DHA induces apoptosis via both extrinsic and intrinsic apoptosis pathways.

<p>(A) DHA induced activation of caspase-8 and -9 assessed by fluorometric assay. Cells were treated with DHA for 36 h. **<i>P</i><0.01, compared with control. (B) DHA induced caspase-8- and -9-dependent caspase-3 activation by fluorometric assay. Cells were treated with DHA for 48 h in the presence or absence of zIETD-fmk and zLEHD-fmk, respectively. <i>**P</i><0.01, compared with control; <i>^##P</i><0.01, compared with DHA treatment alone. (C) DHA induced caspase-8- and -9-dependent cytotoxicity assessed by CCK-8 assay. Cells were treated with DHA for 24 and 48 h in the presence or absence of zIETD-fmk and zLEHD-fmk, respectively. *8<i>P<0.01</i>, compared with control; ^$<i>P<0.05</i>, ^$$<i>P<0.01</i> and ^&<i>P</i><0.05, compared with DHA treatment alone. (D) DHA ROS-mediated apoptosis assessed by FCM. **<i>P<0.01,</i> compared with control; ^##<i>P<0.01</i>compared with DHA alone. (E) DHA induced ROS-dependent caspase-8 activation. **<i>P<0.01</i>, compared with control; ^##<i>P<0.01</i>, compared with DHA treatment alone. (F) DHA induced ROS- and caspase-8-dependnent loss of Δψ_m determined by FCM analysis. <i>**P</i><0.01, compared with control; ^##<i>P<0.01</i>, compared with DHA treatment alone. (G) DHA induced caspase-8-dependent caspase-9 activation. *<i>P<0.05</i> and **<i>P<0.01</i>, compared with control; ^#<i>P<0.05</i>, compared with DHA treatment alone. (H) Typical fluorescence images of Bid translocation to mitochondria inside single living cell after DHA treatment for 36 h. Control cells show the uniform distribution of Bid, while DHA-treated cells show the co-localization between Bid and mitochondria. Scale Bar: 5 µm.</p

IR synergistically enhances DHA-induced G₂/M arrest and apoptosis.

<p>(A) FCM analysis of cells cycle after low-dose IR treatment for 24 h and 36 h in the presence or absence of DHA. (B and C): IR potentiated the DHA-induced G₂/M arrest at 24 h (B) and apoptosis at 36 h (C) analyzed by FCM. <i>**P</i><0.01, compared with treatment with control; <i>^##P</i><0.01, compared with DHA treatment alone, <i>^$$P</i><0.01 compared with 2 Gy IR treatment; <i>^&&P</i><0.01, compared with 4 Gy IR treatment. Cells treated with different doses of IR were cultured with 20 µg/ml of DHA for indicated time and then stained with 5 µg/ml of PI before being analyzed by FCM.</p

ROS-dependent G2/M cell cycle arrest by DHA and IR respectively.

<p>(A) Dynamical fluorescence images of ROS generation in living cells after DHA treatment. Cells were incubated with 20 µM DCFH-DA, an oxidation-sensitive fluorescent probe, for 30 min in the dark and then treated with DHA. The levels of intracellular ROS were monitored by a confocal microscope. Scale bar: 20 µm. (B) Dynamics of DHA-induced ROS generation corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059827#pone-0059827-g002" target="_blank">Figure 2</a> (A). (C and D) FCM assay of ROS generation at 30 min (C) and 120 min (D) after IR, DHA and combination treatment, respectively. (E and F) ROS-dependent G₂/M arrest induced by IR (E) and DHA (F) respectively analyzed by FCM. Cells were irradiated with IR or DHA in the presence or absence of NAC, and then stained with 5 µg/ml of PI before being analyzed by FCM. *<i>*P</i><0.01, compared with control; ^##<i>P</i><0.01, compared with DHA treatment alone (E) and ^##<i>P<0.01</i> and ^&&<i>P<0.01</i>, compared with IR treatment alone (F).</p

IR potentiates DHA-induced extrinsic apoptosis pathway.

<p>(A–B): IR did not accelerate the DHA-induced loss of Δψ_m at 24 h (A) and 36 h (B) after treatment assessed by FCM. <i>**P</i><0.01, compared with control. (C) IR did not accelerate DHA-induced caspase-9 activation. <i>**P</i><0.01, compared with control. (D and E) IR accelerated DHA-induced activation of caspase-8 (D) and -3 (E). Cells treated with IR were then cultured with DHA for 36 h. Caspase-8, -9 and -3 activities were measured by the fluorescence substrate Ac-IETD-AFC, Ac-LEHD-AFC and Ac-DEVD-AFC, respectively. <i>**P</i><0.01, compared with control, <i>^##P</i><0.01, compared with treatment with DHA alone.</p

AFM ultrastructural data of control chondrocytes.

