normal human epithelial breast cell line mcf10a  (ATCC)

Journal: iScience

Article Title: Inhibition of acid or neutral sphingomyelinases differentially impacts RNA and protein cargo sorting to extracellular vesicles

doi: 10.1016/j.isci.2025.112440

Figure Lengend Snippet: ASM-inhibited EVs enhance protein translation in recipient cells (A) Quantification of total EV proteins within the equal volumes of EVs utilized for co-culture experiments. The EVs used are present within equivalent isolation volumes purified from similar cell numbers that have been incubated under both control and SMase inhibitor conditions. (B) Representative light microscope images of wounded MCF10A cells incubated with MCF7 EVs, obtained under control or SMase inhibitor conditions, as captured using the Incucyte Live-Cell Analysis System. The images were taken at the initiation (0 h 00 m) and conclusion (48 h 00 m) of the co-culture experiment. The Incucyte software applied a wound mask. The scale bar represents 700 μm. (C) Quantification of the percentage relative wound density (%RWD) in wounded MCF10A cells over a 48-h period. The cells were incubated with MCF7 EVs obtained under control or SMase inhibitor conditions. Images were captured by an automated camera using the Incucyte live-cell analysis system, and %RWD was calculated using its Scratch Wound Analysis Software Module. %RWD measures the spatial cell density in the wound area relative to the spatial cell density outside of the wound area at each time point. (D) Schematic of the cell proliferation assay through the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) in newly synthesized DNA, followed by the chemoselective ligation of Alexa Fluor 488 picolyl azide by a click chemistry reaction. (E) Percentage of MCF10A cells in the total population labeled by 5-ethynyl-2′-deoxyuridine (EdU) after a 24-h incubation with EdU and MCF7 EVs obtained under control or SMase inhibitor conditions. EdU incorporates into newly synthesized DNA, and quantification is based on high-content microscopy imaging. (F) Schematic of the protein translation assay through the puromycylation of nascent proteins at the ribosome with O-propargyl puromycin (OPP), followed by the chemoselective ligation of Alexa Fluor 488 picolyl azide by a click chemistry reaction. (G) Representative high-content screening microscopy images displaying nuclear stain (NuclearMask) and OPP-labeled protein foci (Alexa Fluor (AF)-488). Red arrows point to example OPP-labeled foci. (H) Distribution of quantified mean intensities (arbitrary units – a.u.) of O-propargyl puromycin (OPP)-labeled foci in MCF10A cells following a 24-h co-culture with MCF7 EVs obtained under control or SMase inhibitor conditions. OPP integrates into the nascent peptides of newly translated proteins, and quantification is performed based on images captured with high-content screening microscopy. For all graphs, unless otherwise stated, the error bars represent the standard error of the mean (SEM) and the statistics were calculated by ordinary one-way ANOVA with Tukey post-hoc test (where ns = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, and ∗∗∗∗ = p ≤ 0.0001) on GraphPad Prism. See also Figures S5 and .

Article Snippet: MCF10A Human Normal Breast Epithelial Cells , ATCC , CRL-10317.

Techniques: Co-Culture Assay, Isolation, Purification, Incubation, Control, Light Microscopy, Cell Analysis, Software, Proliferation Assay, Synthesized, Ligation, Labeling, Microscopy, Imaging, High Content Screening, Staining

Journal: Molecular Medicine Reports

Article Title: CD8 + T cell‑related KCTD5 contributes to malignant progression and unfavorable clinical outcome of patients with triple‑negative breast cancer

doi: 10.3892/mmr.2024.13290

Figure Lengend Snippet: KCTD5 expression and prognosis in TNBC samples. (A) Expression of KCTD5 in TNBC and normal samples of GSE38959 dataset. (B) KCTD5 expression in cancers based on Tumor IMmune Estimation Resource database. (C) Kaplan-Meier OS curve. (D) Comparison of KCTD5 expression levels between TNBC and normal cell line determined by qPCR using a human normal breast epithelial cell line MCF10A and two human TNBC cell lines, MDA-MB-231 and MDA-MB-453. (E) Comparison of KCTD5 protein levels between TNBC and normal cell line detected by western blot based on normal cell line MCF10A and two human TNBC cell lines, MDA-MB-231 and MDA-MB-453. *P<0.05, **P<0.01 and ***P<0.001 vs. normal samples. TPM, transcripts per million; KCTD5, potassium channel tetramerization domain 5; TNBC, triple negative breast cancer.

Article Snippet: A human normal breast epithelial cell line MCF10A and two human TNBC cell lines, MDA-MB-231 and MDA-MB-453, were purchased from Procell Life Science & Technology Co., Ltd.

Techniques: Expressing, Comparison, Western Blot