Journal: Scientific Reports

Article Title: Plumbagin sensitizes breast cancer cells to tamoxifen-induced cell death through GRP78 inhibition and Bik upregulation

doi: 10.1038/srep43781

Figure Lengend Snippet: ( A ) Effects of plumbagin on the expression levels of GRP78. MCF-7 and T47D cells were treated with plumbagin (0–2.5 μM) for 24 h and protein levels were determined with Western blot analysis. ( B ) Silencing of GRP78 in MCF-7 and T47D cells. Cells were transiently transfected with GRP78 siRNA and after 24 h GRP78 levels were determined with Western blot analysis. ( C ) Overexpression of GRP78 was performed by transiently transfecting cells with a GRP78 CRISPR/dCas9 activation plasmid. 24 h after transfection, GRP78 levels were determined with Western blot analysis. ( D ) Influence of GRP78 silencing and ( E ) GRP78 upregulation on the induction of apoptosis by plumbagin. 24 h-post transfection cells were treated with plumbagin (0.5 and 1 μM) for 24 h after which cells were stained with Annexin V-PE/7-AAD, and analyzed by flow cytometry. Values represent mean ± SE of three independent experiments. p < 0.05 (*) indicates differences between control and GRP78-downregulated and upregulated cells treated with plumbagin.

Article Snippet: The overexpression of GRP78 was carried out with a GRP78 CRISPR/dCas9 activation plasmid (Santa Cruz, Germany).

Techniques: Expressing, Western Blot, Transfection, Over Expression, CRISPR, Activation Assay, Plasmid Preparation, Staining, Flow Cytometry, Control

Journal: Scientific Reports

Article Title: Plumbagin sensitizes breast cancer cells to tamoxifen-induced cell death through GRP78 inhibition and Bik upregulation

doi: 10.1038/srep43781

Figure Lengend Snippet: MCF-7 and T47D cells were transiently transfected with ( A ) GRP78 siRNA or ( B ) Bik siRNA. 24 h-post transfection cells were treated for an additional 24 h with plumbagin and tamoxifen. Cells were stained with Annexin V-PE/7-AAD and analyzed by flow cytometry. Values represent mean ± SE of three independent experiments. p < 0.05 (*) indicates differences between combination-treated control and GRP78 or Bik-downregulated cells. ( C ) Influence of combination treatment with plumbagin and tamoxifen on expression levels of GRP78 and Bik. Cells were treated for 24 h with plumbagin (0.5 μM) and tamoxifen (1 μM) and GRP78 or Bik levels were determined with Western blot analysis. ( D ) Influence of GRP78 silencing on plumbagin-mediated Bik induction. 24 h-post transfection cells were treated with plumbagin (0.5 μM) for 24 h and the expression levels of Bik were determined with Western blot analysis.

Article Snippet: The overexpression of GRP78 was carried out with a GRP78 CRISPR/dCas9 activation plasmid (Santa Cruz, Germany).

Techniques: Transfection, Staining, Flow Cytometry, Control, Expressing, Western Blot

Journal: Theranostics

Article Title: ATF4 destabilizes RET through nonclassical GRP78 inhibition to enhance chemosensitivity to bortezomib in human osteosarcoma

doi: 10.7150/thno.36818

Figure Lengend Snippet: GR P 78 disrupts the binding of ATF4 to RET . A, GRP78 colocalizes with RET in the cytoplasm. Confocal images of immunofluorescence staining of GRP78 and RET in human BTZ-resistant OS cells. Scale bars, 10 μm. B, U-2 OS cells were transiently transfected with Myc-RET or HA-GRP78 with or without the vector pcDNA3.1 for 2 days with MG132 (20 μM) treatment for 4 h, and whole-cell lysates were immunoprecipitated with anti-Myc antibody and blotted with the indicated antibodies. C, GRP78 interacts with the TK domain of RET to improve RET stability but interacts with the CLD1 domain for its degradation. Expression vectors encoding HA-GRP78 and Myc-RET mutants were transfected into U-2 OS cells as indicated. Cell lysates were immunoprecipitated with anti-Myc antibody, and the indicated proteins were detected by immunoblotting. Five percent of the HA-GRP78-transfected cell lysate for IP was used as input. D, U-2 OS cells were co-transfected with FLAG-ATF4, Myc-RET and HA-GRP78. Cell extracts were immunoprecipitated using an anti-Myc antibody and blotted with the indicated antibodies. E, Western blotting analysis of U-2 OS/BTZ cells showing RET downregulation and nuclear translocation after ATF4 overexpression in contrast to control vector. F , U-2 OS/BTZ and HOS/BTZ cells were transiently transfected with control (siCon), siATF4 or siGRP78 for 2 days. Protein lysates were harvested and subjected to immunoblotting analysis using the indicated antibodies. G, Equal amounts of protein lysates from a panel of U-2 OS/BTZ cells expressing control (siCon or vector), siRET, siATF4, siATF4 or ATF4 vectors were loaded for SDS-PAGE and western blotting analysis to detect the expression of the indicated proteins. GAPDH was used as a loading control. H, Colony formation assay was performed using paired OS and OS/BTZ cells transfected with control or siGRP78, GRP78, ATF4 and RET vectors with BTZ (100 nM) treatment for the first two days of the experiment. The cells were fixed, stained, and photographed after 14 days. I, Transcriptional activity of HSPA5 in ATF4-expressing and control OS cell lines. Cells were transfected with the ATF4 expression plasmid and the HSPA5 promoter (-457 to +1) luciferase reporter plasmid, and luciferase activity was determined 24 h after transfection. J, ATF4-mediated HSPA5 promoter repression in BTZ-resistant OS requires the first box of ERSE. U-2 OS/BTZ cells were transfected with plasmids encoding the firefly luciferase gene driven by the illustrated promoter together with control vector (white bar) or plasmids encoding ATF4 (black bar). After 24 h, cells were harvested and analysed for luciferase activity. Data are shown as the mean ± SEM. Statistical significance was determined by Student's t-test. * P < 0.05; n.s., non-significant.

