Fig. 4

Down-regulated trophic factor expression in response to ALS-CSF. Graphs a, b denote the down-regulation in the mRNA levels of GDNF (**p < 0.01 NC and ^# p < 0.05 NALS v/s ALS; n = 5 in triplicates) and VEGF (***p < 0.001 NC and ^## p < 0.01 NALS v/s ALS; n = 5 in triplicates), respectively, upon exposure to ALS-CSF as compared to normal and disease controls. The down-regulation in mRNA expression was followed by a reduction in the protein expression for both the proteins, as observed by western blotting as well as immunofluorescence. The representative confocal images and quantitative analysis of MFI indicate a down-regulated expression of GDNF (c–e, graph i; **p < 0.01 NC and NALS v/s. ALS; n = 5 in duplicates) and VEGF (f–h, graph j, **p < 0.01 NC and NALS vs. ALS; n = 5 in duplicates), respectively, in the cultures exposed to ALS-CSF as compared to the normal controls. Scale bars are indicated. The dysregulation was further validated by the semi-quantitative western blot (k) analysis suggesting a decreased expression of both, GDNF (graph l; *p < 0.05 NC v/s, and ^## p < 0.01 NALS v/s. ALS; n = 3 in duplicates), as well as VEGF (graph m; *p < 0.05 NC and NALS v/s. ALS; n = 3 in duplicates) in response to ALS-CSF. Analysis of significance was carried out using one-way ANOVA followed by Tukey’s post hoc test