Journal: Oxidative Medicine and Cellular Longevity

Article Title: MHY2233 Attenuates Replicative Cellular Senescence in Human Endothelial Progenitor Cells via SIRT1 Signaling

doi: 10.1155/2019/6492029

Figure Lengend Snippet: List of primer sequences used for qRT-PCR.

Article Snippet: The membranes were blocked with 5% skim milk for one hour at room temperature and were incubated with primary antibodies against SIRT1 (sc-74504, Santa Cruz), p53 acetylated on K382 (Ac-p53) (ab75754, Abcam), CDKN2A/p16 INK4a (ab108349, Abcam), p21 (ab109199, Abcam), FOXO3a (#2497, Cell Signaling), cyclin D1 (sc-8396, Santa Cruz), cyclin E (sc-481, Santa Cruz), eNOS (#9586, Cell Signaling), endothelial nitric oxide synthase (eNOS) phosphorylated on S1177 (p-eNOS) (#9571, Cell Signaling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-25778 and sc-47724, Santa Cruz) at 4°C overnight.

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MHY2233 Attenuates Replicative Cellular Senescence in Human Endothelial Progenitor Cells via SIRT1 Signaling

doi: 10.1155/2019/6492029

Figure Lengend Snippet: Characterization of senescent EPCs: (a) passage 8 EPCs were considered young, and passage 18 EPCs were considered senescent. Young cells and senescent cells were cultured in medium, and SA- β -gal staining was performed. Representative images of SA- β -gal-stained young and senescent EPCs are shown. SA- β -gal-positive cells are stained a green color. Scale bars: 25 μ M and 5 μ M. (b) Quantification of SA- β -gal-positive cells from three replicates ( n = 3 per group). (c) Young and senescent EPCs were seeded in 96-well plates, and cell proliferation capacity was determined using the WST-8 assay. Absorbance was measured at 0, 12, 24, and 48 h at 450 nM wavelength ( n = 6 per group). (d) Young and senescent cells were seeded in the upper chamber of a Transwell® culture plate. After 6 h, migrated cells were stained and images were taken under a microscope at 100x magnification ( n = 3 wells per group). (e) Quantification of the number of migrated cells among young and senescent EPCs using ImageJ software. (f) For tube formation capacity, young and senescent EPCs were seeded in 96-well plates containing Matrigel®. After 6 h, representative images were taken under a microscope at 40x magnification ( n = 3 per group). Scale bars: 25 μ M. (g) Quantification of the tube length of branches per field in μ M and (h) number of tube branches per field using ImageJ software ( n = 3). (i) Cells were harvested, and total cell lysates were subjected to western blot analysis for protein expression of SIRT1, acetylated p53, p16 INK4a , and p21 for young and senescent EPCs. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.0005 vs. young EPCs by unpaired Student's t -test.

Article Snippet: The membranes were blocked with 5% skim milk for one hour at room temperature and were incubated with primary antibodies against SIRT1 (sc-74504, Santa Cruz), p53 acetylated on K382 (Ac-p53) (ab75754, Abcam), CDKN2A/p16 INK4a (ab108349, Abcam), p21 (ab109199, Abcam), FOXO3a (#2497, Cell Signaling), cyclin D1 (sc-8396, Santa Cruz), cyclin E (sc-481, Santa Cruz), eNOS (#9586, Cell Signaling), endothelial nitric oxide synthase (eNOS) phosphorylated on S1177 (p-eNOS) (#9571, Cell Signaling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-25778 and sc-47724, Santa Cruz) at 4°C overnight.

