Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Protein ERp46 in Renal Cell Carcinoma

doi: 10.1371/journal.pone.0090389

Figure Lengend Snippet: ( a ) Immunocytochemical staining for ERp46 (I, green), or AdipoR1 (II, red). The merged image (III) demonstrates yellow signal which indicates co-localization. Cells were counterstained with DAPI (blue). ( b ) Subcellular protein fractionation. Equal portions of each fractionated cellular extract were analyzed by Western blot using specific antibodies against AdipoR1 and ERp46. Antibodies directed against Hsp90 (cytoplasmic), calreticulin (plasma membrane) and HDAC2 (nuclear) served as fractionation controls. AdipoR1 is detected in the cytoplasmic and plasma membrane fractions, ERp46 in the nuclear soluble and membrane fraction. The asterisk indicates an ERp46 degradation product or possibly the shorter ERp46 isoform 3. ( c ). Western blot analyses for AdipoR1 and ERp46-specific extraction and isolation from 3×10 6 786-O cells. Presence of AdipoR1 in the elution fraction confirms that AdipoR1 is cell surface exposed. Absence of ERp46 in the elution fraction indicates most likely that it is either not cell surface exposed or not tightly bound to a cell surface protein. ( d ) Bacterial adenylate cyclase-based two-hybrid assay (BACTH) used to determine the interaction between ERp46 and AdipoR1. The N-termini of AdipoR1 and ERp46 were expressed as fusion to the T18 and T25 domains of the adenylate cyclase. Interaction was quantified via cAMP/CAP-induced β-galactosidase activity. The pUT18C strain producing AdipoR1 N fused to T25 domain of adenylate cyclase served as negative control. Other controls (plasmid, ERp46 N with linker, ERp46 N fused to T18) were also negative. No interaction between the N-termini of ERp46 and AdipoR1 was observed. T18 and T25 fused to interacting leucine zippers from GCN4 served as positive control. Data are from three independent repetitions and error bars indicate standard deviation. Depending on the fusion direction of AdipoR1 N and ERp46 N to T18 and T25, a “parallel” and “anti-parallel” orientation of the N-termini is captured, shown schematically.

Article Snippet: The following primary antibodies were employed: goat anti-ERp46 (1∶1,000; Santa-Cruz), rabbit anti-AdipoR1 (1∶1,000; AbBiotech), rabbit anti-HDAC2 (1∶1500; Santa-Cruz), rabbit anti-Hsp90 (1∶1,000; Cell Signalling), rabbit anti-calreticulin (1∶1,000; Cell Signaling).

Techniques: Staining, Fractionation, Western Blot, Isolation, Two Hybrid Assay, Activity Assay, Negative Control, Plasmid Preparation, Positive Control, Standard Deviation

Journal: PLoS ONE

Article Title: Wogonin Induced Calreticulin/Annexin A1 Exposure Dictates the Immunogenicity of Cancer Cells in a PERK/AKT Dependent Manner

doi: 10.1371/journal.pone.0050811

Figure Lengend Snippet: Gastric carcinoma cell line MKN-45 cells were either left untreated or treated with wogonin 50, 100, 200 µM, respectively (A), Two-dimensional gel electrophoresis was performed and proteins were identified by mass spectrometry. Note a obvious induction of p22, Annexin A1 and HMGB1 after 24 hours, expression level of those proteins was quantified (B). MFC cells were treated with wogonin (100 µM) for 1, 2 and 4 hours, followed by immunoblot detection of CRT, p22, Annexin 1, EGFR and actin in the plasma membrane protein fraction and in whole cell lysate (C), pre-cleared 600-µg aliquots of cell lysates for the 4 hour time point were incubated with anti-CRT, followed by Western blotting analysis with anti-CRT, p22, Annexin A1 and IgG respectively (D). MFC cells transfected with control or p22 siRNA for 48 hours, p22 expression in whole cell lysate were detected by Western blot. Successful p22 knockdown cells were then treated with wogonin (100 µM) for 2 hours, followed by immunoblot detection of CRT, p22, Annexin A1, EGFR and actin in the plasma membrane protein fraction, p22, Annexin A1 and actin in cell lysate were also tested (E). The effect of p22 siRNA on CRT translocation was also confirmed by confocal immune-fluorescence microscopy assay(F). MCFs cells transfected with control or PERK siRNA (200 nM, for 48 hours) were treated with wogonin (100 µM) for 2 and 4 hours, followed by immunoblot detection of p22 and Annexin A1 in the plasma membrane protein fraction and in total cell lysate (G). WT and AKT1/2 double knockout MEFs were treated with wogonin (100 µM) for 4 hours, p22 and Annexin A1 in both plasma membrane protein fraction and total cell lysate were tested (H). MFC cells were pretreated with AKT specific inhibitor X (AKTi 100 nM) or PI3K/AKT inhibitor LY294002 (100 nM) for 2 hours, followed by wogonin (100 µM) treated for 2 and 4 hours, p22 and Annexin A1 in both plasma membrane protein fraction and total cell lysate were tested (I). * P<0.05 vs. group without wogonin treatment.

