Hypericin uptake and intracellular localization.

<p>Cells were exposed to 3 µM hypericin for 4 h without light activation. (A) Hypericin uptake assay. Data is shown as mean±SEM relative fluorescent units per microgram of protein (RFU/µg of protein, n = 3, *p<0.05). (B) Live confocal fluorescent microscopy images of melanoma cells indicate the intracellular localization of hypericin (red) in relation to the endoplasmic reticulum (ER-YFP), mitochondria (OTC-GFP), lysosomes (Lysotracker yellow) and mature melanosomes (MyosinVa-GFP). Nuclei were counterstained with Hoechst (blue). Profiles taken at different locations through the cell indicate co-localization of the fluorophores. A representative result is shown (n = 3, scale bars: 10/20 µm).</p

Melanoma response mechanisms to hypericin-PDT.

<p>Hypericin (HYP) was taken up by melanoma cells and localized to various intracellular organelles, including the endoplasmic reticulum, mitochondria, lysosomes and melanosomes. Light activation (yellow arrow) of hypericin, in the presence of oxygen (O₂), resulted in loss of structural details of various intracellular organelles, phosphatidylserine (PS) exposure, loss of melanoma cell membrane integrity, cell shrinkage and an increase in granularity/pigmentation. Hypericin-PDT furthermore initiated caspase-dependent apoptotic modes of cell death of both extrinsic (caspase 8 (CASP8)) and intrinsic (caspase 3 (CASP3)) nature, as well as a caspase-independent apoptotic mode that did not involve apoptosis inducing factor (AIF). Both caspase-dependent and caspase-independent apoptotic modes of cell death resulted in the cleavage of poly(ADP-ribose)polymerase 1 (PARP1).</p

Cell death protein expression in response to hypericin-PDT.

<p>Melanoma cells were treated with 3 µM light-activated hypericin and analyzed for protein expression of caspase 3 (CASP3), caspase 8 (CASP8), apoptosis inducing factor (AIF) and poly(ADP-ribose)polymerase 1 (PARP1) at 1, 4, 7 and 24 hours after treatment by Western blot analyses (−: uncleaved, +: cleaved).</p

Hypericin-PDT induced expression of apoptotic proteins.

<p>(A) Caspase 3 (CASP3), (B) caspase 8 (CASP8), (C) poly(ADP-ribose)polymerase 1 (PARP1) and (D) apoptosis inducing factor (AIF) Western blot analyses of whole cell lysates detected at 1, 4, 7 and 24 h after treatment. Data is shown as mean±SEM normalized OD ratio (n≥3, ***p<0.0001, **p<0.01, *p<0.05, CTRL: vehicle-treated control, HYP: hypericin and L: light).</p

Hypericin-PDT induced phosphatidylserine exposure and loss of cell membrane integrity.

<p>(A) Annexin V (phosphatidyl serine exposure) and VIVID (loss of cell membrane integrity) median fluorescent intensities (MFI) normalized to the vehicle-treated, sham-irradiated control (Control −Light) at 30 min, 1, 4, 7 and 24 h after treatment. Flow fluorocytometric data is shown as the median±SEM (n≥3, ***p<0.0001, **p<0.01, *p<0.05, L: light). (B) Percentage gated cells of 4 different populations labeled with Annexin V and VIVD: live (AV− VIVD−), early apoptotic (AV+ VIVID−), necrotic (AV− VIVID+) and late apoptotic/necrotic (AV+ VIVID+) at 30 min, 1, 4, 7 and 24 h after treatment. Data is shown as mean±SEM of gated cells (n≥3).</p

Phenotypic heterogeneity of melanoma cells.

<p>(A) Phase contrast images. A representative result is shown (n≥3, scale bars: 20 µm, inset: higher magnification). (B) Cell pellets. A representative result is shown (n≥3). (C) Mean±SEM tyrosinase activity in counts per minute (cpm)/120 µg protein (n = 3, ***p<0.0001, *p<0.05). MCF7 breast cancer cells were included as a negative control.</p

Hypericin-PDT induced loss of structural details of calreticulin positive structures (endoplasmic reticulum).

<p>Cells expressing calreticulin-YFP (ER-YFP) were exposed to 3 µM hypericin (red) for 4 h, followed by light-activation and imaging using Super-resolution structured illumination microscopy (SR-SIM). (A) Control (hypericin-treated, sham-irradiated). (B) 30 min post PDT. (C) 60 min post PDT. Images are shown at lower magnification (top panel, scale bars: 5 µm) and higher magnification (zoom, lower panel, scale bars: 1/2 µm.) Co-localization plots indicate co-localization of the fluorophores. A representative result is shown (n = 2).</p

Organelle-specific G/YFP-fusion plasmids and dyes used to visualize intracellular organelles.

<p>A summary of the experimental conditions, functions and sources of the organelle-specific G/YFP-fusion plasmids and dyes used in this study.</p

Hypericin-PDT induced loss of structural details of LAMP1 positive structures (endosomes, lysosomes and melanosomes).

<p>Cells expressing LAMP1-YFP were exposed to 3 µM hypericin (red) for 4 h, followed by light-activation and imaging using Super-resolution structured illumination microscopy (SR-SIM). (A) Control (hypericin-treated, sham-irradiated). (B) 30 min post PDT. (C) 60 min post PDT. Images are shown at lower magnification (top panel, scale bars: 5 µm) and higher magnification (zoom, lower panel, scale bars: 1/2 µm.) Co-localization plots indicate co-localization of the fluorophores. A representative result is shown (n = 2).</p

Hypericin-PDT reduced cellular size and increased cellular granularity/pigmentation.

<p>(A) Melanoma cell forward scatter (FS) as an indication of cell size/cell death mechanisms and (B) melanoma cell side scatter (SS) as an indication of cell granularity/pigmentation; normalized to the vehicle-treated, sham-irradiated control (Control −Light) at 30 min, 1, 4, 7 and 24 h after treatment. Flow cytometric data is shown as median±SEM (n≥3, ***p<0.0001, L: light).</p