Techniques: Cell Culture, Staining, Microscopy, Software, Western Blot, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MHY2233 Attenuates Replicative Cellular Senescence in Human Endothelial Progenitor Cells via SIRT1 Signaling

doi: 10.1155/2019/6492029

Figure Lengend Snippet: SIRT1 deacetylase activity of MHY2233: (a) schematic diagram to show isolation of EPCs from the human umbilical cord blood, treatment with drugs, and other basic experimental designs. (b) Chemical structure of MHY2233. (c) EPCs were seeded in 96-well plates and treated with different concentrations of MHY2233 for 24 h ( n = 6 per group). Cytotoxicity assay of MHY2233 at different concentrations was determined using WST-8 assay ( n = 6 per group). (d) Relative mRNA levels of SIRT1 to SIRT7 in young and senescent EPCs were evaluated by qRT-PCR ( n = 3 per group). (e) Relative mRNA expression of SIRT1 to SIRT7 in senescent EPCs treated with DMSO and MHY2233 ( n = 3 per group). (f) SIRT1 deacetylase activity was performed using a commercially available kit. Measurement of SIRT1 deacetylase activity of MHY2233 along with positive and negative controls ( n = 6 per group). ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.0005 vs. DMSO (control) and indicated groups by a one-way ANOVA test.

Article Snippet: The membranes were blocked with 5% skim milk for one hour at room temperature and were incubated with primary antibodies against SIRT1 (sc-74504, Santa Cruz), p53 acetylated on K382 (Ac-p53) (ab75754, Abcam), CDKN2A/p16 INK4a (ab108349, Abcam), p21 (ab109199, Abcam), FOXO3a (#2497, Cell Signaling), cyclin D1 (sc-8396, Santa Cruz), cyclin E (sc-481, Santa Cruz), eNOS (#9586, Cell Signaling), endothelial nitric oxide synthase (eNOS) phosphorylated on S1177 (p-eNOS) (#9571, Cell Signaling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-25778 and sc-47724, Santa Cruz) at 4°C overnight.

Techniques: Histone Deacetylase Assay, Activity Assay, Isolation, Cytotoxicity Assay, Quantitative RT-PCR, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MHY2233 Attenuates Replicative Cellular Senescence in Human Endothelial Progenitor Cells via SIRT1 Signaling

doi: 10.1155/2019/6492029

Figure Lengend Snippet: Roles of MHY2233 in SIRT1 signaling and rescue of replicative senescence in EPCs: (a) EPCs were cultured with DMSO or MHY2233 (10 nM, 100 nM, or 1 μ M) for replicative senescence, and SA- β -gal staining was performed. Representative images of SA- β -gal stain in senescent EPCs. Cells stained green are SA- β -gal-positive cells. (b) Quantification of SA- β -gal-positive cells ( n = 3 per group). (c) Cells were harvested, and total cell lysates were used in western blot analysis for protein expression of SIRT1 in senescent EPCs upon treatment with different concentrations of MHY2233. (d) After chronic treatment of senescent EPCs with DMSO, 10 nM MHY2233, 100 nM resveratrol, or 100 nM EX527, mRNA and total protein were isolated. Measurement of relative mRNA levels of SIRT1, (e) p16, (f) p21, and (g) p53 was analyzed using qRT-PCR ( n = 3 per group). (h) Whole cell lysates were subjected to western blot analysis for determining protein levels of FOXO3a, SIRT1, acetylated p53, p16 INK4a , and p21 in senescent EPCs. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.0005 vs. DMSO (control) by a one-way ANOVA test.

Article Snippet: The membranes were blocked with 5% skim milk for one hour at room temperature and were incubated with primary antibodies against SIRT1 (sc-74504, Santa Cruz), p53 acetylated on K382 (Ac-p53) (ab75754, Abcam), CDKN2A/p16 INK4a (ab108349, Abcam), p21 (ab109199, Abcam), FOXO3a (#2497, Cell Signaling), cyclin D1 (sc-8396, Santa Cruz), cyclin E (sc-481, Santa Cruz), eNOS (#9586, Cell Signaling), endothelial nitric oxide synthase (eNOS) phosphorylated on S1177 (p-eNOS) (#9571, Cell Signaling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-25778 and sc-47724, Santa Cruz) at 4°C overnight.

