Journal: Biology

Article Title: Selective Targeting of Senescent FHs74Int Cells by Human Breast Milk Free Fatty Acids

doi: 10.3390/biology14101355

Figure Lengend Snippet: Detection of senescent cells in FHs74 Int cells. ( A ) Representative β-galactosidase staining of FHs74 cells showing the presence of senescent cells. ( B ) Representative histogram of the expression of p16INK4a (left) and p21 (right) assessed by flow cytometry. Data are presented as Mean ± standard error of the mean (SEM), n = 4. ( C ) Representative flow cytometry dot plot image of Pyronin Y/7-AAD dual staining of freshly thawed FHs74.

Article Snippet: The FHs74 Int cells (FHs74) were obtained from the American Type Culture Collection (ATCC; CCL-241; Manassas, VA, USA).

Techniques: Staining, Expressing, Flow Cytometry

Journal: Biology

Article Title: Selective Targeting of Senescent FHs74Int Cells by Human Breast Milk Free Fatty Acids

doi: 10.3390/biology14101355

Figure Lengend Snippet: Impact of HBM on FHs74 Int cells. ( A ) Representative β-galactosidase staining of FHs74. cells exposed or not to NonCyt or Cyt-HBM. ( B ) Flow cytometry cell counts of viable (7-AAD negative) FHs74 cells recovered after exposure to different HBM samples. ( C ) Flow cytometry expression of Annexin V. Data are presented as Mean ± standard error of the mean (SEM); * p ≤ 0.05, ** p ≤ 0.01. n = 4–5 different HBM samples. Note: The difference in sample size between the FHs74-only condition (n = 4) and the HBM-treated conditions (n = 5) is due to the experimental design, in which multiple HBM donors were tested simultaneously against a single FHs74Int control within the same experiment. FHs74 = FHs74 intestinal cells alone; HBM = Human breast milk; FHs74 + FM = FHs74 intestinal cells exposed to formula milk; FHs74 + HBM = FHs74 intestinal cells exposed to human breast milk.

Article Snippet: The FHs74 Int cells (FHs74) were obtained from the American Type Culture Collection (ATCC; CCL-241; Manassas, VA, USA).

Techniques: Staining, Flow Cytometry, Expressing, Control

Journal: Biology

Article Title: Selective Targeting of Senescent FHs74Int Cells by Human Breast Milk Free Fatty Acids

doi: 10.3390/biology14101355

Figure Lengend Snippet: Quantification of β-galactosidase activity in FHs74 cells. ( A ) Luminescence signal measured by the beta-glo assay following exposure to different HBM samples. ( B ) Two categories of HBM based on their β-galactosidase-reducing activity. Data are presented as Mean and standard error of the mean (SEM); ** p ≤ 0.01; **** p ≤ 0.0001. n = 14 different donors of HBM. Note: Quantification of β-galactosidase activity in FHs74Int cells exposed to different HBM samples. Error bars are not visible for the FHs74Int and Noncyt-HBM conditions due to the absence of variation between samples (100% signal in both cases). FHs74 = FHs74 intestinal cells alone; HBM = Human breast milk; FHs74 + HBM = FHs74 intestinal cells exposed to human breast milk.

Article Snippet: The FHs74 Int cells (FHs74) were obtained from the American Type Culture Collection (ATCC; CCL-241; Manassas, VA, USA).

Techniques: Activity Assay, Glo Assay

Journal: Biology

Article Title: Selective Targeting of Senescent FHs74Int Cells by Human Breast Milk Free Fatty Acids

doi: 10.3390/biology14101355

Figure Lengend Snippet: Expression of signaling molecules after HBM exposure. Expression of phosphorylated p38 ( A ) and NFκB ( B ) in FHs74 cells following treatment with Cyt- or Noncyt-HBM. Data are presented as Mean and standard error of the mean (SEM); **** p ≤ 0.0001. n = 5–9 different donors of human breast milk. Note: The difference in sample size between the FHs74-only condition (n = 4–5) and the HBM-treated conditions (n = 5–9) is due to the experimental design, in which multiple HBM donors were tested simultaneously against a single FHs74Int control within the same experiment . FHs74 = FHs74 intestinal cells alone; FHs74 + FM = FHs74 intestinal cells exposed to formula milk; FHs74 + HBM = FHs74 intestinal cells exposed to human breast milk.

