visualized using DAPI as the counterstain (blue).

Article Snippet: Primary human mammary epithelial cells (HMEC) (#830-05a, Cell Applications, San Diego, CA) were cultured in HMEC medium (#815-500, Cell Applications).

Techniques: Isolation, Gene Expression, Quantitative RT-PCR, Control, Standard Deviation, Expressing, Immunostaining, Staining, Bioprocessing

Journal: Frontiers in Oncology

Article Title: Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway

doi: 10.3389/fonc.2021.654940

Figure Lengend Snippet: IBC cell lines were chemosensitive to Saburinal treatment. (A) Effect of Phenylbutyrate treatment on cytotoxicity. HMEC, SUM149PT, and SUM190PT cells were untreated or treated with Phenylbutyrate at varying concentrations for different time periods, as indicated. Supernatants were collected from the cells to measure the amount of LDH released using spectrophotometry at 490 nm. Each point represents the average ± the standard deviation of three experiments. (B) Effect of Salubrinal treatment on cytotoxicity. HMEC, SUM149PT, and SUM190PT cells were untreated or treated with Salubrinal at varying concentrations for different time points, as indicated. Supernatants were collected from the cells to measure the amount of LDH released using spectrophotometry at 490 nm. Each point represents the average ± the standard deviation of three experiments.

Article Snippet: Primary human mammary epithelial cells (HMEC) (#830-05a, Cell Applications, San Diego, CA) were cultured in HMEC medium (#815-500, Cell Applications).

Techniques: Spectrophotometry, Standard Deviation

Journal: Frontiers in Oncology

Article Title: Salubrinal Exposes Anticancer Properties in Inflammatory Breast Cancer Cells by Manipulating the Endoplasmic Reticulum Stress Pathway

doi: 10.3389/fonc.2021.654940

Figure Lengend Snippet: Salubrinal treatment induces caspase-3 activation and PARP cleavage in IBC cells. (A–C) HMEC and IBC cell lines, SUM149PT and SUM190PT, were treated with various concentrations of Salubrinal for different time intervals. A luminogenic caspase-3 substrate is added, and the luminescence is measured in relative light units (RLU) as an index for caspase-3 activity. Each reaction was done in triplicate, and each bar represents the mean ± SD for three experiments. (D–F) HMEC and IBC cell lines, SUM149PT and SUM190PT, were treated with various concentrations of Salubrinal for different time intervals. Cells were fixed, permeabilized, and incubated with a particular anti-cleaved PARP primary antibody followed by an HRP-labeled secondary antibody. Absorbance (OD) was measured at 450 nm as an index of PARP cleavage. Each reaction was done in triplicate, and each bar represents the mean ± SD for three experiments. (*), p < 0.05, (**) p < 0.01, (***) p < 0.005 indicate a statistically significant difference compared with cells treated for 0h. ns, not significant. (G) SUM149PT and SUM190PT cells were cultured with or without 10M Salubrinal for 24 and 48 hours. Lysates prepared and tested for caspase-3, cleaved caspase-3 and cleaved PARP as indicated by Western blot analysis. Blots were reprobed with anti--actin antibody as a loading control. The level of proteins in untreated samples was considered one for fold activation or down-regulation using the quantification method as described in the Methods section.

Article Snippet: Primary human mammary epithelial cells (HMEC) (#830-05a, Cell Applications, San Diego, CA) were cultured in HMEC medium (#815-500, Cell Applications).

Techniques: Activation Assay, Activity Assay, Incubation, Labeling, Cell Culture, Western Blot, Control