Fig 1.
Macroscopic changes of the thymi upon cuprizone treatment.
Four week-old male mice were treated with cuprizone for one week. Representative photographs (A) of the open chest of a control (Cont.) and a cuprizone-treated (CPZ) animal are demonstrated. Arrows point to the thymi of the mice. The scale bar indicates 5 mm. Thymus mass (B), body mass (C) and relative thymus mass (thymus tissue mass/ body mass) (E) of control (grey bars) and cuprizone-treated (black bars) animals are presented as bar diagrams, mean + SEM (n≥9). * denotes a significant difference from control p<0.05.
Fig 2.
Characterisation of cuprizone-induced cell death.
Four week-old male mice were treated with cuprizone for one week. Type of cell death was determined using flow cytometry following double staining with FITC-labelled AnnexinV and propidium iodide thymus suspensions of untreated (Control, grey bars) and cuprizone-treated (CPZ, black bars) mice. Results are presented as representative dot-plots (A) and bar diagrams (B), mean + SEM (n≥9). Significant difference from control; **p<0.01, *** p<0.001. DN: live cells (lower left quadrant); AnnexinV: early apoptotic cells (lower right quadrant); PI: necrotic cells (upper left quadrant); DP: late apoptotic cells (upper right quadrant).
Fig 3.
Effect of cuprizone on thymic epithelial cells.
Four week-old male mice were treated with cuprizone for one week, then immune-staining (A) with FITC-labelled anti-EpCAM1 (green) and PE-labelled anti-Ly-51 (red) antibodies was performed on thymic sections of untreated (Control) and cuprizone-treated (CPZ) mice. Representative images (A) are presented of the green channel (top panels), the red channel (middle panels) and the merged channels (bottom panels) of three independent experiments, including at least three animals in each group for each experiment. Fluorescent photographs were taken using a 10x objective. The scale bar indicates 200 μm. In a parallel experiment, thymic MHC II and AIRE mRNA expression (B) was determined by using qPCR analysis in untreated (grey bars) and cuprizone-treated (black bars) mice. Results are presented as fold change, mean + SEM (n≥9). Significant difference from control; *p<0.05.
Fig 4.
Effect of cuprizone treatment on thymocytes.
Four week-old male mice were treated with cuprizone for one week, then immune-staining (A) with FITC-labelled anti-CD4 (green) and Alexa647-labelled anti-CD8 (red) antibodies was performed on thymic sections of untreated (Control) and cuprizone-treated (CPZ) mice. Representative images (A) are presented of the green channel (top panels), the red channel (middle panels) and the merged channels (bottom panels) of three independent experiments including at least three animals in each group for each experiment. Fluorescent photographs were taken using a 10x objective. The scale bar indicates 200 μm. In a parallel experiment, flow cytometry was performed on thymus suspensions of untreated (Control, grey bars) and cuprizone-treated (CPZ. black bars) mice following double staining with PE-labelled CD4 and CyChrome (CyC)-labelled CD8 antibodies. Results are presented as representative dot-plots (B) and bar diagrams (C), mean + SEM (n≥9). Significant difference from control; **p<0.01, *** p<0.001. DN: CD4-/CD8- cells (lower left quadrant); CD4: CD4+ cells (lower right quadrant); CD8: CD8+ cells (upper left quadrant); DP: CD4+/CD8+ cells (upper right quadrant); DP/DN: ratio of DP and DN cells; CD4/CD8: ratio of CD4+ and CD8+ cells; CD4/DN: ratio of CD4+ and DN cells. Please note that the x-axis is broken, and the ratios are measured on the right y-axis.
Fig 5.
Effect of cuprizone treatment on thymocyte subpopulations.
Four week-old male mice were treated with cuprizone for one week, then flow cytometry was performed on thymus suspensions on thymus suspensions of untreated (Control, grey bars) and cuprizone-treated (CPZ, black bars) mice following triple staining with FITC-labelled CD3, PE-labelled CD4 and CyChrome (CyC)-labelled CD8 antibodies. Based on CD3 positivity (A, B) the data on CD4 and CD8 were allocated to low (CD3low; C, D) and high (CD3high; E, F) CD3 positivity subgroups (the ranges are indicated in A). Results are presented as representative line charts (A), dot-plots (C, E) and bar diagrams (B, D, F), mean + SEM (n≥9). Significant difference from control; **p<0.01, *** p<0.001. DN: CD4-/CD8- cells (lower left quadrant); CD4: CD4+ cells (lower right quadrant); CD8: CD8+ cells (upper left quadrant); DP: CD4+/CD8+ cells (upper right quadrant); CD3high/CD3low: ratio of CD3high and CD3low cells. Please note that the x-axis in B is broken, and the ratio is measured on the right y-axis.
Fig 6.
Effect of cuprizone treatment on subcellular morphology.
Four week-old male mice were treated with cuprizone for one week. Subcellular morphology was assessed in ultrathin thymic sections of control (A) and cuprizone-treated (B-E) mice. Representative images are presented of three independent experiments including at least three animals in each group for each experiment. Arrows indicate normal (A) and enlarged (B) mitochondria, a large lipid droplet (D), and large lysosomes packed with darkly stained material (E). Horizontal thin and vertical thick arrows in (C) point to myelin body and a degraded mitochondrion, respectively. Scale bars indicate 200 nm.
Fig 7.
Effect of cuprizone treatment on death pathway and signalling proteins in the thymus.
Four week-old male mice were treated with cuprizone for three days. Steady-state cytoplasmic levels of cytochrome C (Cyt C) (A), caspase 3 (casp 3) (A), cleaved caspase 3 (cleav. casp 3) (A) and nuclear apoptosis inducing factor (AIF) content (A), as well as cellular levels of Bad (B), BimEL (B), BimL (B) and Bax (B) were assessed in the thymi of untreated (Cont., grey bars) and cuprizone-treated (CPZ, black bars) mice by using specific primary antibodies and immunoblotting. The activation state of Bad (p-Bad) (B), JNK (p-JNK) (C), ERK (p-ERK) (C) and p38 MAPK (p-p38) (C) was also determined by using phosphorylation-specific primary antibodies and immunoblotting. GAPDH (A-C) and histone H1 (His H1) (A) were used as loading controls for cytoplasmic/cellular and nuclear fractions, respectively. Results are presented as representative immunoblots and bar diagrams, mean + SEM (n≥9). Significant difference from control; *p<0.05. Please note that the y-axis in B is broken to accommodate the very high Bax value.