Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: dDNMT activates TRAIL-death receptor signaling in VHL -deficient ccRCC cells (A) Volcano plot of SGI1027-induced and -repressed genes in RCC10 cells ( n = 2 biological replicates). FC, fold change. (B) Biocarta pathway enrichment analysis of SGI1027-induced genes in RCC10 cells. (C) RT-qPCR analysis of TNFSF10 , TNFRSF10A , TNFRSF10B , and TNFRSF10D mRNA levels in RCC10 cells treated with vehicle or SGI1027 for 2 days ( n = 3 biological replicates). (D and E) Immunoblot analysis of TRAIL, DR4, DR5, DcR2, pro-caspase-10, and cleaved caspase-10 (C-caspase-10) proteins in isogenic RCC10 cells treated with vehicle, SGI1027 (D and E, n = 2 biological replicates), MS1129 (E, n = 2 biological replicates), or decitabine (E, n = 2 biological replicates) for 2 or 7 days. (F) Global m5C levels in RCC10 cells treated with vehicle or SGI1027 for 2 days by ELISA assay ( n = 3 biological replicates). (G–J) MeDIP-qPCR assay in RCC10 cells treated with vehicle or SGI1027 for 2 days ( n = 3 biological replicates). (K–M) DNMT1, DNMT3A, and DNMT3B ChIP-qPCR assay in RCC10 cells ( n = 3 biological replicates). (N) Scheme of dDNMT-activated apoptotic pathway. Data represent mean ± SEM. p value was determined by bioinformatics with edgeR (A) or gene set enrichment analysis (B), unpaired 2-tailed Student’s t test (C and F), two-way ANOVA with Tukey’s test (G–I), and one-way ANOVA with Dunnett’s test (K–M). See also and .

Article Snippet: The following antibodies were used: anti-HIF-1α antibody (A300-286A, Bethyl Laboratories), anti-HIF-2α antibody (home-made), anti-DNMT1 antibody (24206-1-AP, Proteintech), anti-DNMT3A antibody (20954-1-AP, Proteintech), anti-DNMT3B antibody (26971-1-AP, Proteintech), anti-DNMT3L antibody (sc-393603, Santa Cruz), anti-cleaved-caspase-3 antibody (9661S, Cell Signaling Technology), anti-cleaved-caspase-7 antibody (8438S, Cell Signaling Technology), anti-PARP1 antibody (9542S, Cell Signaling Technology), anti-caspase-10 antibody (14311-1-AP, Proteintech), anti-caspase-8 antibody (13423-1-AP, Proteintech), anti-TRAIL antibody (27064-1-AP, Proteintech), anti-DR4 antibody (24063-1-AP, Proteintech), anti-DR5 antibody (69400S, Cell Signaling Technology), anti-DcR2 antibody (16781-1-AP, Proteintech), anti-β-actin (81115-1-RR, Proteintech), and anti-α-tubulin (66031-1-Ig, Proteintech).

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Methylated DNA Immunoprecipitation, ChIP-qPCR

Journal: Cell Reports Medicine

Article Title: HIF-activated priming of TRAIL-induced cell death determines epigenetic vulnerability in kidney cancer

doi: 10.1016/j.xcrm.2026.102630

Figure Lengend Snippet: dDNMT specifically kills patient-derived VHL -deficient ccRCC in mice (A) Tumor growth curves of VHL -deficient UTSW-PDX206, UTSW-PDX258, UTSW-PDX490, and UTSW-PDX26 in mice treated with vehicle (Veh) or SGI1027 for 10 days. (B) Tumor growth curves of VHL -WT UTSW-PDX416 and UTSW-PDX143 in mice treated with vehicle or SGI1027 for 10 days. (C) Kaplan-Meier survival curve of UTSW-PDX490-bearing mice ( n = 10 biological replicates). (D and E) Global m5C levels in UTSW-PDX206 (D) or UTSW-PDX258 (E) tumors harvested from mice after treatments by ELISA assay ( n = 5 biological replicates). (F) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, TRAIL, DR4, DR5, procaspase-10, C-caspase-3, and C-caspase-7 proteins in UTSW-PDX258 tumors harvested from mice after treatments ( n = 5 biological replicates). (G) Representative C-caspase-3 IHC in UTSW-PDX258 tumors. Scale bar, 100 μm. (H) Quantification of C-caspase-3-positive cells in (G) ( n = 5 biological replicates). (I) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, TRAIL, DR4, DR5, C-caspase-3, and VHL proteins in UTSW-PDX416 tumors harvested from mice after treatments ( n = 5 biological replicates). (J) Immunoblot analysis of DNMT1, DNMT3A, DNMT3B, and procaspase-10 proteins in UTSW-PDX206, UTSW-PDX258, and UTSW-PDX26 tumors ( n = 4–5 biological replicates). Data represent mean ± SEM. p value was determined by two-way ANOVA with Tukey’s test (A), log rank test (C), and unpaired 2-tailed Student’s t test (D, E, and H). See also and ; .

