Transient increase in apoptosis in ARPE-19 cells after photocoagulation.

<p><b>A)</b> Representative images showing apoptotic ARPE-19 cells visualized by TUNEL-staining (brown) at various time-points after <i>in vitro</i> photocoagulation. Scale bar represents 100 µm; magnification 10X. <b>B)</b> Summarized data showing measurements of cytoplasmic histone-associated DNA fragments in cell homogenates and culture media at various time-points after <i>in vitro</i> photocoagulation. Data is expressed as mean absorbance ratio between the photocoagulated samples and the corresponding non-irradiated controls for each time-point (apoptosis enrichment factor). Six to 8 samples were analyzed for each time-point. ***<i>p</i><0.001 vs 12 h and vs 48 h in the cell-homogenates; **<i>p</i><0.01 vs 48 h and vs 168 h in the medium.</p

Confocal immunofluorescence images showing PCNA (proliferating cell nuclear antigen) expression (red) in ARPE-19 cells at various time-points after <i>in vitro</i> photocoagulation.

<p>Nuclei are stained with SYTOX Green. Expression of PCNA in non-irradiated control cells at 6 h is also shown for comparison. Scale bar represents 200 µm.</p

A) Visualization of dead (red) and live (green) ARPE-19 cells after photocoagulation.

<p>Loss of plasma membrane integrity results in uptake of red-fluorescent ethidium homodimer-1 in dead cells (middle panels), while living cells stain positive for intracellular esterase activity labelled with green-fluorescent calcein-AM (upper panels). Lower panels show merged images for the two fluorophores. Scale bar represents 100 µm. <b>B)</b> Early transient increase in necrosis in ARPE-19 cells, as assessed by quantification of lactate dehydrogenase activity in the culture medium at various time-points after photocoagulation. Data is expressed as mean absorbance ratio between the photocoagulated samples and the corresponding non-irradiated controls for each time-point. Four samples were analyzed at each time-point; *<i>p</i><0.05 vs non-irradiated controls at 30 min, 2 h and 6 h.</p

Photocoagulation results in changes in gene expression.

<p><i>IL1β, IL8, HMGA2, TGFBR2, ADAMTS6, TIMP3, HSPA6, IL33</i> and <i>ANKRD1</i> mRNA expression in ARPE-19 cells 24 h after photocoagulation and in control non-irradiated cells. Measurements were performed by qPCR using cyclophilin B as an endogenous control. ***<i>p</i><0.001; **<i>p</i><0.01 and *<i>p</i><0.05 vs. control; <i>n</i>≥6.</p

Both cell migration and proliferation contribute to lesion repair.

<p><b>A)</b> Representative H&E images showing ARPE-19 cells that had been cultured with and without docetaxel (1 nM) or mitomycin C (1 µM) for 48 h after laser photocoagulation. Scale bar represents 100 µm. <b>B)</b> Summarized data from experiments as in A, showing the effects of culture with and without docetaxel (1 nM) or mitomycin C (1 µM) for 12 h, 24 h or 48 h on the diameter of the cell-free region of the lesion. For the 72 h time-point, two concentrations of docetaxel (1.0 and 0.1 nM) and of mitomycin C (1.0 and 0.3 µM) were tested. Four samples were analyzed for each condition or time-point; ***<i>p</i><0.001 and **<i>p</i><0.01 vs. untreated controls for the corresponding time-points.</p

Haematoxylin & Eosin stained ARPE-19 cells at various time points after <i>in vitro</i> photocoagulation.

<p>The following settings were used: 200 µm, 300 mW, 0.1 s. Scale bar represents 100 µm; magnification 10X. Control cells at time 0 (non-laser) are shown for reference.</p

Altered cell-cell connections and insulin granule distribution in beta cells from OPN^-/- mice.

<p>(A) Ultrastructural images from a section of a WT (left) and OPN^-/- (right) islet. Scale bar 5 μM. (B) Ultrastructural images from a section of an OPN^-/- islet. The atypical structure on the beta cell is marked with a black arrow. Scale bar 2μM. (C, D, and E) Histogram of the calculated volume density (C), surface density (D), and distribution of the insulin containing large dense-core vesicles (LDCVs; E) in pancreatic beta cells from WT and OPN^-/- mice. Data are given as mean ± SEM from 24–26 cells taken from 3 animals per condition. ** p≤ 0.01; *** p≤ 0.001.</p

Example traces from intracellular Ca²⁺ measurements in WT and OPN^-/- islets.

<p>Representative examples of Ca²⁺ traces obtained from an islet from a WT (A) or OPN^-/- (B) mouse. Marked on the traces are the measurements mentioned in the text. C₀ is the area of the Ca²⁺ dip that initially occurs upon glucose-stimulation. C₁ is the amplitude of the first peak. 16.7 G is 16.7 mM glucose and 2.8 G is 2.8 mM glucose. The baseline is marked with a dashed line. Histogram of the cytoplasmic Ca²⁺ concentration (measured as area under the curve but above the baseline; C), C₀ (D), C₁ (E), and the frequency of ocillations during the sustained phase (F) in islets from WT (white bars) and OPN^-/- (black bars) mice. Data are obtained from 16–21 islets from 3 mice per condition. ** p≤ 0.01; *** p≤ 0.001.</p

Pancreatic slices from OPN^-/- and WT mice have similar histology.

<p>Representative images from (A) eosin and hematoxylin stainings and (B) insulin stainings on pancreatic slices from WT (left) and OPN^-/- (right) mice. Scale bar 100 μm.</p

Deletion of OPN has modest effects on metabolism.

<p>(A) Body weight in WT and OPN^-/- mice measured at 12 weeks. (B) Non-fasted blood glucose levels from WT and OPN^-/- mice measured at 12 weeks. (C) Insulin content in isolated islets from WT and OPN^-/- mice. (D) Glucose-induced insulin secretion at 11.1 mM glucose from isolated WT and OPN^-/- islets described as fold increase over basal insulin secretion at 2.8 mM glucose with and without the addition of 200 ng/ml OPN and/or 100 nM GIP. (E) Same experiment as in (D) but displaying the non-processed data as ng/islet/h. Data are given as mean ± SEM from 28–30 animals (A and B) or from 4 biological experiments with 3 technical replicates in each experiment (C, D, and E). * p≤ 0.05.</p