Research Paper Volume 11, Issue 16 pp 6175—6198

Cortical neurons develop a senescence-like phenotype promoted by dysfunctional autophagy

Figure 9. Autophagy modulation alters cortical cells senescence. (A) Experimental design. (B) Early inhibition of autophagy with Spautin1 increased the number of cells with SA-β-gal activity, while early induction of autophagy by adding trehalose reduced them. Spautin1 or trehalose were added during periods of several days, at the indicated time intervals in days of culture (DIV); after 26 DIV all cultures (including control with no treatment) were fixed to quantify the percentage of cells showing high SA-β-gal activity. Scale bars represent 500 μm. The bars in graphs represent the mean of each independent experiment, each done by triplicates. Three fields from each replica were scored (9 fields per experiment), each dot represent the percentage of SA-β-gal positive cells per field. Data were analyzed by two-way RM ANOVA, followed by Dunnett´s multiple comparison test. ***p<0.001 Spautin1 added during 6-13 DIV in comparison with control; **** p< 0.0001 Spautin1 added during 13-19 DIV in comparison with control, and Trehalose treatments in comparison with control. (C) Autophagy induction with trehalose reduced the abundance of intranuclear fold with Lamin-A/C. Trehalose were added during periods of several days, at the indicated time intervals in days of culture (DIV); after 26 DIV all cultures (including control with no treatment) were fixed to detect Lamin-A/C by immunofluorescence. Scale bar represents 5 μm. Representative images of three independent experiments are shown. At least 60 cells per treatment were quantified as described in Methods. Bars in the bottom graph represents mean. Data were analyzed by two-way RM ANOVA, followed by Dunnett´s multiple comparison test. ***p<0.001; **** p< 0.0001 with respect to control.