Fig 1: Proteasome inhibitor bortezomib augments the antitumor efficiency of bevacizumab in vitro and in vivo. a Combined effect of bortezomib and bevacizumab in A549 cells. After incubating cells with increasing concentrations of bortezomib and bevacizumab, cell viability was determined, and the combination index (CI) was calculated by CompuSyn software. b The mouse body weight of the five groups was measured every 3 days after the indicated treatment. c Representative tumors from the five groups are shown (n = 5). d Tumor volume from homografts in different treatment groups was recorded every 3 days. Data are represented as the mean ± S.E.M. (n = 5). e Tumor weight was detected at the time of sacrifice for different treated groups. f Representative images of H&E staining and immuno-histochemical staining of the different treated groups. Scale bar = 50 µm. g Ki67-positive rates in each group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the negative control. Data are shown as the mean ± S.E.M. h Western blot analysis of AGR2, PARP, LC3 expression in differentially treated groups. i As liver function tests, ALT and AST were examined at the time of sacrifice for different treated groups. Data are shown as the mean ± S.E.M (n = 5). j, k Locomotor activity of different treated groups was detected at 24 h after each injection. Data are shown as the mean ± S.E.M (n = 15). **p < 0.01 compared with the negative control
Fig 2: Proteasome inhibition decreases the AGR2 expression. a Kaplan–Meier survival curves of lung adenocarcinoma patients are plotted for AGR2. The survival rate of the patients in the AGR2-lower group was significantly higher than that of patients in the AGR2-higher group (log-rank test, p= 0.02). b The baseline expression of AGR2 in several lung cancer cell lines. GAPDH served as a loading control. **p < 0.01 and ***p < 0.001 compared with the HBE cells. c The expression change of AGR2 in MG132-treated A549 cells was estimated by western blotting analysis. d MTT assay and e annexin staining was used to evaluate the effect of MG132 on A549 cells at 24 h. All values are expressed as the means ± SD of three independent experiments. f The levels of p53, p27, and AGR2 were measured by western blotting with 5 µM MG132 or 10 µM bortezomib for indicated time. g QRT-PCR analysis of AGR2 mRNA levels with 5 µM MG132 for 24 h. **p < 0.01 compared with the negative control. h The AGR2 promoter (0.5 or 1.4 kb) reporter constructs were transfected into A549 cells for 24 h, and then cells were treated with 5 µM MG132 for another 24 h. Luciferase activity was determined and was normalized to Renilla luciferase activity. ***p < 0.001 compared with the basic reporter
Fig 3: Autophagy stimulated by MG132 is required for the degradation of AGR2. a A549 cells were incubated with autophagy inhibitor 3-MA (5 mM) and CQ (10 µM) for 2 h prior to MG132 treatment. LC3B and AGR2 expression were determined by western blot analysis. b, c The expression changes of AGR2 in A549 cell, knockdown of b Atg5, and c Atg7 by siRNA for 48 h and then treated with MG132. d The level of AGR2 in Rapamycin (100 nM)-treated A549 cells. e A549 cells were treated with CHX and Rapamycin for 24 h. f The baseline expression of AGR2 and autophagy in normal bronchial epithelial cell line (HBE) and several lung cancer cell lines. GAPDH served as a loading control. g The expression changes of AGR2 with CQ treatment for 24 h. h, i The correlative analysis of AGR2 and p62/Atg5 in human lung cancer datasets (GSE27262) from GEO database was assessed by Pearson’s correlation test
Fig 4: Suppression of AGR2 by MG132 is independent of the induced ER stress and ROS. a Analysis of the effect of MG132 on proteins associated with ER stress by western blot. b A549 cells were incubated with ER stress inhibitor 4-PBA (10 mM) for 2 h prior to MG132 (5 µM) treatment of 24 h. p-eIF2a and AGR2 expression were determined by western blot analysis. c The level of AGR2 was detected with 10 µM salubrinal treatment for 24 h. d The expression changes of IRE1a and AGR2 in MG132-treated A549 cells. e Knockdown of ATF4 by siRNA. f The expression change of ATF4 in 5 µM MG132-treated A549 cells was estimated by western blotting analysis. g Knockdown of ATF4 by siRNA. h QRT-PCR and western blotting analysis of mRNA and protein levels of AGR2 in TG (2 µM)/TM (2 mg/ml)/MG132 (5 µM)-treated A549 cells. GAPDH served as a loading control. i A549 cells were treated with ER stress inducer TG/TM/MG132 treatment at indicated time
Fig 5: Repression of E2F1 by MG132 contributes to reduced AGR2 transcription. a The expression changes of E2F1 and AR in MG132-treated A549 cells were estimated by western blotting analysis. GAPDH served as a loading control. b A549 cells were transfected with E2F1 expression plasmid for 24 h prior to MG132 treatment. AGR2 mRNA and protein were investigated by QRT-PCR and western blotting analysis, *p < 0.05 and **p < 0.01 compared with the negative control. Data shown are the means ± SD, n = 3. c Impact of knockdown of E2F1 by siRNA on the AGR2 mRNA and protein. d The AGR2-promotor reporter constructs were co-transfected with E2F1 expression plasmid into A549 cells for 48 h prior to MG132 treatment. Luciferase activity was determined after MG132 treatment for 24 h and was normalized to Renilla luciferase activity. Data shown are the means ± SD, n = 3. e Knockdown of AR by siRNA in A549 cells. *p < 0.05. f The change of AGR2 mRNA and protein induced by overexpression of AR in MG132-treated A549 cell. Data shown are the means ± SD, n = 3
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