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Figures
- Fig. 1.
Autophagosomes associated with collagen. (A) Representative confocal images of collagen (left), LC3 (middle), and merged images (right). Lower panels show enlarged images of the boxed regions in the upper panels. The fibroblasts in these images were cultured on FITC-labeled collagen and stained with anti-LC3 antibody. Scale bars: 5 µm (upper) and 1 µm (lower). (B) Representative image of orthographic project. Fibroblasts were cultured on FITC-labeled collagen and stained with a CellTracker orange probe. (C) Collagen deposition with (right) and without (left) HCQ treatment. Fibroblasts were cultured on FITC-labeled collagen for 120 min. Scale bar: 20 µm. (D) Quantification of the area of FITC-labeled collagen per cell. Data are mean and s.e.m. in control and HCQ-treated cells (n=3 fields of control; five fields for HCQ-treated cells). *P<0.001, Student's t-test. Three independent similar experiments are shown.
- Fig. 2.
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).
- Fig. 3.
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.
- Fig. 4.
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.
- Fig. 5.
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.
- Fig. 6.
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.
- Fig. 7.
Autophagy-deficient cells exhibited more adherence than control cells. (A) Control and Atg5−/− MEFs were cultured on collagen for 30 min and stained with 0.5% crystal violet. Scale bar: 50 µm. (B) Adhesion assay in control and Atg5−/− MEFs. Crystal violet staining in these cells was eluted and absorbance of the resulting solution at 550 nm was examined. Data presented are from three experiments. *P<0.01, Student's t-test. (C) Representative crystal violet staining images of control and Atg7−/− MEFs. Scale bar: 50 µm. (D) Adhesion assay in control and Atg7−/− MEFs. Data presented are from three experiments. *P<0.01, Student's t-test. (E) Representative images of phalloidin staining for F-actin and DAPI staining of control and Atg5−/− MEFs. The cells were cultured on collagen for 120 min. Scale bar: 10 µm.
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