Journal: Cell Death Discovery

Article Title: Disulfide bond-disrupting agents activate the tumor necrosis family-related apoptosis-inducing ligand/death receptor 5 pathway

doi: 10.1038/s41420-019-0228-9

Figure Lengend Snippet: a MDA-MB-468 cells were treated with DMSO or 5 µM tcyDTDO for 2 h. Total RNA was isolated from each sample and converted to cDNA using reverse transcriptase. The mRNA levels of DR5 were measured using real-time qPCR, and relative mRNA expression was calculated. Fold change was calculated by normalizing all values to the untreated group. T test showed p = 0.0395. b Protein synthesis was assessed by 3 H-leucine incorporation in MDA-MB-468 cells treated with the indicated concentrations of tcyDTDO or cycloheximide (CHX). c Immunoblot analysis of MDA-MB-468 cells engineered to express DR5 in a tetracycline-inducible manner (468/tet-DR5) and the corresponding control cell line (468/tet-fLuc) after the indicated 24-h treatments. d Immunoblot analysis of 468/tet-DR5 cells treated separately or with combinations of 1 µg/ml doxycycline, 12.5 ng/ml TRAIL, and 5 µM tcyDTDO for 24 h. e Immunoblot analysis of 468/tet-DR5 cells treated with the indicated agents for 24 h. f Upper panel. Diagram based on DR5 crystal structures showing the presence of seven intramolecular disulfide bonds and an unpaired cysteine residue in the extracellular domain of DR5. Lower panel. Immunoblot analysis of DR5 from 468/tet-DR5 cells treated for 24 h as indicated under nonreducing and reducing conditions. Cell extraction in the presence of N -ethylmaleimide NEM (100 mM) was used to limit thiol–disulfide exchange under nonreducing conditions.

Article Snippet: Reporter assays were performed as described previously using the DR5 reporter construct obtained from Addgene (Plasmid 16012 ).

Techniques: Isolation, Expressing, Western Blot

Journal: Cell Death Discovery

Article Title: Disulfide bond-disrupting agents activate the tumor necrosis family-related apoptosis-inducing ligand/death receptor 5 pathway

doi: 10.1038/s41420-019-0228-9

Figure Lengend Snippet: a , b MTT cell viability assays performed on MDA-MB-468 and BT474 cells, respectively, after 72 h of the indicated treatments (upper panel) and analyzed using the Chou–Talalay method to calculate combination indices (CIs) (lower panel). [CIs: =1, >1, and <1 represent additivity, antagonism, and synergy, respectively.] Graphs represent the average of quadruplicate determinations ± standard deviation. c Left panel. Immunoblot analysis of BT474 control and DR5-knockout (DR5-KO) clones after a 24-h treatment with vehicle or 10 µM tcyDTDO. Right panel. MTT cell survival assays performed on BT474 control and DR5 -knockout clones after 72 h of the indicated treatments. d Immunoblot analysis showing comparison of DR4 oligomerization in MDA-MB-468 and BT474 vector control or DR5-knockout clones. e The indicated T47D cell lines were treated in the presence or absence of 5 µM tcyDTDO for 24 h. Samples were collected under reducing and nonreducing conditions for immunoblot analyses.

Article Snippet: Reporter assays were performed as described previously using the DR5 reporter construct obtained from Addgene (Plasmid 16012 ).

Techniques: Standard Deviation, Western Blot, Knock-Out, Clone Assay, Plasmid Preparation

Journal: Cell Death Discovery

Article Title: Disulfide bond-disrupting agents activate the tumor necrosis family-related apoptosis-inducing ligand/death receptor 5 pathway