Article Snippet: Precleared samples were incubated with anti-Calreticulin (CRT) Antibody (cs-2891, Cell Signaling Tech), or anti-DNA-PKcs (sc-9051, Santa Cruz antibodies) in lysis buffer overnight at 4°C.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Mass Spectrometry, Expressing, Western Blot, Incubation, Transfection, Translocation Assay, Fluorescence, Microscopy, Double Knockout

Journal: PLoS ONE

Article Title: Wogonin Induced Calreticulin/Annexin A1 Exposure Dictates the Immunogenicity of Cancer Cells in a PERK/AKT Dependent Manner

doi: 10.1371/journal.pone.0050811

Figure Lengend Snippet: Gastric carcinoma cell line MKN-45 cells were either left untreated or treated with wogonin 50, 100, 200 µM, respectively (A), Two-dimensional gel electrophoresis was performed and proteins were identified by mass spectrometry. Note a obvious induction of p22, Annexin A1 and HMGB1 after 24 hours, expression level of those proteins was quantified (B). MFC cells were treated with wogonin (100 µM) for 1, 2 and 4 hours, followed by immunoblot detection of CRT, p22, Annexin 1, EGFR and actin in the plasma membrane protein fraction and in whole cell lysate (C), pre-cleared 600-µg aliquots of cell lysates for the 4 hour time point were incubated with anti-CRT, followed by Western blotting analysis with anti-CRT, p22, Annexin A1 and IgG respectively (D). MFC cells transfected with control or p22 siRNA for 48 hours, p22 expression in whole cell lysate were detected by Western blot. Successful p22 knockdown cells were then treated with wogonin (100 µM) for 2 hours, followed by immunoblot detection of CRT, p22, Annexin A1, EGFR and actin in the plasma membrane protein fraction, p22, Annexin A1 and actin in cell lysate were also tested (E). The effect of p22 siRNA on CRT translocation was also confirmed by confocal immune-fluorescence microscopy assay(F). MCFs cells transfected with control or PERK siRNA (200 nM, for 48 hours) were treated with wogonin (100 µM) for 2 and 4 hours, followed by immunoblot detection of p22 and Annexin A1 in the plasma membrane protein fraction and in total cell lysate (G). WT and AKT1/2 double knockout MEFs were treated with wogonin (100 µM) for 4 hours, p22 and Annexin A1 in both plasma membrane protein fraction and total cell lysate were tested (H). MFC cells were pretreated with AKT specific inhibitor X (AKTi 100 nM) or PI3K/AKT inhibitor LY294002 (100 nM) for 2 hours, followed by wogonin (100 µM) treated for 2 and 4 hours, p22 and Annexin A1 in both plasma membrane protein fraction and total cell lysate were tested (I). * P<0.05 vs. group without wogonin treatment.

Article Snippet: Cancer cells with indicated treatment were fixed and blocked with 10% BSA in PBS for 30 min at room temperature and then incubated with 1∶100 rabbit anti-calreticulin (CRT) Antibody (cs-2891, Cell Signaling Tech) for 1 h(testing CRT translocation) followed by FITC–anti-rabbit secondary antibody (Cell Signaling Tech, MA) at 1∶100 for 30 min and CRT immune-fluorescence was observed in a confocal microscopy(Leica TCS SMD FCS, Leica, Germany), Hoechst 33342 was used to stain the nuclear.

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Mass Spectrometry, Expressing, Western Blot, Incubation, Transfection, Translocation Assay, Fluorescence, Microscopy, Double Knockout

Journal: Autophagy

Article Title: Necroptosis promotes autophagy-dependent upregulation of DAMP and results in immunosurveillance

doi: 10.1080/15548627.2017.1386359

Figure Lengend Snippet: SK induced DAMP release and DAMP ectolocalization by 4T1-luc2 cells. (A) Effect of SK on the release of test DAMPs and LDHA from tumor cells. 4T1-luc2 cells were treated with increasing concentrations of SK for 24 h and test culture media were harvested. Proteins in conditioned media were fractionated by SDS-PAGE and subjected to western blotting using anti-HSP90AA1, HSPA1A, and CALR Ab. LDHA was used as a control. (B) Imaging of DAMP ectolocalization in SK-treated cells. The expression levels of a single surface DAMP (white triangle) were visualized by confocal microscopy and compared among treatment groups. Ectolocalizations of HSP90AA1, HSPA1A, and CALR were compared. Bar: 20 µm. (C) Time course and dosage effect of SK on 4T1 cells. Test cells treated with different concentrations of SK for 1 to 24 h were subjected to the corresponding antibody staining and subjected to flow cytometry analysis. Percentages of cells expressing respective ectoDAMP were determined. (D) The dosage-dependent effect of SK on the ectolocalization of HSPA1A/90 and CALR. Test cells treated for 24 h with the indicated SK dosage were analyzed for the ectolocalization of DAMPs. Data are expressed as mean ± SEM of triplicate determinations. P value was determined by a one-way ANOVA with Tukey's test. Data presented are from one of 3 representative experiments. MFI, mean fluorescence intensity.

