Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) mRNA was extracted from WT or IRE1 DN-expressing U87 cells and from empty vector (EV), IRE1WT- or IRE1Q780*-expressing RADH85 and RADH87 cells. CD95 mRNA was quantified by RT-qPCR and normalized to GAPDH. Mean ± SEM, n = 3–5. ( B ) CD95 cell surface level expression was evaluated by flow cytometry. Mean of MFI ratio ± SEM, n = 3–4. Unpaired t -test for U87 (** p = 0.001126); one-way ANOVA with Tukey multiple comparison correction for RADH85 (* p = 0.018416 and ** p = 0.003549) and RADH87 (* p = 0.0263 and ** p = 0.0054). ( C , D ) U87 cells pre-treated for 2 h with MKC-8866 (30 μM) as indicated were further treated with 500 ng/mL tunicamycin for the indicated times. Lysates were analysed using western blot. ( C ) One representative experiment out of three independent ones is shown. ( D ) Quantification for three independent experiments is depicted. Mean ± SEM. ( E , F ) U87 cells pre-treated for 2 h with MKC-8866 (30 μM) as indicated were further treated with 50 nM thapsigargin for the indicated times. Lysates were analysed using western blot. ( E ) One representative experiment out of three independent ones is shown. ( F ) Quantification for three independent experiments is depicted. Mean ± SEM. .

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Flow Cytometry, Comparison, Western Blot

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) CD95 protein level was evaluated using western blot on lysates from the indicated cells. One representative experiment out of three independent experiments is presented. ( B ) U87 or SUM159 cells were pre-incubated for 1 h with 1 μg/mL of actinomycin D and further treated with 10 μM MKC-8866 for 1 h followed by 2 h treatment with 10 μM MG-132 as indicated. CD95 mRNA expression level, normalized to GAPDH, was expressed as fold of value obtained for control (actinomycin-only treated samples). Mean ± SEM, n = 3–4. Unpaired t -test (for comparing MG-132 and MG-132 + MKC-treated group), **** p = 0.0003. ( C , D ) U87 or SUM159 cells were pre-incubated for 1 h with 1 μg/mL of actinomycin D and further treated with 10 μM MKC-8866 or 10 μM Z4 for 1 h followed by 2 h treatment with 1μg/mL tunicamycin as indicated. CD95 mRNA expression level, normalized to GAPDH, was expressed as fold of value obtained for control (actinomycin-only treated samples). Mean ± SEM, n = 3–4. Unpaired t -test (for comparing TM and TM + MKC groups for ( C ) or TM and TM + Z4 groups for ( D )), ( C ) * p = 0.0461 for U87, * p = 0.0437 for SUM159, ( D ) * p = 0.0241 for U87, (ns, p = 0.0538 for SUM159).

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques: Western Blot, Incubation, Expressing, Control

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) RNA (2 μg) extracted from U87 cells was incubated with the indicated amounts of recombinant IRE1 for 1 h. CD95 mRNA was then quantified by RT-qPCR and normalized to GAPDH. Mean ± SEM, n = 3. One-way ANOVA with Dunnett multiple comparison correction, *** p = 0.0003 (0 vs 0.5 μg IRE1 groups), *** p = 0.0007 (0 vs 1 μg IRE1 groups) ( B ) Predicted folded structure of CD95 mRNA. The two predicted cleavage sites within hairpin loops are highlighted. ( C , D ) RNA (2 μg) extracted from U87 cells was incubated with the indicated amounts of recombinant IRE1 for 1 h. 136-bp ( C ) and 121-bp ( D ) parts of CD95 mRNA including the indicated potential cleavage sites were then quantified by RT-qPCR and normalized to GAPDH. Mean ± SEM, n = 3. One-way ANOVA with Dunnett multiple comparison correction, ( D ) * p = 0.0331, ** p = 0.0096. .

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques: Incubation, Recombinant, Quantitative RT-PCR, Comparison

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) U87 were transfected with siRNA control or targeting CD95. 48 h later, cells were treated for 48 h with the indicated ER stress inducers. Viability was determined using an MTT assay. The relative IC50 calculated for each independent experiment is represented (see also Appendix Fig. S ). Mean ± SEM, n = 3–4. ** p = 0.013, unpaired t -test ( B ). U87 WT or expressing IRE1DN were treated with 1 μg/mL CD95L for 12 h. % of cell death was defined as the % of Cytotox red-positive cells as detected by the Incucyte. Mean ± SEM of three independent experiments. ( C ) U87 WT or expressing IRE1DN were treated with 250 ng/mL CD95L for the indicated times. Lysates were analysed using western blot. One experiment representative of three independent ones is shown. ( D ) RADH85 control (EV), stably expressing IRE1Q780* or IRE1WT were treated with 500 ng/mL CD95L for 12 h. % of cell death was defined as the % of Cytotox red-positive cells as detected by the Incucyte. Mean ± SEM of three independent experiments. ( E ) Empty vector (EV), IRE1WT- or IRE1Q780*-expressing RADH85 were treated with 500 ng/mL CD95L for the indicated times. The DISC was immunoprecipitated using an anti-CD95 antibody prior to western blot analysis. One experiment representative of two independent ones is shown. *Indicates an unspecific band. .