doi: 10.1038/s41420-019-0228-9

Figure Lengend Snippet: a Kaplan–Meier plot showing that patients with tumors overexpressing both EGFR and MYC are associated with a significantly worse survival than are patients with tumors expressing low levels of both EGFR and MYC. Patients ranked based on the expression of EGFR and MYC were classified into four groups, named “low EGFR low MYC ( N = 359)”, “low EGFR high MYC ( N = 220)”, “high EGFR low MYC ( N = 225)”, and “high EGFR high MYC ( N = 372)”. Overall survival (OS) was compared among these groups. The F test was used to compare the variance between groups ( P > 0.05, ns; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001). b Micrographs showing the morphology of the indicated MCF10A stable cell lines after 24 h of treatment with 5 µM tcyDTDO, 12.5 ng/ml TRAIL, tcyDTDO + TRAIL, tcyDTDO + TRAIL + 10 µM Q-VD-OPH, or 10 µM Lapatinib. c Immunoblot analysis of stable MCF10A cell lines treated for 24 h as in b . d Model for how DDAs activate TRAIL/DR5-induced cell death in an oncogene-dependent manner. In the context of EGFR or HER2 overexpression, DDAs elevate ER stress resulting in transcriptional upregulation of DR5. Through a second mechanism, DDAs alter DR5 disulfide bonding to promote DR5 protein stabilization, oligomerization, and activation of pro-apoptotic signaling. Cytotoxicity of DDAs and TRAIL is also potentiated in MYC-overexpressing cells.

Article Snippet: Reporter assays were performed as described previously using the DR5 reporter construct obtained from Addgene (Plasmid 16012 ).

Techniques: Expressing, Stable Transfection, Western Blot, Over Expression, Activation Assay

Journal: The Journal of Cell Biology

Article Title: ATG conjugation–dependent/independent mechanisms underlie lysosomal stress–induced TFEB regulation

doi: 10.1083/jcb.202307079

Figure Lengend Snippet: APEX1 is required for TFEB expression and stability. (A) Quantification of the relative expression of TFEB, TFE3, and MITF by qPCR in HeLa cells transfected with siLuc or si APEX1 under nutrient conditions ( n = 3 biologically independent samples). (B) Representative immunoblots of TFEB, p-TFEB (Ser211), APEX1, and GAPDH in WT HeLa cells transfected with EGFP stop (negative control) or EGFP::APEX1, either untreated (control) or treated with 1 mM LLOMe for 1 h. (C) Quantification of 2×CLEAR (TFEB RE)-luciferase reporter assays in HeLa cells stably expressing mNG or APEX1::mNG transfected under 1 mM LLOMe treatment for 3-h conditions ( n = 3 biologically independent samples). (D) Representative immunoblots of TFEB, APEX1, and GAPDH in WT HeLa cells that were either untreated (control) or treated with MG132 for 3 h. Cells were transfected with siLuc or si APEX1 . (E) Quantification of the image data shown in D ( n = 3 biologically independent samples). (F) Representative immunoblots of TFEB and GAPDH in WT HeLa cells treated with chloroquine for 4 h. Cells were transfected with si APEX1 . (G) Quantification of image data shown in F ( n = 3 biologically independent samples). (H) Representative immunofluorescence images of HeLa cells stained using an antibody against endogenous γH2AX (green). Cells were transfected with siLuc or si APEX1 . (I) Representative immunoblots of γH2AX and GAPDH in WT HeLa cells that were transfected with siLuc or si APEX1 . (J) Quantification of image data shown in I ( n = 3 biologically independent samples). (K) Schematic representation of APEX1 mutant constructs. (L) Representative fluorescence images of HeLa cells stably expressing mNG::APEX1 WT or mNG::APEX1 mutants. (M) Results of the PLA using WT HeLa cells stably expressing mNG::stop, mNG::APEX1 WT, or mNG::APEX1 mutants. Cells were transfected with si APEX1 . The indicated plasmids were transiently transfected and were either untreated (control) or treated with 1 mM LLOMe for 1 h followed by the PLA procedure. (H, L, and M) Scale bars, 50 μm. (E) P values were determined by ANOVA with Tukey’s multiple comparison test; *P < 0.05. (A and C) P values were determined by a t test; *P < 0.05, **P < 0.01. Source data are available for this figure: .

Article Snippet: Briefly, 2×CLEAR (TFEB RE)-luciferase reporters (plasmid #81120; Addgene) and plasmid encoding pMCX-β-galactosidase (gift from Dr. H. Ogawa, The Research foundation for Microbial disease of Osaka University, Suita, Japan) were cotransfected into HeLa cells.

Techniques: Expressing, Transfection, Western Blot, Negative Control, Control, Luciferase, Stable Transfection, Immunofluorescence, Staining, Mutagenesis, Construct, Fluorescence, Comparison