Fig. 1.
Construction of FoxG1 stable expression cell lines and FoxG1 induces autophagy in N2A cells. Stable expression of pCDH-FoxG1-m-FLAG-CopGFP-T2A-Puro in mouse N2A cell lines. N2A was stably transfected with pCDH-FoxG1-m-FLAG-CopGFP-T2A-Puro or pCDH-CopGFP-T2A-Puro and selected with puromycin-containing medium. Cell lines were named as FoxG1/N2A and pCDH/N2A cells, respectively. A Green fluorescent proteins are screened positive clones after using 1 μg/mL puromycin 7 days, 14 days, and 21 days, respectively. Scale bar = 100 μm. Expression patterns and qualification of FoxG1 expression in the FoxG1/N2A cell lines and pCDH/N2A cell lines were studied by (B, D [t11 = 3.496, P = 0.025]) western blotting across the protein bands. C Anti-FLAG antibody was also used to check the successful construction of FoxG1 stably expressing cell lines after 14–21 days of puromycin screening. GAPDH or Tubulin was used as the relative loading control. E pCDH/N2A cells were treated with 20 nM Rap for 24 h, protein was then harvested for the detection of Beclin1 and LC3 and SQSTM1/P62 by Western blot in pCDH/N2A cells and FoxG1/N2A cells and Rap treating pCDH/N2A cells. (G [F2,15 = 15.92, P = 0.004]) Bar chart indicates the relative expression of LC3 II/LC3 I in (E). (F [F2,15 = 16.03, P = 0.0039], H [F2,15 = 10.32, P = 0.0114]) Bar chart indicates the relative expression of these proteins in E. GAPDH was used as loading control. I N2A cells transiently transfected with siRNA controller siRNA-FoxG1 for 24 h were treated with 20 nM Rap for 24 h. After treatment, cells were harvested for the detection of Beclin1 and LC3 and SQSTM1/P62 by Western blot. (K [F3,20 = 16.71, P = 0.0008]). Bar chart indicates the expression of LC3 II/LC3 I in (I). (J [F3,20 = 9.676, P = 0.0049], L [F3,20 = 15.44, P = 0.0011]) Bar chart indicates the relative expression of these proteins in I. GAPDH was used as loading control. M Cells were infected with mcherry-GFP-LC3 adenovirus and transfected with vector or FoxG1 plasmid or treated with Rap. Yellow dots indicate autophagosomes and red dots indicate autolysosomes. (N [F2,12 = 11.17, P = 0.0095]) Quantification of autophagosomes in (M). (O [F2,12 = 59.95, P = 0.0001]) Quantification of autolysosomes in (M). Scale bar = 5 μm. For all experiments, different letters indicate statistical differences in mean values across groups (P < 0.05). Values are expressed as means ± SEM. *P < 0.05, **P < 0.01,***P < 0.001. For each group, n = 6/group