Volume 125, Issue 1, Pages 70-79 (July 2003) Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility  Roberto De giorgio, Monica Bovara, Giovanni Barbara, Marco Canossa, Giovanni Sarnelli, Fabrizio De ponti, Vincenzo Stanghellini, Marcello Tonini, Silvia Cappello, Eleonora Pagnotta, Eduardo Nobile-Orazio, Roberto Corinaldesi  Gastroenterology  Volume 125, Issue 1, Pages 70-79 (July 2003) DOI: 10.1016/S0016-5085(03)00664-4

Figure 1 Representative confocal photomicrograph showing SH-Sy5Y cells with intense HuD immunolabeling throughout the cytoplasm (A, with the anti-HuD antibody from Santa Cruz Biotechnology; B, with the anti-HuC/D antibody from Molecular Probes Inc.). Note the absence of HuD immunoreactivity in cells (C ) exposed to control sera or (D) incubated with a mixture in which the HuD antibodies were preadsorbed with the homologous peptide. Bar: A, C, and D, 20 μm; B, 40 μm. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 2 Representative confocal photomicrographs of cultured myenteric neurons showing simultaneous visualization of (A ) MAP-2 and (B) HuD immunoreactivities. Arrows indicate the colocalization of MAP-2 and HuD immunofluorescence in the perikaryon (A and B). The concomitant immunostaining with MAP-2 and anti-HuD antibodies preadsorbed with the homologous peptide resulted in (D) a complete absence of HuD immunoreactivity (C ) without affecting MAP-2 labeling. Bar = 20 μm. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 3 Neuronal apoptosis induced by anti-HuD-positive sera from patients with paraneoplastic dysmotility in SH-Sy5Y cells. Representative confocal picture double labeled with (A ) TUNEL (green fluorochrome) and (B) Hoechst 33342 dye (blue fluorochrome). Arrows indicate TUNEL-positive neuroblastoma cells with condensed nuclei after incubation with anti-HuD-positive sera. Bar = 20 μm. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 4 Quantitative analysis of neuroblastoma cells exposed to anti-HuD-positive sera showed a significantly greater percentage of TUNEL-positive nuclei compared with controls. HuD-positive sera and dopamine (positive control) induced apoptosis to a similar extent. Data are expressed as mean ± SEM (n = 5); ∗P <0.001 vs. controls. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 5 Apoptosis in SH-Sy5Y cells induced by anti-HuD antibodies. (A ) A time-course analysis showing a significantly greater number of apoptotic neuroblastoma cells evoked by anti-HuD (Santa Cruz Biotechnology, gray bars) and anti-HuC/D (Molecular Probes Inc., black bars) at 24, 48, and 72 hours compared with controls. Data are expressed as mean ± SEM; ∗P <0.05 and ∗∗P <0.001 vs. controls (FCS, light gray bars). (B) Dilution curve showing concentration-dependent apoptosis evoked by the anti-HuD antibody. Data are expressed as mean ± SEM; ∗P <0.05 and ∗∗P <0.001 vs. controls. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 6 Representative confocal images showing activated (A–C ) caspase-3 and (D–F ) apaf-1 immunofluorescence in SH-Sy5Y cells exposed to (C and F ) control sera, (A and D) anti-HuD-positive sera, or (B and E ) commercial anti-HuD antibody. Neuroblastoma cells exposed to (A and D) anti-HuD-positive sera and to (B and E ) anti-HuD antibody showed intense cytoplasmic immunofluorescence for both activated caspase-3 and apaf-1; (C and F ) both immunoreactivities were absent in cells exposed to control sera. Bar = 20 μm. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 7 Quantitative analysis showing the percentage of neuroblastoma cells displaying activated (A ) caspase-3 and (B) apaf-1. Activated caspase-3 and apaf-1 immunoreactivities were significantly greater in cultures exposed to anti-HuD-positive sera or commercial anti-HuD antibody compared with controls. Each bar represents the mean ± SEM; ∗P <0.001 vs. controls. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 8 Representative confocal double-labeling images showing (A ) TUNEL positivity (green fluorochrome) in (A and C ) MAP-2-identified cultured myenteric neurons (red fluorochrome). Exposure of myenteric neurons to commercial anti-HuD antibody evoked an intense TUNEL positivity in nuclei (arrows, A ); in contrast, TUNEL positivity was absent in MAP-2-immunostained myenteric neurons exposed to FCS (arrow, C ). B shows myenteric neurons labeled with Hoechst 33342 dye (blue fluorochrome), which identifies simultaneous nuclear condensation (arrows) in MAP-2/TUNEL-positive cells exposed to commercial anti-HuD antibody. Nuclear condensation was not identified in MAP-2-immunostained neurons (arrow, C ) incubated with FCS (arrow, D). Bar = 20 μm. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 9 Quantitative data of TUNEL-positive/MAP-2-identified myenteric neurons exposed to commercial anti-HuD antibody or FCS. Anti-HuD antibody induced a percentage of TUNEL-positive cells significantly greater than that evoked by FCS. Data are expressed as mean ± SEM; ∗P <0.001 vs. controls. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)

Figure 10 Activated caspase-3 immunoreactivity induced by (A and D) anti-HuD antibody in (B and E ) myenteric neurons identified with MAP-2 (arrows). (A ) Anti-HuD antibody induces the expression of activated caspase-3 (green fluorochrome; arrows). (D) In contrast, MAP-2-identified myenteric neurons exposed to FCS were negative for activated caspase-3. C and F show myenteric neurons labeled with Hoechst 33342 dye (blue fluorochrome; arrows); in C, nuclear condensation in MAP-2/caspase-3-positive cells after exposure to anti-HuD antibody is observed. Conversely, nuclear condensation was absent (arrows, F ) in MAP-2-immunostained neurons (arrows, E ) incubated with FCS. Bar = 20 μm. Gastroenterology 2003 125, 70-79DOI: (10.1016/S0016-5085(03)00664-4)