<p>(A1–A3) Control chondrocytes. (B1–B3) Chondrocytes treated with 1.5 mM SNP for 12 h. (C1–C3) The chondrocytes were pretreated with 100 μM of RV for 24 h, and then treated with 1.5 mM of SNP for 12 h. Scanning area: 2×2 μm². (A1), (B1), (C1) was topography mode. (A2), (B2), (C2) 3-D mode of (A1), (B1) and (C1), respectively. (A3), (B3), (C3) was contour map of (A1), (B1) and (C1), respectively. (D1) and (D2) were histograms of average roughness (Ra) of chondrocytes which were analyzed in 5×5 μm² and 2×2 μm², respectively. In (D1) and (D2), ten cells in each group were selected to measure the values of Ra, statistical analysis was performed using Student's <i>t</i>-test. P<0.05 was regarded as statistically significant.</p

Protection effects of RV on SNP-induced apoptosis of chondrocytes.

<p>Cells were pretreated with different concentrations (0, 25, 50 and 100 mM) of RV for 24 h, and then treated with 1.5 mM of SNP for 12 h. After that, the cell viability was assayed using CCK-8 (comparing with control group, *P<0.05, **P<0.01; comparing with SNP treated group, #P<0.05, ##P<0.01, ###P<0.001).</p

Alterations in nanobiotechnology of chondrocytes detected by AFM.

<p>(A1–A5) isolation of chondrocytes: (A1) Cartilage collected from the bilateral joints of the knees, hips, and shoulders. (A2) The joints were minced into small pieces, treated with 0.015% trypsin for 30 min, and subsequently digested. (A3) Morphology of primary joint chondrocytes. (A4) The morphology of primary joint chondrocytes cultured for 7 days. (A5) The AFM tip was employed to detect the morphology and biomechanics of chondrocytes. (A6) Typical force-distance curve detected using AFM: (1) The tip is approaching the surface of sample, (2) the tip is just in contact with the surface of cells, (3) the tip is further put into repulsive contact with the cellular surface, (4) lastly, the tip-sample contact is retracted. (A7–A9) are the representative force-distance curves obtained on control chondrocytes (A7), chondrocytes treated with 1.5 mM SNP for 12 h (A8), and chondrocytes pretreated with RV and the induce with SNP (A9), respectively. The elasticity maps, histogram of elasticity, adhesion force map and histogram of adhesion force of control chondrocytes (B1–B4), chondrocytes treated with 1.5 mM SNP for 12 h (C1–C4), and chondrocytes pretreated with RV and then cotreated with SNP (A4), respectively.</p

Cytotoxicity of SNP in chondrocytes.

<p>(A) <i>C</i>ell viability of chondrocytes treated by different concentrations of SNP for 24 h. (B) <i>C</i>ell viability of chondrocytes treated by 1.5 mM of SNP for different time periods (comparing with control group, *P<0.05, **P<0.01, ***P<0.001). The results indicated the killing effects of SNP on chondrocytes were in a dose- and time-dependent manner.</p

Evaluation on covalent and noncovalent linking of peptide to graphene oxide for MMP-9 detection

<p>FITC-labeled peptide (Pep-FITC) containing the cleavage site of matrix metalloproteinase-9 (MMP9) was non-covalently or covalently linked to graphene oxide (GO) to form non-covalent or covalent nanoprobes (nGO/Pep-FITC and nGO-Pep-FITC) for MMP9 detection. nGO-Pep-FITC was prepared by formation of amide bonds between the carboxyl groups of c-nGO and the amine groups on Pep-FITC with the addition of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC). Pep-FITC was physically adsorbed onto the surface of c-nGO through π-π stacking and electrostatic interactions to produce nGO/Pep-FITC. Both nGO/Pep-FITC and nGO-Pep-FITC exhibited good selectivity for MMP9 detection. nGO/Pep-FITC and nGO-Pep-FITC showed 29.61 and 32.53 pM of detection limits for MMP9, respectively. nGO-Pep-FITC was much more resistant to bovine serum albumin (BSA) than nGO/Pep-FITC, thus may be applicable to analyze clinical samples especially containing proteins.</p

Supplementary document for Im-SCC-FRET: Improved single-cell-based calibration of a FRET system - 6747398.pdf

Im-SCC-FRET: Improved single-cell-based calibration of a FRET system: supplemental documen