Figure 2
From: LC3A Silencing Hinders Aggresome Vimentin Cage Clearance in Primary Choroid Plexus Carcinoma
Increased autophagy flux in CCHE-45 cells is not blocked by lysosome inhibitor chloroquine. (A) Western blot analysis of CCHE-45 cells cultured under normal condition or serum starved for 2 or 6 hours in HBSS. β-actin was used as a loading control. (B) Immunofluorescence staining of CCHE45 cells. Cells were cultured under normal condition or serum starved in HBSS for 5 hours or serum starved and treated with 50 µM chloroquine. CCHE-45 cells were immunostained with rabbit anti-vimentin (green) and mouse anti-LC3B (red), or rabbit anti-LC3B (green) and mouse anti-LAMP2 (red) and visualized using Alexa Fluor 488 goat anti-rabbit antibody or Alexa Fluor 555 goat anti-mouse. White arrow heads show LC3B positive autophagosomes. (C) Flow cytometry-based profiling of CYTO-ID Autophagy detection for CCHE-45 and SH-SY5Y cells. Mean fluorescent intensity comparison between CCHE-45 and SH-SY5Y is representative of three independent experiments. Statistical analysis was performed using paired student’s t test. The level of significance was set at p-value of 0.05. Error bars represent average ± SEM. (D) Western blot analysis of soluble and insoluble protein fractions collected from CCHE-45 cells cultured under normal conditions or treated with HBSS for 1, 2 or 6 hours. Ctrl = control, SS = serum starved, Rapa = rapamycin, CLQ = chloroquine, Sol = soluble protein fraction, Insol = insoluble protein fraction.