Techniques: Cell Culture, Staining, Western Blot, Expressing, Isolation, Quantitative RT-PCR, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MHY2233 Attenuates Replicative Cellular Senescence in Human Endothelial Progenitor Cells via SIRT1 Signaling

doi: 10.1155/2019/6492029

Figure Lengend Snippet: Possible working model of MHY2233 in preventing senescence in EPCs: possible mechanism of MHY2233 to prevent vascular senescence in EPCs. MHY2233 increases SIRT1 activity and, upon aging or stress, increases the activity of FOXO3a, which translocates into the nucleus and upregulates the expression of SIRT1. SIRT1 increases the activity of eNOS and reduces the ROS levels by producing nitric oxide. Similarly, SIRT1 downregulates the SASPs and p16 ink4a and reduces EPC senescence. Moreover, SIRT1 activity deacetylates p53 and downregulates the expression of p21, thereby reducing EPC senescence.

Article Snippet: The membranes were blocked with 5% skim milk for one hour at room temperature and were incubated with primary antibodies against SIRT1 (sc-74504, Santa Cruz), p53 acetylated on K382 (Ac-p53) (ab75754, Abcam), CDKN2A/p16 INK4a (ab108349, Abcam), p21 (ab109199, Abcam), FOXO3a (#2497, Cell Signaling), cyclin D1 (sc-8396, Santa Cruz), cyclin E (sc-481, Santa Cruz), eNOS (#9586, Cell Signaling), endothelial nitric oxide synthase (eNOS) phosphorylated on S1177 (p-eNOS) (#9571, Cell Signaling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-25778 and sc-47724, Santa Cruz) at 4°C overnight.

Techniques: Activity Assay, Expressing

Journal: Frontiers in Microbiology

Article Title: Novel silver nanoparticle-based biomaterials for combating Klebsiella pneumoniae biofilms

doi: 10.3389/fmicb.2024.1507274

Figure Lengend Snippet: Cell viability and Cytotoxicity of AgNPs@chitosan and AgNPs@chitosan-NaF on normal lung fibroblast WI-38 cells (A,B) , and lung carcinoma epithelial A549 ATCC-185 cells (C,D) for 24 h. The results were taken from replicated ( n = 3) (Mean ± SD).

Article Snippet: Further evaluation of the AgNPs@chitosan and AgNPs@chitosan-NaF showed the lowest level of cytotoxic activity against WI-38 cells against WI-38 cells at a concentration of less than 1,000 μg mL −1 (>59.25%),and at a concentration of less than 62.5 μg mL −1 (>59.10%), respectively ( , ), and cytotoxic activity against A549 ATCC-185 cells at a concentration of less than 1,000 μg mL −1 (>57.45%),and at a concentration of less than 15.62 μg mL −1 (>73.67%), respectively ( , ).

Techniques:

Journal: Frontiers in Microbiology

Article Title: Novel silver nanoparticle-based biomaterials for combating Klebsiella pneumoniae biofilms

doi: 10.3389/fmicb.2024.1507274

Figure Lengend Snippet: Morphological features on lung carcinoma epithelial A549 cells of (A) AgNPs@chitosan and (B) AgNPs@chitosan-NaF, the images were taken from the cells were treated with an average size of 10 nm for 24 h.

Article Snippet: Further evaluation of the AgNPs@chitosan and AgNPs@chitosan-NaF showed the lowest level of cytotoxic activity against WI-38 cells against WI-38 cells at a concentration of less than 1,000 μg mL −1 (>59.25%),and at a concentration of less than 62.5 μg mL −1 (>59.10%), respectively ( , ), and cytotoxic activity against A549 ATCC-185 cells at a concentration of less than 1,000 μg mL −1 (>57.45%),and at a concentration of less than 15.62 μg mL −1 (>73.67%), respectively ( , ).

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