Figure 1.
TN induces the expressions of CLU and GRP78 in HCC cell lines in vitro.
(A–C) SMMC7721, HCCLM3 and HepG2 cells were treated for 24 h with TN for the indicated doses. Protein extracts were analyzed for CLU, GRP78 and GAPDH. TN treatment induced both CLU and GRP78 expressions and the molecular weight of CLU was decreased after TN treatment.
Figure 2.
CLU knockdown enhances TN treatment induced apoptosis in SMMC7721 and HCCLM3 cells.
(A–B) SMMC7721 and HCCLM3 cells were infected twice for a total of 2 days (1 day for each infection). The expression of CLU in SMMC7721 and HCCLM3 cells were inhibited through lentivirus-mediated shRNA interfering followed by 1 µg/ml of TN treatment for 24 h. Protein extracts were analyzed for CLU, GRP78 and GAPDH. (C–D) CCK8 assay was performed to determine cell viability after TN treatment with indicated concentrations for 24 h. The results indicated a synergistic effect of CLU knockdown with TN treatment. The values of relative survival rate were normalized with untreated cells. *P<0.05, versus mock group. TN-induced cell apoptosis in SMMC7721 and HCCLM3 cells were measured by Western blot analysis of PARP cleavage (CF, cleaved form) (E–F) and flow cytometry (sub-G1) (G–J). All these data were from a representative experiment and the representative result was from at least three independent experiments.
Figure 3.
CLU induction following TN treatment is cytoprotective via increasing the expression of GRP78.
(A) Control, mock and CLU-HepG2 cells were treated with 1 µg/ml of TN for 24 h. Protein extracts were analyzed for CLU, GRP78 and GAPDH. (B) CCK8 assay was performed to determine cell viability after TN treatment with indicated concentrations for 24 h. The values of relative survival rate were normalized with untreated cells. *P<0.05, versus mock group. (C–E) TN-induced cell apoptosis in HepG2 cells were measured by Western blot analysis of PARP cleavage (CF, cleaved form) and flow cytometry (sub-G1). The results indicated that CLU played a protective role on HCC cells under ER stress condition. (F) HepG2 cells were transfected with scramble siRNA or siRNA specific for GRP78 followed by 1 µg/ml of TN treatment for 24 h. Protein extracts were analyzed for GRP78 and GAPDH. Knockdown of GRP78 expression in HepG2 inhibited TN induced GRP78 expression. (G) CLU overexpressed HepG2 cells were transfected with scramble siRNA or siRNA specific for GRP78 followed by 1 µg/ml of TN treatment for 24 h. Protein extracts were analyzed for CLU, GRP78 and GAPDH. (H–J) TN-induced cell apoptosis in HepG2 cells were measured by Western blot analysis of PARP cleavage (CF, cleaved form) and flow cytometry (sub-G1). The results suggested GRP78 may be important in mediating the protective effect of CLU under ER stress condition. All these data were from a representative experiment and the representative result was from at least three independent experiments.
Figure 4.
CLU associates with GRP78 under ER stress conditions.
(A) Western blot confirmed the presence of GRP78 in CLU co-IP. HCC cell lines (SMMC7721, HCCLM3 and HepG2) were treated with 1 µg/ml TN for 24 h. Pre-cleared protein from whole cell lysates were incubated with goat anti-CLU antibody and then detected with anti-GRP78. (B–D) HCC cell lines (SMMC7721, HCCLM3 and HepG2) were treated with 1 µg/ml TN for 24 h and then co-immunostained with anti-CLU (green) and anti-GRP78 (red). The signals were detected with A1R MP Multiphoton Confocal Microscope. (E) CLU-HepG2 cells were treated with 1 µg/ml of TN for 24 h, the GRP78 was detected by immunostaining with anti-GRP78 (red). Ectopically expressed CLU is tagged with GFP (green). All these data were from a representative experiment and the representative result was from at least three independent experiments.
Figure 5.
Silencing of CLU induces GRP78 down-regulation and cell apoptosis in vivo.
(A) Immunohistochemistry staining with anti-CLU and anti-GRP78 were performed on serial sections of tumor specimen (×400). (B–C) The apoptotic cells were identified by TUNEL staining. The number of TUNEL-positive cells in CLU-shRNA group was significantly elevated compared with mock group (× 400). (D) Total proteins were extracted from the xenograft tumors and CLU, GRP78 and PARP cleavage (CF, cleaved form) were analyzed by Western blotting.
Figure 6.
There was a correlation between the levels of CLU and GRP78 in clinical HCC specimens.
(A–B) Representative immunostaining for CLU and GRP78 is shown for three patient samples (A: ×50; B: ×200). (C) Positive correlation between CLU expression and GRP78 level was examined in tumor tissues derived from 96 patients (r = 0.294, P<0.01).