Supplementary Figure 4: Construction and characterization of ALK5 shRNA retroviruses that decrease the survival of infected newborn neurons in C57BL/6 mouse dentate gyrus.

(a) Design of the retroviral vectors. MCS, multiple cloning site. (b) Immunoblot of lysates from HEK293T cells co-transfected with an expression vector for ALK5 and different shRNAs probed with antibodies against ALK5 and actin. Full-length blots are presented in Supplementary Figure 10. (c,d) Representative confocal images of retrovirus-injected dentate gyrus of 2-month-old C57BL/6 mice immunostained for eGFP and ALK5 at 7 dpi in (c) or eGFP and p-Smad2 at both 7 dpi and 14 dpi in (d). DAPI stains nuclei. Green or blue dashed circles highlight the cell bodies of eGFP+ or eGFP– neurons. Scale bar is 10 μm. (e) Quantification of p-Smad2 signal intensity from (d). P-Smad2 signals were normalized to signals in uninfected cells in (e). n = 20, 20, 23, 23 neurons from four mice total for eGFP–/shRNA-C, eGFP+/shRNA-C, eGFP–/shRNA-A1, eGFP+/shRNA-A1 group at 7 dpi, respectively. n = 15, 15, 14, 14 neurons from four mice total for eGFP–/shRNA-C, eGFP+/shRNA-C, eGFP–/shRNA-A1, eGFP+/shRNA-A1 group at 14 dpi, respectively. (f) Quantification of the number of virus-infected mCherry only and eGFP-labeled (eGFP only and eGFP/mCherry double-labeled) newborn neurons in the dentate gyrus from stereotaxically injected 2-month-old female C57BL/6 mice at 7 (n = 5 mice), 14 (n = 4 mice, P = 0.0485), and 21 dpi (n = 5 mice, P = 0.0021). Four sections per mouse. Cell numbers were normalized to values at 7 dpi. Data are presented as mean + s.d. in (e) and mean + s.e.m. in (f). *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA, Tukey's post-hoc test (e); Student's t-test (f).