Immunolocalization of PKCζ and occludin in retinal pigment epithelium (RPE) flatmounts.

<p>In 6-month-old controls, PKCζ staining (red) appeared as focal spots within the loops depicted by occludin (green) protrusions (a). In age-matched diabetic conditions, no PKCζ labeling (box and green arrow) was observed (b). After 12 month of diabetes marked disruption of intercellular junctions (arrows) was evidenced (c). At this stage the PKCζ at the junctions (TJ)/total amount of PKCζ ratio was significantly decreased for diabetic conditions (Image J software, National Institutes of Health, Bethesda, MD) (d) and occludin internalization from the cell membrane into the cytoplasm was found (e). The colocalization of PKCζ (red) and PKCζ-P (green) found in 12-month-old controls (f) was not present in age-matched diabetics (g). Furthermore the activated PKCζ-P form exhibited a discontinuous staining along the cell membrane (g).</p

<i>In-vivo</i> PKCζ inhibition in 6-month-old-diabetic rats partially restores PKCζ staining pattern in cone outer segments.

<p>The phosphorylated active form PKCζ-P (green) was found in IS of both control (CTL) and diabetic (DIA) rats (a). Diabetic inhibitor (DIA+IZ) treated rats showed partial restoration of OS PKCζ (red) staining (a). We performed laser micro dissection of the photoreceptor layer on cryosections (b) to study layer-specific protein level by Western blotting analysis. The PKCζ-P immunoreactivity increase found in diabetic rats (DIA) was partially restored in treated rats (DIA+IZ).</p

Alteration of PKCζ distribution in the outer retina is associated with cone outer segment and OLM disruption in diabetic rats.

<p>PKCζ staining was located in inner segments (IS) of rod and cones and exclusively in cone OS as evidenced by PKCζ-PNA double labeling in 6-month-old control rats (a). In 6-month-old diabetic rats, no OS staining was detected (a). Furthermore PKCζ staining (red) was lost in S-cones, specifically marked by Blue opsin staining (green), as compared to controls. Some S-cone OS also showed marked structural alterations, suggesting early photoreceptor degeneration (b). In 12-month-old diabetic rats OLM discontinuity (arrows) was evidenced (c). OLM tight-junction disruption was further confirmed on retinal flatmounts by an occludin/PKCζ double staining (d, white arrows).</p

Characteristics of the study animals.

<p>a: diabetes versus controls, P value<0.05.</p

Diabetes destabilizes and down regulates PAR3/PAR6 PKCζ-associated protein complex.

<p>a) Upper panels: In 12-month-old diabetic rats (DIA) PAR6 distribution in flatmount RPE showed a clear cytoplasmic relocation of PAR6 compared to age-matched control rats (CTL) where PAR6 appeared as relative regular punctiform staining at the TJ levels. The same applied to PAR3 staining distribution (Lower panels). b) PAR6 and PAR3 immunoblotting on RPE cell extracts from 12-month-old rats showed a significant decrease of both protein levels in diabetic conditions (statistical analysis was performed on the PAR3 180 KDa isoform).</p

PKCζ specifically regulates NF-κB signaling pathway which participates to diabetes-induced cone photoreceptor degeneration.

<p>In 6-month-old rat cryosections, the p65-P subunit of NF-κB (red) was only detected in diabetic (DIA) conditions in some nuclei of the outer nuclear layer (<i>arrows</i>) and not in controls (CTL) or treated rats (DIA+IZ). The triple staining with addition of PNA (green), a specific cone marker, confirmed that the nuclear translocation p65-P subunit of NF-κB exclusively occurred in cones (a). In age-matched rat cryosections, TUNEL assay confirmed apoptosis of photoreceptors in diabetic (DIA) conditions (b).</p

Diabetes stage-specific retinal pigment epithelium (RPE) level of PKCζ and its activated phosphorylated form PKCζ-P T410.

<p>No significant changes were observed for PKCζ level at any stage between control and diabetic conditions. To the contrary, PKCζ-P T410 immunoreactivity was significantly (*P<0.05, Mann–Whitney test) increased (by around 40%) at 6 months and then decreased (by around 60%) at 12 months of diabetes compared to controls.</p

Diabetes induces outer retinal edema and loss of cones photoreceptors.

<p>Diabetic retina (DIA) demonstrated increased extracellular spaces within the outer nuclear layer (ONL) and photoreceptor segment disorganization, consistent with an edematous aspect of the outer retina (Historesin (a) and semithin sections (b)). Peanut agglutinin labeling, a specific marker of cone extracellular matrix, evidenced a marked decreased in cell cone density (c). Whole retina quantification of cones evaluated the net loss to be around 20% (d) as compared to controls (p = 0.002, Mann–Whitney test).</p

MART-1 cross-presentation to M27 CD8⁺ T cell clone.

<p>Cross-presentation assay was performed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040311#s2" target="_blank">Materials and Methods</a>. For co-cultures, a 3∶1 ratio iDC (panel A) or MAK (panel B) to gamma-MEL-XY3 target cells was used and incubation was done for the indicated times. After co-culture, M27 CD8⁺ T cells were added and cells were further incubated overnight. Positive controls were either live MEL-XY3 cells or MART-1 peptide loaded mDC or MAK, incubated with M27 cells overnight. As negative controls, gp100 peptide or IIB-BR-G cells were used. MAK, DC and gamma-MEL-XY3 were also tested alone and with M27 cells to measure basal IFN-gamma levels. Results are expressed as mean ±SD pg/mL IFN-gamma released into the supernatant. One representative experiment performed in triplicate is shown. Differences between basal levels (M27 clone+gamma-irradiated tumor cells) and co-culture-stimulated M27 cells (either with DC:gamma-MEL-XY3 or MAK:gamma-MEL-XY3) were statistically significant from 3 to 48 hours (* p<0.05; ** p<0.005; *** p<0.0005).</p

Immunohistochemical staining of melanoma tumor tissues with the 2A9 mAb.

<p>2A9 mAb staining of a primary tumor and of one metastasis from the same melanoma patient is shown in (A) and (B), respectively; staining of melanocytes present in epidermis is shown in (C), while no staining of keratinocytes is observed. Black arrows indicate melanocytes. No staining is observed with a breast carcinoma tumor biopsy (D). Original magnification: A and C = 1000×; B and D = 400×.</p