Figure 9.

Detachment-induced upregulation of ATG7 contributes to autophagosome formation in detached intestinal epithelial cells. (A) IEC-18 cells IEC-18 cells were transfected with 100 nM control RNA (cont RNA) or ATG7-specific small interfering RNA (ATG7siRNA)4 (left) or 19 (center) and cultured detached from the ECM for 20 h (left, center). IEC-18 cells transfected as above were cultured attached to the ECM for 20 h in case of the control RNA or detached from the ECM for 20 h in case of ATG7siRNAs 4 and 19 and assayed for ATG7 expression by western blot. (B) IEC-18 cells were transfected (+) or not transfected (−) as in (A), then transiently transfected with a GFP-LC3B expression vector, detached from the ECM for 12 h and assayed for the presence of GFP-LC3B puncta as in Figure 1. The results represent the average of the number of GFP-LC3B puncta per cell observed for 60–120 cells plus the SE. This experiment was repeated twice with similar results. * indicates that the p-value was less than 0.05. (C) IEC-18 cells were transfected as in (A), cultured for 16 h detached from the ECM and assayed for GABARAP expression by western blot. Positions of GABARAP-I and GABARAP-II on the gel are indicated. ACTB was used as a loading control in (A and C).