Article Snippet: The FHs74 Int cells (FHs74) were obtained from the American Type Culture Collection (ATCC; CCL-241; Manassas, VA, USA).

Techniques: Expressing, Control

Journal: Biology

Article Title: Selective Targeting of Senescent FHs74Int Cells by Human Breast Milk Free Fatty Acids

doi: 10.3390/biology14101355

Figure Lengend Snippet: Cellular stress and apoptosis markers. ( A ) Caspase 3/7 activity in FHs74 cells exposed to Cyt- and Noncyt-HBM. ( B ) γH2AX expression levels indicate DNA damage. ( C ) Mitochondrial membrane potential measured by DiOC 2 (3) staining. Data are presented as Mean and standard error of the mean (SEM); * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. n = 5 different donors of human breast milk. Note: The difference in sample size between the FHs74-only condition (n = 4) and the HBM-treated conditions (n = 5) is due to the experimental design, in which multiple HBM donors were tested simultaneously against a single FHs74Int control within the same experiment. FHs74 = FHs74 intestinal cells alone; FHs74 + FM = FHs74 intestinal cells exposed to formula milk; FHs74 + HBM = FHs74 intestinal cells exposed to human breast milk.

Article Snippet: The FHs74 Int cells (FHs74) were obtained from the American Type Culture Collection (ATCC; CCL-241; Manassas, VA, USA).

Techniques: Activity Assay, Expressing, Membrane, Staining, Control

Journal: Biology

Article Title: Selective Targeting of Senescent FHs74Int Cells by Human Breast Milk Free Fatty Acids

doi: 10.3390/biology14101355

Figure Lengend Snippet: Fatty acid fraction and HBM cytotoxicity. ( A ) Quantification of FFA levels across HBM samples ( n = 10). ( B ) Correlation between FFA concentration and Beta-glo signal ( n = 10). ( C ) Beta-glo signal after exposure to whole HBM, aqueous, and fatty fractions ( n = 4). Data are presented as Mean and standard error of the mean (SEM); ** p ≤ 0.01; **** p ≤ 0.0001. FHs74 = FHs74 intestinal cells alone; FHs74 + FM = FHs74 intestinal cells exposed to formula milk; FHs74 + HBM = FHs74 intestinal cells exposed to human breast milk.

Article Snippet: The FHs74 Int cells (FHs74) were obtained from the American Type Culture Collection (ATCC; CCL-241; Manassas, VA, USA).

Techniques: Concentration Assay

Journal: Biology

Article Title: Selective Targeting of Senescent FHs74Int Cells by Human Breast Milk Free Fatty Acids

doi: 10.3390/biology14101355

Figure Lengend Snippet: FFA concentration and β-galactosidase signal. ( A ) Quantification of FFA levels in Noncyt-HBM samples prior and after 4 days at 37 °C. ( B ) Beta-glo signal of FHs74 intestinal cells to Noncyt-HBM samples prior and after 4 days at 37 °C. Data are presented as Mean and standard error of the mean (SEM); * p ≤ 0.05; *** p ≤ 0.001. n = 4 different donors of human breast milk. HBM = human breast milk; FFA = Free fatty acids.

Article Snippet: The FHs74 Int cells (FHs74) were obtained from the American Type Culture Collection (ATCC; CCL-241; Manassas, VA, USA).

Techniques: Concentration Assay

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin

doi: 10.1186/s13071-024-06244-2

Figure Lengend Snippet: Primary cells used in the current study

Article Snippet: FHs74 , ATCC , CCL-241 , .

