Fig. 3: Effect of RTN-1C down regulation on autophagy and endoplasmic reticulum morphology.
From: Modulation of autophagy by RTN-1C: role in autophagosome biogenesis
a Immunoblot analysis of RTN-1C and LC3 in SH-SY5Y control cells (Ctrl), overexpressing RTN-1C for the indicated times (6–18 h) or starved for 6 h (Stv) in the presence of 20 μM cloroquine. Actin was used as loading control. A representative experiment among 3 is shown. b, c Densitometric analysis of RTN-1C (B) and LC3II (C) expression in SH-SY5Y control cells (Ctrl) and overexpressing RTN-1C for the indicated times (6–18 h). (*) (P < 0.05 vs. Ctrl group, Student’s t test). d SH-SY5Y control cells (Ctrl) and overexpressing RTN-1C for 6 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: 7 μm. e Immunoblot analysis of RTN-1C protein levels in SH-SY5Y wild-type cells treated with scramble siRNAs (Scr) or siRNA specific for RTN-1C at the indicated times. Actin was used as loading control. A representative experiment among three is shown. f Immunoblot analysis of LC3 protein in SH-SY5Y control cells (Ctrl), in cells treated with scramble siRNA (Scr) and treated with siRNA for RTN-1C for 24 h (siRTN-1C) in the absence or presence of CQ. GADPH was used as loading control. A representative experiment among three is shown. g Densitometric analysis of LC3II expression in control cells (Scr), and in cells treated with siRNA for RTN-1C for 24 h (siRTN-1C) in the presence of CQ. Results represent the mean ± SD of three independent determinations. (**) (P < 0.01 vs. Scr group, Student’s t test). h SH-SY5Y control cells (scramble, Scr) starved for 6 h (Stv) or treated with siRNA for RTN-1C for 24 h and starved for 6 h (siRTN-1C + Stv) were stained with anti-LC3 antibody and analyzed by confocal microscopy. Scale bars: 20 μm (left panels), 7 μm (right panels). Nuclei were controstained by using the fluorescence dye Hoescht-H 33342. i Quantification (means ± SD) of autophagy is reported in the bar graph (n = 150). (**) (P < 0.01 vs. Stv group, Student’s t test). l–n Ultrastructural analyses of SH-SY5Y neuroblastoma controls cells (scramble, Scr) and cells treated with siRNA for RTN-1C for 24 h (siRTN-1C). Arrows indicate endoplasmic reticulum fragmentation. m mitochondria, er endoplasmic reticulum. Scale bars: 1 μm.
