Journal: Communications Biology

Article Title: Repeat DNA methylation is modulated by adherens junction signaling

doi: 10.1038/s42003-024-05990-4

Figure Lengend Snippet: a – c Mean square displacement (MSD) of mVenus spots was measured in sparse and dense MCF10A-BiAD cultures. a Shown are representative trajectories integrated over a lag time of 3 s. N = 20. b Representative violin plot showing the distribution of mVenus spot movement (lag time of 1 s) in data collected from one biological repeat for sparse (blue) and dense (orange) seeded cells. N = 50–140, statistical testing was determined with a two-tailed unpaired t test, **** p < 0.0001. c Quantification of the MSD experiments representatively shown in ( b ). n = 3, N = 50–140. Statistical analysis was done with a two-tailed paired t test, * p < 0.05. d RT-qPCR analysis of α-satellite transcripts showing increased transcription in 5-aza-dC 24 h after drug treatment. n = 3. Statistical analysis was done with a two-tailed unpaired t test, ** p < 0.01. e RT-qPCR analysis of α-satellite transcripts was performed on RNA isolated from MCF10A cells grown for 24 h in either sparse or confluent conditions. n = 3. Statistical analysis was done with a two-tailed unpaired t test, ** p < 0.01. f RT-qPCR was used to analyze transcription from α-satellite DNA in MCF10A cells that had been treated for 24 h with the E-cadherin blocking antibody. n = 3. Statistical analysis was done with a two-tailed unpaired t test, ** p < 0.01. g Western blot analysis of cell lysates isolated from MCF10A, SK-BR-3 and SUM159 cells and probed for E-cadherin. GAPDH was used as loading control. h MSRE-qPCR analysis of the α-satellite locus on genomic DNA isolated from SK-BR-3 and SUM159 cells, which were cultured for 24 h under either sparse or dense conditions. n = 3. Statistical analysis was done with a two-tailed unpaired t test, ns not significant. i qPCR analysis of the α-satellite transcripts on RNA isolated from SK-BR-3 and SUM159 cells, which were cultured for 24 h under either sparse or dense conditions. n = 3. Statistical analysis was done with a two-tailed unpaired t test, ns: not significant. j Scheme of newly discovered pathway linking the methylation status and transcription state of satellite DNA to cell density, mediated by E-cadherin. The figure was generated using Biorender. c , d , e , f , h , i In the dot blots, each dot is the mean value obtained for one biological repeat, the line indicates the mean of all biological repeats, and error bars represent their standard deviation. Paired measurements are indicated with the color coding.

Article Snippet: SUM159 (provided by Dr. Thordur Oskarsson, DKFZ) and SK-BR-3 cells (CLS Cell Lines Service GmbH) were cultured in DMEM medium (Gibco) with 10% fetal calf serum (Gibco).

Techniques: Two Tailed Test, Quantitative RT-PCR, Isolation, Blocking Assay, Western Blot, Control, Cell Culture, Methylation, Generated, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O- glycans that can be modified by ABO blood group determinants

doi: 10.1074/jbc.RA118.001934

Figure Lengend Snippet: The O-glycome of recombinant CEL is dramatically altered in cancer cell lines. A–D, the O-glycan pool of recombinant CEL expressed in HEK293 (A), pancreatic cancer (B, PANC-1; C, MIA PaCa-2), and breast cancer (D, SK-BR-3) cell lines were investigated by MALDI-TOF MS after alkaline β-elimination and permethylation. The O-glycan population varied with the cellular expression system employed. No ABO blood group antigens were detected in any cell line. See Fig. 8 legend for further information about the spectra.

Article Snippet: Human breast cancer cell line SK-BR-3 (ATCC) was grown and maintained in McCoy's 5A medium with 10% FBS and 100 units/ml penicillin/streptomycin.

Techniques: Recombinant, Expressing

Journal: Cancers

Article Title: High Antitumor Activity of the Dual Topoisomerase Inhibitor P8-D6 in Breast Cancer

doi: 10.3390/cancers14010002

Figure Lengend Snippet: Target control. MCF-7 ( A ) and SkBr3 ( B ) cells were treated with 10 µM P8-D6 (fluorophore: 462Ex/530Em) or control (PBS) for 10 h. P8-D6 were localized in vitro. ( i ): Cells were stained by CellTracker TM Deep Red Dye and Hoechst 33342. ( ii ) After fixation, topoisomerase I were detected. Fluorescence images show the fluorophore P8-D6 in green, membrane staining ( i ) or topoisomerase expression ( ii ) in red and nucleus staining in white. Scale bars, 50 µm. ( C ) Fluorescence intensity of P8-D6 in the nuclei was compared to PBS control. ( D ) BC cell lines and primary cells were lysed and protein expression was analyzed using western blot. HSP 90 was used as loading control.

Article Snippet: MCF-7 (1000/well), SkBr3 (1000/well), MDA-MB231 (2500/well), MDA-MB468 (500/well), BT-20 (18,750/well) and UF-182 (20,000/well) cells were seeded onto a 96-well Ultra-Low Attachment plate (Corning #4520).