Article Snippet: Plasmids encoding the luciferase reporter gene under the control of the GRP78 promoter were purchased from General Biosystems (Anhui, China) and were purified using the High Pure Plasmid Miniprep Kit (Real-Times, China).

Techniques: Binding Assay, Immunofluorescence, Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Western Blot, Translocation Assay, Over Expression, Control, SDS Page, Colony Assay, Activity Assay, Luciferase

Journal: Theranostics

Article Title: ATF4 destabilizes RET through nonclassical GRP78 inhibition to enhance chemosensitivity to bortezomib in human osteosarcoma

doi: 10.7150/thno.36818

Figure Lengend Snippet: ATF4 activator screening from a compound library . A, Proliferation of control or piperine and ribociclib-treated OS and OS/BTZ cells was evaluated by colony formation assays. B, BTZ-resistant OS sublines were cultured in medium containing the indicated concentrations of piperine and ribociclib for 2 days, and lysates were blotted for the indicated proteins. C and D, ATF4 was knocked down in U-2 OS/BTZ cells, and then the cells were treated with piperine or ribociclib. Lysates were harvested from cells for immunoprecipitation with anti-RET antibody. IP samples were used to study ubiquitinated RET levels by western blotting with anti-FLAG antibody. E, Model indicating that the regulation of RET signalling by ATF4 leads to drug sensitivity in osteosarcoma. In wild-type OS cells, ATF4 promotes Cbl-c transcription and recruits it to synergistically to bind to RET by inducing the nuclear translocation of RET, thus resulting in the degradation of RET and inactivation of downstream signalling. Meanwhile, ATF4-transactivated GRP78 competitively interacts with RET, which is beneficial for RET stabilization. In contrast, ATF4 specifically transcriptionally inhibits GRP78 in BTZ-resistant OS cells, represents reinforced function of overwhelming RET signaling for the prevention of cancer malignancy drug resistance.

Article Snippet: Plasmids encoding the luciferase reporter gene under the control of the GRP78 promoter were purchased from General Biosystems (Anhui, China) and were purified using the High Pure Plasmid Miniprep Kit (Real-Times, China).

Techniques: Drug discovery, Control, Cell Culture, Immunoprecipitation, Western Blot, Translocation Assay

Journal: Cell Death & Disease

Article Title: MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer

doi: 10.1038/s41419-018-0950-x

Figure Lengend Snippet: a Illustration of the GRP78 3′-UTR-containing reporter constructs. Mutations were generated at two predicted miR-495-3p binding sites located in the GRP78 3′-UTR. b Representative luciferase activity in SGC7901 cells co-transfected with wild-type or mutated reporter plasmids and nc, miR-495-3p mimic. c Western blot showed the changes in GRP78 protein levels after transient transfection of miR-495-3p inhibitor or mimic with indicated concentration gradient as compared to the negative controls (nc or anti-nc, respectively). d Real-time PCR determined the GRP78 level in SGC7901 after transfection with miR-495-3p inhibitor/mimic or anti-nc/nc. NS is no significance. All values were expressed as mean ± SD, n = 3 for each group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For the reporter gene assay, the cells were plated in 24-well plate and transfected with 0.5 μg GRP78 3′-UTR (untranslated region) luciferase reporter plasmids and GRP78 3′-UTR mutant1 and mutant2 luciferase reporter plasmids (Land biolog Co. Ltd, Guangzhou, China) using lipofectamine 2000 (Invitrogen, USA).

