Fig 2: C1q targeted DDR1 in HepG2 cells. Cells treated with or without C1q were harvested and DDR1 mRNA level was assessed by real-time PCR (A). Cells were treated with the indicated doses of C1q for 18 h and DDR1 expressions were analyzed by flow cytometry (B) and Western blot (C). Levels of phosphor-DDR1 and total DDR1 in cells treated with C1q for the indicated times were assessed by immunoprecipitation followed by western blotting analysis (D). *P < 0.05, **P < 0.01.

Fig 3: Knockdown of DDR1 attenuated the C1q-induced migration and invasion of HepG2 cells. Evaluation of DDR1 protein levels by western blotting after transfection for 36 h with scrambled (siCtrl) or DDR1-specific siRNAs (A). Control or DDR1 siRNA-transfected cells were cultured in 48-well plastic plates and allowed to adhere. A linear wound was then created in each well using a pipette and cells were treated with or without C1q to initiate migration (B). Transfected cells were cultured in transwell inserts in the presence or absence of C1q for 18 h and invading cells were counted as described in Materials and Methods (E). Bar diagrams representing migration distances (C), and counts of migrating cells (D) and invading cells (F). *P < 0.05, **P < 0.01 vs siCtrl + C1q, #P < 0.05 vs siCtrl.

Fig 4: C1q regulated epithelial-mesenchymal transition (EMT) via DDR1 in HepG2 cells. The protein levels of the EMT markers E-cadherin, vimentin, and N-cadherin were assessed in cells treated with or without C1q by western blotting. (A) Cells were treated with the indicated doses of C1q for 18 h and E-cadherin, vimentin, N-cadherin, and actin expressions were analyzed by Western blot. (B) Bar graphs of E-cadherin/Actin, (C) N-cadherin/Actin, (D) Vimentin/Actin and ratios. (E) Cells were transfected with control (siCtrl) or siDDR1 for 36 h, treated with or without C1q for 6 h, and Western blotted for E-cadherin, vimentin, N-cadherin and actin. (F) Bar graphs of E-cadherin/Actin, (G) N-cadherin/Actin, (H) Vimentin/Actin and ratios. *P < 0.05, **P < 0.01.

Fig 5: C1q bound directly to DDR1 in HepG2 cells. The interaction between C1q with DDR1 was investigated by ELISA and co-immunoprecipitation (Co-IP). (A) Binding of C1q with DDR1 by ELISA. Plates were coated with equal amounts of DDR1 (BSA was used as negative control) and incubated with diluted recombinant C1q. Absorbance was measured at 450 nm. C1q was found to dose-dependently bind to DDR1 with OD values higher than those of the control (BSA). (B) Co-immunoprecipitation of C1q and DDR1 examined by Western blotting. HepG2 cells were grown in 10 cm plates, treated with or without C1q for 1 h, lysed, and subjected to Co-IP. The lysates were with anti-DDR1 antibody or anti-IgG. The immunoprecipitates obtained were western blotted using anti-C1q or anti-DDR1 antibodies. As a protein input control, 5% of cell lysates were used. *P < 0.05, **P < 0.01.