Techniques: Control, In Vitro, Staining, Fluorescence, Membrane, Expressing, Western Blot

Journal: Cancers

Article Title: High Antitumor Activity of the Dual Topoisomerase Inhibitor P8-D6 in Breast Cancer

doi: 10.3390/cancers14010002

Figure Lengend Snippet: Antitumor responses in BC 2D monolayers. MCF-7, SkBr3, MDA-MB231, MDA-MB468, BT-20 (cell line) and UF-182 (primary cells) were treated with P8-D6, cisplatin, etoposide, topotecan and PBS as control. The relative caspase activity representing the rate of apoptosis were measured 48 h after treatment in MCF-7 ( A ), SkBr3 ( B ), MDA-MB231 ( C ), MDA-MB468 ( D ), BT-20 ( E ) and UF-182 ( F ) cells. Additionally, the anti-proliferative effects in the cells were visualized by microscopy after 24 h treatment. Scale bars, 50 µm ( G ). Heat map presents the IC50 values calculated by viability ( H ). P8-D6 was compared to cisplatin, epirubicin and topotecan. Data are means + SD one-way ANOVA, * ( p < 0.05), ** ( p < 0.01), *** ( p < 0.001), **** ( p < 0.0001), ns (non-significant).

Article Snippet: MCF-7 (1000/well), SkBr3 (1000/well), MDA-MB231 (2500/well), MDA-MB468 (500/well), BT-20 (18,750/well) and UF-182 (20,000/well) cells were seeded onto a 96-well Ultra-Low Attachment plate (Corning #4520).

Techniques: Control, Activity Assay, Microscopy

Journal: Cancers

Article Title: High Antitumor Activity of the Dual Topoisomerase Inhibitor P8-D6 in Breast Cancer

doi: 10.3390/cancers14010002

Figure Lengend Snippet: Growth changes in BC spheroids. For 3D culture, MCF-7, SkBr3, MDA-MB231, MDA-MB468, BT-20 (cell line) and UF-182 (primary cells) cells were cultured in ULA plates for 96 h. Subsequently, spheroids were treated with P8-D6 (10 µM, 1 µM, 0.5 µM, 0.1 µM), topotecan (10 µM, 1 µM) and PBS for 48 h. For the monitoring of growth changes and morphological changes, spheroids were imaged every 24 h by microscopy. MCF-7 ( A ), SkBr3 ( B ), MDA-MB231 ( C ), MDA-MB468 ( D ), BT-20 ( E ) and UF-182 ( F ). Scale bars, 500 µm.

Article Snippet: MCF-7 (1000/well), SkBr3 (1000/well), MDA-MB231 (2500/well), MDA-MB468 (500/well), BT-20 (18,750/well) and UF-182 (20,000/well) cells were seeded onto a 96-well Ultra-Low Attachment plate (Corning #4520).

Techniques: Cell Culture, Microscopy

Journal: Cancers

Article Title: High Antitumor Activity of the Dual Topoisomerase Inhibitor P8-D6 in Breast Cancer

doi: 10.3390/cancers14010002

Figure Lengend Snippet: Apoptosis induction in BC spheroids. For 3D culture, MCF-7, SkBr3, MDA-MB231, MDA-MB468, BT-20 (cell line) and UF-182 (primary cells) cells were cultured in ULA plates for 96 h. Subsequently, spheroids were treated with P8-D6 (10 µM, 1 µM, 0.5 µM, 0.1 µM), topotecan (10 µM, 1 µM) and PBS for 48 h. After treatment, the viability and caspase activity were analyzed in MCF-7 ( A ), SkBr3 ( B ), MDA-MB231 ( C ), MDA-MB468 ( D ), BT-20 ( E ) and UF-182 ( F ) spheroids. Data are means + SD ( n = 3) one-way ANOVA, * ( p < 0.05), ** ( p < 0.01), *** ( p < 0.001), **** ( p < 0.0001), ns (non-significant).

Article Snippet: MCF-7 (1000/well), SkBr3 (1000/well), MDA-MB231 (2500/well), MDA-MB468 (500/well), BT-20 (18,750/well) and UF-182 (20,000/well) cells were seeded onto a 96-well Ultra-Low Attachment plate (Corning #4520).

Techniques: Cell Culture, Activity Assay

Journal: Cancers

Article Title: High Antitumor Activity of the Dual Topoisomerase Inhibitor P8-D6 in Breast Cancer

doi: 10.3390/cancers14010002

Figure Lengend Snippet: Cell toxicity, live-dead staining and morphological changes in BC spheroids. For 3D culture, MCF-7, SkBr3, MDA-MB231, MDA-MB468, BT-20 (cell line) and UF-182 (primary cells) cells were cultured in ULA plates for 96 h. Subsequently, spheroids were treated with P8-D6 (10 µM, 1 µM, 0.5 µM, 0.1 µM), topotecan (10 µM, 1 µM) and PBS for 48 h. After treatment, the spheroids were stained with propidium iodide (red), calcein-AM (green) and Hoechst 33342 (blue) and imaged using NYONE ®® Scientific. MCF-7 ( A ), SkBr3 ( B ) and UF-182 ( C ). Scale bars, 500 µm. ( D ) During treatment, the cell toxicity was measured by fluorescence microscopy using CellTox™ Green (24 h, 48 h). Scale bars, 500 µm. These fluorescence signals were quantified (fluorescence intensity RFU) and presented in a heat map ( E ). ( F ) The P8-D6- (1 μM) or PBS-treated SkBr3 and UF-182 spheroids (48 h) were analyzed by scanning electron microscopy (SEM). Scale bars, 20 µm.

Article Snippet: MCF-7 (1000/well), SkBr3 (1000/well), MDA-MB231 (2500/well), MDA-MB468 (500/well), BT-20 (18,750/well) and UF-182 (20,000/well) cells were seeded onto a 96-well Ultra-Low Attachment plate (Corning #4520).

Techniques: Staining, Cell Culture, Fluorescence, Microscopy, CellTox Assay, Electron Microscopy