Techniques: Construct, Generated, Binding Assay, Luciferase, Activity Assay, Transfection, Western Blot, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer

doi: 10.1038/s41419-018-0950-x

Figure Lengend Snippet: GRP78 expression is increased and associated with poor outcomes in GC. a Real-time PCR shows the expression of GRP78 in 15 paired gastric cancer samples as compared to adjacent tumor tissues. b Western blotting indicates the protein level of GRP78 in 13 paired gastric cancer tissues and corresponding adjacent tumor tissues (C is carcinoma, N is normal tissues). Data from TCGA show the transcript per million of GRP78 in normal and primary GC tissues ( c ) based on individual cancer stages ( d ) and tumor grades ( e ). f The expression of GRP78 in normal gastric epithelial cell line and five gastric cancer cell lines and GC MDR cells by Real-time PCR. All values expressed as mean ± SD, n = 3 for each group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For the reporter gene assay, the cells were plated in 24-well plate and transfected with 0.5 μg GRP78 3′-UTR (untranslated region) luciferase reporter plasmids and GRP78 3′-UTR mutant1 and mutant2 luciferase reporter plasmids (Land biolog Co. Ltd, Guangzhou, China) using lipofectamine 2000 (Invitrogen, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cell Death & Disease

Article Title: MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer

doi: 10.1038/s41419-018-0950-x

Figure Lengend Snippet: Downregulation of GRP78 by miR-495-3p inhibits autophagy in GC MDR cells. a Representative images and b Quantification of LC3 puncta (green) in GC MDR cells transfected with si-GRP78 or si-nc. Scale bars: 50 μM. c Representative images and d Quantification of LC3 puncta (green) in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. e The expression level of GRP78, LC3BI/II, and P62 in GC MDR cells transfected with si-GRP78 or si-NC were determined by western blotting. f The protein level of GRP78, LC3BI/II, and P62 in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. All values expressed as mean ± SD, n = 3 for each group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For the reporter gene assay, the cells were plated in 24-well plate and transfected with 0.5 μg GRP78 3′-UTR (untranslated region) luciferase reporter plasmids and GRP78 3′-UTR mutant1 and mutant2 luciferase reporter plasmids (Land biolog Co. Ltd, Guangzhou, China) using lipofectamine 2000 (Invitrogen, USA).

Techniques: Transfection, Mutagenesis, Over Expression, Plasmid Preparation, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer

doi: 10.1038/s41419-018-0950-x

Figure Lengend Snippet: a Western blot analysis of phosphorylated mTOR (p-mTOR) and its main substrates 4E-BP1 (p-4E-BP1) and S6 (p-S6) in SGC7901 transfected with anti-miR-495-3p and GC MDR cells transfected with miR-495-3p. b The phosphorylation status of mTOR, S6K (p-S6K), and 4E-BP1 (p-4E-BP1) transfected with si-GRP78 and si-NC were measured by western blotting ( c ). Western blotting determines the phosphorylation of mTOR, S6K (p-S6K), and 4E-BP1 (p-4E-BP1) in SGC7901/ADR co-transfected with GRP78 3′-UTR-mutant overexpression vector/nc and miR-495-3p/nc. All values expressed as mean ± SD, n = 3 for each group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For the reporter gene assay, the cells were plated in 24-well plate and transfected with 0.5 μg GRP78 3′-UTR (untranslated region) luciferase reporter plasmids and GRP78 3′-UTR mutant1 and mutant2 luciferase reporter plasmids (Land biolog Co. Ltd, Guangzhou, China) using lipofectamine 2000 (Invitrogen, USA).

Techniques: Western Blot, Transfection, Phospho-proteomics, Mutagenesis, Over Expression, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer

doi: 10.1038/s41419-018-0950-x

Figure Lengend Snippet: Expression levels of miR-495-3p and GRP78 in GC specimens. a The expression levels of miR-495-3p (upper) and GRP78 (lower) in peri-tumor and primary GC tissues, scale bars: 500 μm (top) and 200 μm (bottom). b miR-495-3p and c GRP78 expression staining score in peri-tumor and primary GC tissues. Kaplan–Meier survival curves of GC patients expressing miR-495-3p ( d ) and GRP78 ( e ) GC patients were ranked based on the staining score and divided into high-expression (>4) and low-expression (≤4) groups. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For the reporter gene assay, the cells were plated in 24-well plate and transfected with 0.5 μg GRP78 3′-UTR (untranslated region) luciferase reporter plasmids and GRP78 3′-UTR mutant1 and mutant2 luciferase reporter plasmids (Land biolog Co. Ltd, Guangzhou, China) using lipofectamine 2000 (Invitrogen, USA).

Techniques: Expressing, Staining

Journal: Cell Death & Disease

Article Title: MicroRNA-495-3p inhibits multidrug resistance by modulating autophagy through GRP78/mTOR axis in gastric cancer

doi: 10.1038/s41419-018-0950-x

Figure Lengend Snippet: Correlation of miR-495-3p and GRP78 expression with patients’ clinicopathological variables in GC tissues

Article Snippet: For the reporter gene assay, the cells were plated in 24-well plate and transfected with 0.5 μg GRP78 3′-UTR (untranslated region) luciferase reporter plasmids and GRP78 3′-UTR mutant1 and mutant2 luciferase reporter plasmids (Land biolog Co. Ltd, Guangzhou, China) using lipofectamine 2000 (Invitrogen, USA).

Techniques: Expressing