Article Snippet: The following antibodies were used: anti-HIF-1α antibody (A300-286A, Bethyl Laboratories), anti-HIF-2α antibody (home-made), anti-DNMT1 antibody (24206-1-AP, Proteintech), anti-DNMT3A antibody (20954-1-AP, Proteintech), anti-DNMT3B antibody (26971-1-AP, Proteintech), anti-DNMT3L antibody (sc-393603, Santa Cruz), anti-cleaved-caspase-3 antibody (9661S, Cell Signaling Technology), anti-cleaved-caspase-7 antibody (8438S, Cell Signaling Technology), anti-PARP1 antibody (9542S, Cell Signaling Technology), anti-caspase-10 antibody (14311-1-AP, Proteintech), anti-caspase-8 antibody (13423-1-AP, Proteintech), anti-TRAIL antibody (27064-1-AP, Proteintech), anti-DR4 antibody (24063-1-AP, Proteintech), anti-DR5 antibody (69400S, Cell Signaling Technology), anti-DcR2 antibody (16781-1-AP, Proteintech), anti-β-actin (81115-1-RR, Proteintech), and anti-α-tubulin (66031-1-Ig, Proteintech).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: A variety of ER stressors alter DR5 disulfide bonding. A, upper panel. ERp44-deficient HepG2 cells into which vector, wild type or catalytically null ERp44 were reintroduced were treated as indicated for 24 h and analyzed by non-reducing immunoblot. A, lower panel. Expanded region of the DR5 immunoblot showing altered DR5 disulfide bonding in the Thapsigargin/PERKi combination treatment. B. Non-reducing immunoblot of MDA-MB-468 cells treated as indicated for 24 h. C. MDA-MB-468 cells were treated as indicated for 24 h and subjected to non-reducing (DR5, Cleaved Caspase 3, DR4, Actin) or reducing (MET) immunoblot analysis. D. Protein synthesis assays of cells pre-treated for 24 h as indicated before protein synthesis was measured by 3 H-Leucine incorporation over a 2 h pulse. Data are plotted as the average (N = 6), with error bars representing standard deviation. E. HepG2 cells were treated as indicated for 24 h and subjected to non-reducing (DR5, Cleaved Caspase 3, DR4, Actin) or reducing (MET) immunoblot analysis. F. Control and PERK knockout HepG2 cells were treated for 24 h as indicated and subjected to non-reducing immunoblot analysis. G. Non-reducing immunoblot analysis of the indicated cell lines treated as specified for 24 h. Note the DR5 oligomerization in A431 cells when Thapsigargin, Tunicamycin, or dFtcyDTDO are combined with PERK inhibition. H. Non-reducing immunoblot analysis of WM793 cells treated as specified for 24 h. Unless otherwise specified, the following concentrations of compounds were used in A-H above: dFtcyDTDO (2.5 μM), Thapsigargin (400 nM), Tunicamycin (500 ng/ml), Cyclosporine A (10 μM), Dithiothreitol (DTT; (2.5 mM)), ISRIB (200 nM), or PERKi (1μM). O and M represent Oligomeric and Monomeric protein isoforms in panels A, C, E, G, and H.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Plasmid Preparation, Western Blot, Standard Deviation, Control, Knock-Out, Inhibition

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: Genetic disruption of multiple DR5 disulfide bonds induces its stabilization and pro-apoptotic signaling. A. Structural model of DR5 showing its disulfide bonds, and the positive patch autoinhibitory domain described in the literature. B. Non-reducing immunoblot analysis of MDA-MB-468 cells engineered with doxycycline-inducible expression of wild type (WT) DR5 or the indicated Cys to Ser disulfide bond mutants. Cells were treated as indicated for 24 h with 1 μg/ml doxycycline and 2.5 μM dMtcyDTDO. The red arrow denotes DR4 oligomers that coincide with DR5 oligomerization. C. Reducing immunoblot analysis of the indicated MDA-MB-468 stable cell lines. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 10 μM Q-VD-OPH. The catalog numbers of DR5 and DR4 antibodies are shown. D. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type DR4 and DR5, and DR4 and DR5 C-terminal deletion constructs defective in apoptotic signaling. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. E. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type and apoptosis-defective DR5. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. F. Non-reducing immunoblot analysis of the indicated MDA-MB-468 doxycycline-inducible stable cell lines. Cells were treated for 24 h as indicated. G. Non-reducing immunoblot analysis of the indicated MDA-MB-468 cell lines with doxycycline-inducible expression of wild type and apoptosis-defective DR5. Cells were treated for 24 h as specified with 1 μg/ml doxycycline or doxycycline + 2.5 μM dMtcyDTDO. O and M represent Oligomeric and Monomeric protein isoforms in panels B and D-G.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Disruption, Western Blot, Expressing, Stable Transfection, Construct

