Research Paper Volume 12, Issue 24 pp 25020—25034

Figure 5. TRIB3 knockdown induces autophagy flux in GBM cells. (A) Western blot analysis to detect the protein levels of P62, LC3, ATG5, ATG7, and GAPDH (control for loading) in U251 cells treated with Ad-Scr, Ad-siTRIB3, Ad-vector, and Ad-TRIB3 for 48 h. Data are representative of 3 independent experiments. (B) Western blot analysis of LC3, ATG5, ATG7, and GAPDH in U251 cells treated with Ad-Scr+si-NC, Ad-siTRIB3+si-NC, Ad-siTRIB3+si-ATG7, and Ad-siTRIB3+si-ATG5 for 48 h. Data are representative of 3 independent experiments. (C) Fluorescence images of GFP-MAP1LC3B puncta in U251 cells pretreated with autophagy inhibitors (CQ) or vehicle (DMSO) followed by transfection of scramble RNA or TRIB3 siRNA for 48 h. (D) Fluorescence images of GFP-MAP1LC3B puncta in U251 cells transfected with si-NC or ATG5 siRNAs followed by transfection of scramble RNA or TRIB3 siRNA for 48 h. *p<0.05, **p< 0.01, ^##p< 0.01 vs. TRIB3 KD + siNC.