Fig 1.
Structures of thalidomide (racemic form), fluoro-thalidomide (racemic form), and its (R)- and (S)- enantiomers, (R)-fluoro-thalidomide and (S)-fluoro-thalidomide.
Fig 2.
Effects of fluoro-thalidomide on H929 and Oda cells.
(A) Proportion of annexin V-positive cells in H929 cells by thalidomide, fluoro-thalidomides fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide, or cytosine β-D-arabinofuranoside (Data are shown as mean ± SD (n = 3). ***, p < 0.001 **, p < 0.01 *, p < 0.05 vs. untreated, unless otherwise noted). (B) Plots and histograms of annexin V and propidium iodide-positive cells in H929 cells after 24 h treatment with fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide, or cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL. (C) Proportion of annexin V-positive cells in Oda cells by thalidomide, fluoro-thalidomide, (R)-fluoro-thalidomide or (S)-fluoro-thalidomide.
Fig 3.
Effects of fluoro-thalidomide on H929, AGLCL and U937 cells.
(A) Proportion of annexin V-positive cells in AGLCL cells, normal human B cells, by thalidomide, fluoro-thalidomide, (R)-fluoro-thalidomide or (S)-fluoro-thalidomide (Data are shown as mean ± SD (n = 3). ***, p < 0.001 **, p < 0.01 *, p < 0.05 vs. untreated, unless otherwise noted). (B) Proportion of annexin V-positive cells in U937 cells by thalidomide, fluoro-thalidomide, (R)-fluoro-thalidomide or (S)-fluoro-thalidomide. (C) MTT assay measuring (550 nm) the optical density of H929 cells treated with fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide, or cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL for 24 h. (D) Observation of morphological changes in H929 cells after treatment with fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide, or cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL for 24 h using Wright-Giemsa staining protocol. Apoptotic bodies (indicated by arrows) were observed after the treatment.
Fig 4.
Measurement of caspase activity in H929 cells.
(A) Caspase-3 activity (after treatment with fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL for 24 h. Data are shown as mean ± SD (n = 3). ***, p < 0.001 vs. untreated, unless otherwise noted). (B) Caspase-8 activity. (C) Caspase-9 activity. (D) Caspase inhibitor activity. Annexin V expression in H929 cells by fluoro-thalidomide at a concentration of 20 μg/mL for 24 h was inhibited after the addition of inhibitors of caspase-3, -8–9 and caspase-family inhibitor.
Fig 5.
Effects of fluoro-thalidomide on H929 cells.
(A) Detection of cleaved PARP expression (after treatment with fluoro-thalidomide, (R)-fluoro-thalidomide, (S)-fluoro-thalidomide or cytosine β-D-arabinofuranoside at a concentration of 20 μg/mL for 24 h unless otherwise noted). (B) Detection of Fas (CD95) expression. (C) From left to right, detection of DNA fragmentation treated with cytosine β-D-arabinofuranoside, (S)-fluoro-thalidomide, (R)-fluoro-thalidomide, fluoro-thalidomide and untreated sample by agarose gel electrophoresis compared to DNA size marker (100bp DNA Ladder, Promega, Madison, USA). (D) Cell cycle analysis. The proportion of cells in the G1, S, G2/M or sub-G1 phase. Data are shown as mean (n = 3). (E) Phosphorylation expression of Bcl-2 Ser 70. Data are shown as mean ± SD (n = 3). ***, p < 0.001 **, p < 0.01 *, p < 0.05 vs. untreated.
Fig 6.
Fluoro-thalidomide not thalidomide promotes VEGF induced tube formation in HUVECs.
(A) Representative photographs were shown in tube formation. Quantitative analysis of stained tube formation structures was measured by using angiogenesis imaging analyzer, version 2 in five different areas for each well. We measured joint (B), joints (B), paths (C), tube area (D), lengths (E). Data are shown as mean ± SEM (n = 3 or 4). ##, p < 0.01 vs. Control group, and *, p < 0.05 **, p < 0.01 vs. Vehicle-treated group.