Techniques:

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin

doi: 10.1186/s13071-024-06244-2

Figure Lengend Snippet: Toxoplasma gondii arrests primary human and bovine cells in the S-phase 24 h p.i. Three isolates of HUVEC, HFF, FHs74, BSIEC and BCEC were infected with T. gondii tachyzoites, and the samples were collected 24 h p.i. Fixed samples were stained with propidium iodide (PI) and analysed using FACS. Gating was performed by first selecting the cell population according to the shape and granularity. Then, the cell cycle phases were analysed using a histogram of the number of cells versus the PI signal. Cells in the first peak correspond to those in the G1-phase, the second peak to cells in the G2/M-phase, and cells in between the two peaks were cells in the S-phase. The results showed that all human cells were arrested in the S-phase ( A ), whilst only one bovine cell showed no effect on cell cycle progression before T. gondii infection ( B ). Graph bars represent the median ± SD of three biological replicates

Article Snippet: FHs74 , ATCC , CCL-241 , .

Techniques: Infection, Staining

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin

doi: 10.1186/s13071-024-06244-2

Figure Lengend Snippet: Cell cycle arrest in T. gondii -infected cells is independent of the mitosis checkpoint protein control cyclin B1. Three donors of HUVEC, HFF, FHs74, BSIEC and BCEC were infected with T. gondii tachyzoites and fixed 24 h p.i. Cells were stained against cyclin B1 and an isotype control and analysed using FACS. Gating was done first by choosing the cell population and then selecting the same number of cells in each sample ( A ). The cyclin B1 signal was analysed as the mean of fluorescence (MOF) and plotted for non-infected and T. gondii- infected cells. The results showed that cyclin B1 was not affected after infection in any of the cell types studied ( B , C ). Graph bars represent the median ± SD of three biological replicates

Article Snippet: FHs74 , ATCC , CCL-241 , .

Techniques: Infection, Control, Staining, Fluorescence

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin

doi: 10.1186/s13071-024-06244-2

Figure Lengend Snippet: Toxoplasma gondii infection affects the mitosis rate and the chromosome segregation at the mitosis phase. HUVEC, HFF, FHs74, BSIEC and BCEC were infected with T. gondii tachyzoites, and were fixed 24 h p.i. with PFA and stained against DAPI (chromosome marker, blue), γ-tubulin (centrosome marker, magenta) and T. gondii (green, asterisks). The mitosis index was counted as the total number of cells facing mitosis (see scheme) related to the total number of cells in the field of view. This percentage was calculated for the bovine ( A ) and human cell lines ( B ). The results showed that no bovine cell line studied here modified its mitosis rate after infection with T. gondii. However, FHs74 and HFF showed an increased proportion of mitotic cells . C The mitosis phases were followed by chromosome segregation, and the results showed that all cell lines developed chromosome segregation errors mainly due to an abnormal number of centrosomes (white arrows). Thereafter, the proportion of aberrant mitosis was determined in both bovine and human cells by counting the total number of aberrant mitoses relative to the total mitotic cells (normal and aberrant). The scale bar represents 5 µm

Article Snippet: FHs74 , ATCC , CCL-241 , .

Techniques: Infection, Staining, Marker, Modification

Journal: Parasites & Vectors

Article Title: Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin

doi: 10.1186/s13071-024-06244-2

Figure Lengend Snippet: Toxoplasma gondii induces cytokinesis failure in human and bovine host cells. HUVEC, HFF, FHs74, BSIEC and BCEC were infected with T. gondii tachyzoites, and fixed 24 h p.i. with PFA and stained against DAPI (nuclear marker, blue), β-catenin (membrane marker, green) and T. gondii (red). As in the scheme, binucleated cells were those cells with more than one nucleus per cell ( A ). The total number of binucleated cells was normalized to the total number of cells counted and presented as a percentage in the graphs. The results showed that all tested cell lines increased the percentage of binucleated cells after infection with T. gondii ( B , C ). Graph bars represent the median ± SD of three biological replicates

Article Snippet: FHs74 , ATCC , CCL-241 , .

Techniques: Infection, Staining, Marker, Membrane