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques: Transfection, Control, MTT Assay, Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Immunoprecipitation

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) MDA-MB-231 WT or CD95 KO clones were treated for 48 h with the indicated ER stress inducers. Viability was determined using an MTT assay and relative IC50 calculated for each independent experiment (see also Appendix Fig. ). ** p = 0.044, *** p = 0.0002, one-way ANOVA with Tukey multiple comparison correction. ( B ) RADH87 control (EV), stably expressing IRE1Q780* or IRE1WT were pre-treated with 200 nM (2X) of Birinapant for 1 h prior to addition of 1 μg/mL CD95L for 24 h. % of cell death was defined as the % of Cytotox red-positive cells as detected by the Incucyte. Mean ± SEM of three independent experiments.

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques: Clone Assay, MTT Assay, Comparison, Control, Stable Transfection, Expressing

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) Timeline of in vivo experiment 1. Eight mice were divided in two groups of 4 and repeatedly injected as indicated with either vehicle (group 1) or MKC-8866 (group 2). ( B ) IHC staining for CD95 on liver tissue from the two groups of mice described in A. Left: quantification of CD95 staining. Mean ± SEM of n = 4 mice per group; * p = 0.0286, with Mann–Whitney test for comparison of the two groups. Right: representative IHC image for each group. ( C ) IHC staining for cleaved caspase-3 on liver tissue from the two indicated groups of mice described in Fig. . Left: quantification of cleaved caspase-3 staining. Mean ± SD of n = 10 mice per group; five images per liver. **** p = 0.000043 Mann–Whitney test for comparison of the two indicated groups. Right: representative IHC image for each group. .

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques: In Vivo, Injection, Immunohistochemistry, Staining, MANN-WHITNEY, Comparison

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) Timeline of the second in vivo experiment. 40 mice were divided in two groups of 20 and were repeatedly injected with either vehicle or MKC-8866 as indicated. On day 2 at 12 pm, each of this initial groups were further divided in two groups of 10 mice which were injected with either an anti-CD95 antibody or with an isotype control as indicated. ( B ) CD95 expression was evaluated by IHC in mice injected with vehicle or MKC-8866 and the isotype control antibody. One representative image is shown for each of these two groups. ( C ) HES staining was performed on liver tissue sections from mice of each of the four groups described in ( A ). One representative image is shown for each of these groups. ( D ) Western blot analysis of liver lysates from mice treated as indicated.

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques: In Vivo, Injection, Control, Expressing, Staining, Western Blot

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) U87 cells were transfected with siRNA control or targeting XBP1, IRE1 or PERK as indicated. 16 h later, cells were treated with DMSO (D) or 250 nM thapsigargin (TG) for 8 h. Lysates were analysed using western blot. One experiment representative of at least three independent ones is shown. ( B ) U87 cells were transfected with a plasmid coding for FLAG-XBP1s (XBP1s) or an empty vector (EV). 48 h later, cells were treated with the indicated concentrations of CD95L for 48 h. Viability was assessed using MTT assay and normalized to untreated cell values. Mean ± SEM of three independent experiments. Inset: western blot analyses of cell lysates 48 h post-transfection. ( C ) U87 cells were treated with DMSO or MKC-8866 (30 mM) for the indicated times. Lysates were analysed using western blot. One experiment representative of three independent ones is shown. ( D ) U87 cells treated with 30μM MKC-8866 or DMSO were further treated with 1 µg/mL CD95L for the indicated times. Cell death was evaluated using Cytotox red positivity. Mean ± SEM, n = 3. ( E – G ) U87 were transfected with siRNA control or targeting XBP1, IRE1 or PERK as indicated. 72 h later, cell lysates were analysed using western blot. One experiment representative of at least three independent ones is shown. ( H , I ) CD95 expression z-scores of 45 GB and 62 TNBC tumours were plotted according to the RIDD activity score ( H ) and according to the XBP1s activity score ( I ). The distribution of z-score is represented as violin plots. For GB n = 45; for TNBC n = 62. Statistical difference of expression between groups was calculated using Mann–Whitney tests and the p-value is indicated ( H GB *** p = 4.3e−05, TNBC *** p = 4.1e−06; I GB** p = 0.0025, * TNBC p = 0.015). .

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, MTT Assay, Expressing, Activity Assay, MANN-WHITNEY

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: RT-qPCR primers.

