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

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

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