Fig. 1: RTN-1C overexpression affects autophagic process.
From: Modulation of autophagy by RTN-1C: role in autophagosome biogenesis
a SH-SY5Y control cells (Ctrl), starved for 6 h (Stv) or overexpressing RTN-1C for 24 h (RTN-1C) were stained with anti-LC3 antibody and analyzed by confocal microscopy. Nuclei were controstained by using the fluorescence dye Hoescht-H 33342. Scale bars: 20 μm (upper panels) 5 μm (lower panels). b Quantification (means ± SD) of autophagy in SH-SY5Y control cells (Ctrl), overexpressing RTN-1C for 24 h (RTN-1C) or starved for 6 h (Stv), (n = 50). (***) (P < 0.001 vs. Ctrl group, Student’s t test). c Flow cytometry analysis of autophagy in cells overexpressing RTN-1C for 24 h, in the absence or in the presence of 20 μM cloroquine, performed with a Cyto-ID Autophagy Detection Kit. Numbers represent the mean fluorescence intensity. A representative experiments among three is shown. Treatment with lysosomal inhibitor (CQ) increase the fluorescence intensity and is indicative of autophagy activity [17]. d SH-SY5Y control (ctrl) or RTN-1C overexpressing cells (RTN-1C) were transiently transfected with LC3-GFP construct for 24 h and analyzed by confocal microscopy. Quantification (means ± SD) of LC3-GFP signal distribution in RTN-1C cells (n = 33) compared to control (n = 27) is reported. (***) (P < 0.001 vs. Ctrl group, Student’s t test). e SH-SY5Y controls cells or overexpressing RTN-1C for 24 and 48 h were stained with acridine orange and analyzed by flow cytometry. Results are means ± SD of 3 independent determinations. (**) (P < 0.01 vs. Ctrl group, Student’s t test). f Immunoblot analysis of LC3 in SH-SY5Y control cells (Ctrl), starved for 6 h (STV) or overexpressing RTN-1C for the indicated times (18–24 h) in the absence or presence of CQ. Actin was used as loading control. A representative experiment among 3 is shown (g) Immunoblot analysis of LC3 in SH-SY5Y control cells (Ctrl) or overexpressing RTN-1C for 24 h (RTN-1C), starved for different times (STV) in the absence or presence of CQ. Actin was used as loading control. A representative experiment among three is shown.
