Internalized collagen levels were higher in autophagy-deficient cells than in control cells. (A) Left panels show western blot analysis of Atg5 (Atg5–Atg12 conjugate), LC3-I and LC3-II in control and Atg5^−/− MEFs. Right panels show western blot analysis of Atg7, LC3-I, and LC3-II in control and Atg7^−/− MEFs. NS, non-specific. GAPDH was used as an internal control. (B) Internalized FITC-labeled collagen in control (left) and Atg5^−/− (right) MEFs. The cells were cultured on FITC-labeled collagen for 120 min. Scale bar: 20 µm. (C) Quantification of the FITC-labeled collagen area per cell. Data presented are mean and s.e.m. in control and Atg5^−/− cells. *P<0.05, Student's t-test (n=5 fields of control; seven fields for Atg5^−/− cells). Four independent similar experiments are shown. (D) Internalized FITC-labeled collagen in control (left) and Atg7^−/− (right) MEFs. The cells were cultured on FITC-labeled collagen for 120 min. Scale bar: 20 µm. (E) Quantification of the FITC-labeled collagen area per cell. Data presented are mean and s.e.m. in control and Atg7^−/− cells. *P<0.05, Student's t-test (n=10).

Degradation of internalized collagen was decreased in autophagy-deficient cells compared with control cells. (A) Representative confocal images of internalized collagen in control and Atg5^−/− MEFs. These MEFs were pre-cultured on FITC-labeled collagen in the presence of HCQ (40 µM) for 16 h, then were transferred to new chamber slides without FITC-labeled collagen. Subsequently, the cells were cultured for 24 h with (upper panel) or without (lower panel) HCQ. Scale bar: 20 µm. (B) Quantification of the area of FITC-labeled collagen per cell in cells treated with HCQ (upper graph) and after washout of HCQ (lower graph). The bar graph for HCQ shows mean±s.e.m. in control and Atg5^−/− cells and the graph for washout shows mean±s.e.m. in control and Atg5^−/− cells (n=7 fields of control; six fields for Atg5^−/− cells). Three independent similar experiments are shown. *P<0.05, ** P<0.001, Student's t-test. (C) Internalized collagen remaining after washout of HCQ, as a percentage of internalized collagen in HCQ-treated cells. *P<0.01, Student's t-test; mean±s.e.m. (D) Representative confocal images of internalized collagen in control and Atg7^−/− MEFs. After pre-culture on FITC-labeled collagen in the presence of HCQ (40 µM) for 16 h, cells were cultured for 24 h with (upper panel) or without (lower panel) HCQ on new chamber slides without FITC collagen. Scale bar: 20 µm.

Autophagosomes associated with the internalized complexes of focal adhesion (FA) complex. (A) Representative confocal image of GFP-paxillin (left) and endogenous LC3 (middle). Lower panels show enlarged images of the boxed regions in the upper images. Paxillin was surrounded by puncta of LC3. Whole-cell merged image with DAPI counterstaining (right). Scale bar: 10 µm. (B) Fibroblasts were plated, and after 90 min were stained with antibodies against paxillin (left), LC3 (middle) and whole-cell merged image with DAPI counterstaining (right). The lower panels show enlarged images of the boxed regions in the upper images. Scale bar: 10 µm. (C) Representative confocal image of FAK-pY397 (left), LC3 (middle), and whole-cell merged image with DAPI counterstaining (right). Scale bar: 1 µm.

FA complex distribution in autophagy-deficient cells and control cells. (A) Control and Atg5^−/− MEFs were cultured for 120 min and stained with anti-paxillin antibody and with DAPI. Scale bar: 20 µm. (B) Area of paxillin, presented as mean and s.e.m, in control and Atg5^−/− cells (n=8 fields of control; nine fields for Atg5^−/− cells). *P<0.01, Student's t-test. Four independent similar experiments are shown. (C) The number of FAs in control and Atg5^−/− cells, presented as mean and s.e.m, in control and Atg5^−/− cells. *P<0.01, Student's t-test. (D) Representative confocal image of control and Atg7^−/− MEFs, which were cultured for 120 min and stained with anti-paxillin antibody and DAPI. Scale bar: 20 µm.

Inhibiting autophagy attenuated FA kinase (FAK) and enhanced RhoA activity. (A) Control and Atg5^−/− MEFs were plated on collagen I-coated dishes for 0, 15 and 30 min. Cell lysates were collected from dishes at the indicated time points. Lysates were immunoblotted with anti-FAK-pY397, anti-FAK, anti-Src-pY416, and anti-Src antibodies. GAPDH is shown as a loading control. (B) Quantification of the ratio of FAK-pY397 and FAK protein levels. Bar graph shows mean and s.e.m. (n=10 replicates). *P<0.05, ANOVA with Tukey's post hoc test. (C) RhoA activity in control and Atg5^−/− MEFs. Control and Atg5^−/− MEFs were plated on collagen I for 30 min. Cell lysates were assessed by G-LISA RhoA activation assay. The graph shows absorbance at 490 nm from five experiments (n=5 replicates). *P<0.01, Student's t-test. Three independent similar experiments are shown. (D) Western blot analysis of RhoA in control and Atg5^−/− MEFs. Actin was used as a loading control.