Article Snippet: After electrophoresis, proteins were transferred onto Hybond-ECL membranes (GE-Healthcare, RPN2020D) and immunoblotted with primary anti-CASP8/caspase-8 (Cell Signaling Technology, 4790), anti-HSPA1A (Proteintech, 10995-1-AP), anti-HMGB1 (Abcam, ab18256), and anti-CALR (Cell Signaling Technology, 2891) antibodies, and monoclonal antibody against PARP1 (Cell Signaling Technology, 9532), LC3B (Cell Signaling Technology, 3868), HSP90AA1 (Cell Signaling Technology, 4877), ubiquitin (Santa Cruz Biotechnology, sc-271289), and ACTB/actin, beta (Santa Cruz Biotechnology, sc-47778).

Techniques: SDS Page, Western Blot, Imaging, Expressing, Confocal Microscopy, Staining, Flow Cytometry, Fluorescence

Journal: Autophagy

Article Title: Necroptosis promotes autophagy-dependent upregulation of DAMP and results in immunosurveillance

doi: 10.1080/15548627.2017.1386359

Figure Lengend Snippet: Immunogenicity of SK-induced 4T1-luc2 ICD occurs in a cell-to-cell-interaction-dependent manner. (A) Expression of test activation markers on naïve DCs. The expression of CD40, CD80, CD86, and H2-I molecules was determined on 10-d-old freshly purified BM-DCs (thin line/white area). The corresponding isotype controls were also used (gray area). (B) The effect of ectoDAMPs versus the release of DAMPs on DC activation. 4T1-luc2 cells treated in the presence or absence of 5 μM SK for 24 h were centrifuged. The resulting supernatants were collected and used as conditioned media for BM-DC culturing. The expression of 4 activation markers in DCs was analyzed at 24 h postcoculturing with tumor cells (upper panel). 4T1-luc2 cells that were collected were washed, resuspended, and used to coculture BM-DCs using a transwell apparatus. DCs were harvested 24 h after incubation and the cell activation status determined (middle panel). Aliquots of collected 4T1-luc2 cells were also mixed with BM-DCs at a 1:1 ratio and expression of CD40, CD80, CD86, and H2-I molecules were determined by flow cytometry analysis (lower panel). (C) Antibody blocking of ectoDAMP expressions in SK-treated tumor cells suppressed DC activation. SK-treated 4T1 cells were incubated with or without anti-HSPA1A, anti-HSP90AA1, and anti-CALR antibodies (+ Abs) for 2 h before they were cocultured with BM-DCs. After incubation for 24 h, test cells were harvested and the expression of CD40, CD80, CD86 and H2-I molecules on BM-DCs was determined via flow cytometry analysis. (D) The effect of SK-treated cells on expression of specific proinflammatory cytokines in DCs. Conditioned coculture medium of DC + 4T1 cells was harvested and the levels of secreted IL1B, IL6, and IL12B were determined by ELISA. Data are expressed as mean ± SEM of triplicate determinations. P value was determined by a one-way ANOVA with Tukey's test. Data presented are from one of 3 representative experiments.

Article Snippet: After electrophoresis, proteins were transferred onto Hybond-ECL membranes (GE-Healthcare, RPN2020D) and immunoblotted with primary anti-CASP8/caspase-8 (Cell Signaling Technology, 4790), anti-HSPA1A (Proteintech, 10995-1-AP), anti-HMGB1 (Abcam, ab18256), and anti-CALR (Cell Signaling Technology, 2891) antibodies, and monoclonal antibody against PARP1 (Cell Signaling Technology, 9532), LC3B (Cell Signaling Technology, 3868), HSP90AA1 (Cell Signaling Technology, 4877), ubiquitin (Santa Cruz Biotechnology, sc-271289), and ACTB/actin, beta (Santa Cruz Biotechnology, sc-47778).

Techniques: Expressing, Activation Assay, Purification, Incubation, Flow Cytometry, Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Curcumin Enhanced Ionizing Radiation-Induced Immunogenic Cell Death in Glioma Cells through Endoplasmic Reticulum Stress Signaling Pathways

doi: 10.1155/2022/5424411

Figure Lengend Snippet: Curcumin enhanced IR-induced ICD by activating ER stress. (a) Curcumin increased the expressions of ER stress-related proteins in normoxic and hypoxic glioma. Cells were irradiated with 10 Gy/25 Gy IR after treatment with or without 30 μ M curcumin for 24 h, and then, indicated protein expressions were assessed by WB. (b) Cell apoptosis was assessed with or without pretreatment with 2 μ M GSK2606414 or 10 μ M 4u8c for 1 h before irradiation. (c) Calreticulin exposure was analyzed by flow cytometry. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. control; # P < 0.05 and ## P < 0.01 vs. IR.

Article Snippet: Cells were stained with Calreticulin-PhycoErythrin antibody (1 : 50, Cell Signaling) and then washed and resuspended in FACS buffer for assessing with flow cytometry.

Techniques: Irradiation, Flow Cytometry