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: DDAs upregulate TRAIL Decoy Receptor 2, an effect overridden by Cyclosporine A. A. Sequence alignment of the putative autoinhibitory motifs of DR4, DR5, DCR1, and DCR2. B. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle, 5 μM Cyclosporine A or 100 nM FK506. C. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle or 5 μM Cyclosporine A. D. Non-reducing immunoblot analysis of MDA-MB-468 cells treated for 24 h with the indicated concentrations of dFtcyDTDO, combined with DMSO vehicle or 5 μM Cyclosporine A. Data are plotted as the average (N = 3), with error bars representing standard error. Asterisks denote p < 0.05 compared to control using Student’s unpaired t -test. E. Densitometry analysis of the relative levels of total, monomeric, and oligomeric forms of DR5 (left panel) or total levels of Cyclophilin B or DCR2 (right panel) from panels 7B-D. O and M represent Oligomeric and Monomeric protein isoforms in panels B-D.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Sequencing, Western Blot, Control

Journal: bioRxiv

Article Title: DR5 disulfide bonding as a sensor and effector of protein folding stress

doi: 10.1101/2024.03.04.583390

Figure Lengend Snippet: Effects of altered disulfide bonding on DR5 cell surface localization and antibody recognition. A. Flow cytometry analysis of the indicated doxycycline-inducible MDA-MB-468 stable cell lines with an antibody to DR5 (Clone DJR2-4 (7-8)) (top panel) or DR4 (bottom panel). Prior to analysis, cells were treated for 24 h as indicated with 10 μM Q-VD-OPH, 1 μg/ml doxycycline, or 2.5 μM dFtcyDTDO. Dots represent the average values from three independent biological replicates performed in triplicate. * Represents p < 0.05, **** represents p < 0.0001, and ns represents not significant (p > 0.05). B. Non-reducing immunoblot analysis of the indicated MDA-MB-468 stable cell lines treated for 24 h as specified. Note the alternate staining patterns observed with different DR5 antibodies. O and M represent Oligomeric and Monomeric protein isoforms. C. The indicated MDA-MB-468 stable cell lines were treated for 24 h as indicated with 1 μg/ml doxycycline and 2.5 μM dFtcyDTDO and subjected to cell surface protein biotin labeling. Cell surface proteins (External; Ext.) were affinity purified with Streptavidin-agarose, and the unlabeled flow-through (Internal; Int.) proteins were also collected. Both fractions were analyzed by non-reducing immunoblot using the indicated antibodies. D. Cell surface protein labeling experiment as in panel C except that cell lines were treated with the indicated combinations of 1 μg/ml doxycycline, 2.5 μM dFtcyDTDO, and 10 μM Cyclosporine A.

Article Snippet: In order to construct the Tet-DR4 expression vector, DR4 (Addgene plasmid #61382) was amplified using the following primers: 5′-TTTTATCGATCACCATGGCGCCCGTCGCCGTCTGG-3′ and 5′-TTTTGGATCCTCACTCCAAGGACACGGCAG-3′ and cloned into the pRetroX-TetOne-Puro vector with a modified cloning site that incorporates Not I, Bcl I, and Cla I sites 5′ to the BamH I site (Clontech, Mountain View, CA, USA).

Techniques: Flow Cytometry, Stable Transfection, Western Blot, Staining, Labeling, Affinity Purification

Journal: Cell reports

Article Title: Functional genomics identifies N -acetyllactosamine extension of complex N -glycans as a mechanism to evade lysis by natural killer cells

doi: 10.1016/j.celrep.2024.114105

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-human TRAIL-R1 PE , BD Biosciences , 564180, clone S35-934; RRID: AB_2738648.

Techniques: Virus, Cell Isolation, Recombinant, Plasmid Preparation, Selection, CRISPR, Knock-Out, Software