Journal: International Journal of Molecular Sciences

Article Title: Pharmacological Inhibition of Cyclin-Dependent Kinases Triggers Anti-Fibrotic Effects in Hepatic Stellate Cells In Vitro

doi: 10.3390/ijms21093267

Figure Lengend Snippet: CR8-treatment triggers accumulation of immortalized hepatic stellate cells in mitosis. GRX and LX-2 cells were treated for 48 h with increasing concentrations of CR8. Dimethyl sulfoxide (DMSO) treatment alone (0 nM) served as control. ( a , b ) Determination of cellular DNA content in ( a ) murine GRX and ( b ) human LX-2 cells through DAPI staining and flow cytometry (FACS) analysis. Left panels: histograms showing DNA content and the corresponding assignment to the distinct cell cycle phases in untreated ( a ) murine GRX and ( b ) human LX-2 cells as follows: G1: 2n DNA content, S: 2-4n; G2/M: 4n. Right panels: DNA content distribution of CR8-treated ( a ) murine GRX and ( b ) human LX-2 cells at indicated concentrations. ( c , d ) Cells were stained with an antibody directed against phosphorylated Histone H3 (pH3) and analyzed by FACS. ( c ) Representative FACS-plots of cleaved pH3-positive (% of single cells) GRX (upper panels) and LX-2 (lower panels) cells at different concentrations of CR8. ( d ) Quantification of pH3-positive GRX (left panel, n = 4) and LX-2 (right panel) cells (% of single cells) as fold induction compared to controls by FACS. Values are means of at least n = 3 independent experiments, unless otherwise indicated. ** p < 0.01; **** p < 0.0001.

Article Snippet: Co-staining of cells was performed using a mix of fluorescence-labeled antibodies against pH3-AL647 (1:50, Cell Signalling Technology, Danver, MA, USA) and cleaved PARP-PE (1:50, BD Biosciences, San Jose, CA, USA) diluted in FACS-blocking buffer (mixture of 0.66% human/rabbit/mouse serum, Sigma-Aldrich, and 1% Bovine Serum Albumin, Sigma-Aldrich in 1× PBS) for 30 min at 4 °C.

Techniques: Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Niclosamide suppresses acute myeloid leukemia cell proliferation through inhibition of CREB-dependent signaling pathways

doi: 10.18632/oncotarget.23794

Figure Lengend Snippet: HL60 and KG1cells were treated with 1 μM niclosamide for 1d. A. Cells were fixed, and then stained with anti-cleaved PARP (cPARP) antibody and DAPI. Percentages of apoptotic cells are indicated. B. Fixed cells were stained with anti-phospho-CREB(p-CREB) (S133) and anti-phospho-NFkB p65 (S536) antibodies. Plots show the suppression of p-CREB in cells treated with niclosamide as compared with untreated control, but not phospho-NFkB p65. Percentages of p-CREB-negative population are presented as mean ± SEM ( n = 3). C. Cells were stained with anti-phospho-STAT1 (Y701), anti-phospho-STAT3 (Y705), and anti-phospho-STAT5 (Y694) antibodies. HL60 cells: Top panels, KG1 cells: Bottom panels. Negative populations of each antibody were calculated. Values are graphed as mean ± SEM ( n = 3). *, p < .05; **, p < .001.

Article Snippet: The following antibodies were used in intracellular protein staining: PE-conjugated anti-Cyclin A (clone BF683), Alexa Fluor 647-conjugated anti-cPARP (clone F21-852), PE-conjugated anti-pCERB (pS133) (clone J151-21), Alexa Fluor 488-conjugated anti-pSTAT1 (pY701) (clone 4a), PE-conjugated anti-pSTAT3 (pY705) (clone 4/P-STAT3), Alexa Fluor 647-conjugated anti-pSTAT5 (pY694) (clone 47, BD Biosciences); PECy7-conjugated ant-pH3 (pS28) (clone HTA283, Biolegend); Alex Fluor 647-conjugated anti-p-NFkB p65 (pS536) (clone 93H1, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining, Control