Article Snippet: Then, samples were saturated with PBS containing 1% BSA and 1% FCS and human FcR-blocking reagent (Miltenyi Biotec, 130-059-901) for 15 min at 4 °C prior to labelling using an anti-human CD95-APC antibody (clone DX2, Miltenyi Biotec, 130-117-701, AB_2751411) or corresponding IgG1-APC isotype (clone IS5-21F5, Miltenyi Biotec, 130-113-196, AB_2733440) for 30 min at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A CD95L can exist either in a membrane-bound or in a soluble form, the latter resulting from protease cleavage. Key domains include the CD95 binding domain (CD95 Bind), the TNF homology domain (THD) for its homotrimerization, and the proline-rich domain (PRD). B Workflow of the CD95L-IZ construct design, secretory expression, affinity purification, and modification. The recombinant protein consists of a signaling peptide (Sig), an affinity tag (Tag), a cleavage site (Clv), a Cysteine (Cys), an isoleucine zipper domain (IZ) fused via a linker to the extracellular CD95L domain. In method 1 transient CD95-IZ expression was used. Here, a His-tag was used as Tag and a TEV protease sequence as Clv. Affinity purification was done by anti-His-tag antibody coupled agarose beads. In method 2 a cell line stably expressing CD95-IZ was used. Here, a Twin-Strep-Tag was used as Tag and a PreScission cleavage site as Clv. Affinity purification was done by Strep-TactinXT4Flow beads. C Upper panel: examination of cell morphology before and up to three days after transfection. Lower panel: photos of cell culture and transfection using a 10-layer cell factory in a cell culture hood (left) or incubator (right). A T175 cell culture flask was seeded with the same cell density as in a 10-layer cell factory to check the cell condition and confluency. D CD95L-IZ protein expression and secretion was verified by a Western blot of the cell culture medium using anti-CD95L antibody. The image contrast was adjusted for better visibility.

Article Snippet: Apoptosis dynamics assays were performed to demonstrate the killing efficiency of CD95L-IZ-X (CD95L-IZ-His/CD95L-IZ/CD95L-IZ-biotin) and its inhibition by CD95-His (Sino Biological) or enhancement by anti-His-tag antibody (BioLegend).

Techniques: Membrane, Binding Assay, Construct, Expressing, Affinity Purification, Modification, Recombinant, Sequencing, Stable Transfection, Strep-tag, Transfection, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A ELISA analysis of CD95L-IZ-biotin and CD95L-Enzo binding to CD95. B Comparison of representative bright field images showing morphological changes of cells treated with CD95L-IZ-X or soluble CD95L (Enzo Life Science) after 2, 6, and 15 h. Red asterisks indicate the position of apoptotic cell. Scale bars are 50 µm. C Apoptosis dynamics analysis of CD95L-IZ apoptosis induction at different stages: post-purification, post-His-tag cleavage, post-biotinylation, and comparison with CD95L-Enzo. D The efficiency of CD95L-IZ-His apoptosis induction is increased by anti-His-tag antibody crosslinking. E The presence of CD95-His (in 10x, 50x, 100x, and 200x molar excess in comparison to CD95L-IZ-biotin) as competitive binders to the natural CD95 on the cell membrane, successively inhibits CD95L-IZ-biotin apoptosis induction. In panels C, D, and E data points are marked with dots. Hill equation fit curves are represented by solid lines, and standard deviations are indicated with shaded regions.

Article Snippet: Apoptosis dynamics assays were performed to demonstrate the killing efficiency of CD95L-IZ-X (CD95L-IZ-His/CD95L-IZ/CD95L-IZ-biotin) and its inhibition by CD95-His (Sino Biological) or enhancement by anti-His-tag antibody (BioLegend).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, Purification, Membrane

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A CD95L can exist either in a membrane-bound or in a soluble form, the latter resulting from protease cleavage. Key domains include the CD95 binding domain (CD95 Bind), the TNF homology domain (THD) for its homotrimerization, and the proline-rich domain (PRD). B Workflow of the CD95L-IZ construct design, secretory expression, affinity purification, and modification. The recombinant protein consists of a signaling peptide (Sig), an affinity tag (Tag), a cleavage site (Clv), a Cysteine (Cys), an isoleucine zipper domain (IZ) fused via a linker to the extracellular CD95L domain. In method 1 transient CD95-IZ expression was used. Here, a His-tag was used as Tag and a TEV protease sequence as Clv. Affinity purification was done by anti-His-tag antibody coupled agarose beads. In method 2 a cell line stably expressing CD95-IZ was used. Here, a Twin-Strep-Tag was used as Tag and a PreScission cleavage site as Clv. Affinity purification was done by Strep-TactinXT4Flow beads. C Upper panel: examination of cell morphology before and up to three days after transfection. Lower panel: photos of cell culture and transfection using a 10-layer cell factory in a cell culture hood (left) or incubator (right). A T175 cell culture flask was seeded with the same cell density as in a 10-layer cell factory to check the cell condition and confluency. D CD95L-IZ protein expression and secretion was verified by a Western blot of the cell culture medium using anti-CD95L antibody. The image contrast was adjusted for better visibility.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Membrane, Binding Assay, Construct, Expressing, Affinity Purification, Modification, Recombinant, Sequencing, Stable Transfection, Strep-tag, Transfection, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: High yield purification of an Isoleucine zipper modified CD95 Ligand with either biotin or DNA-oligomer binding domain for efficient Cell Apoptosis Induction

doi: 10.1101/2024.10.12.618001

Figure Lengend Snippet: A ELISA analysis of CD95L-IZ-biotin and CD95L-Enzo binding to CD95. B Comparison of representative bright field images showing morphological changes of cells treated with CD95L-IZ-X or soluble CD95L (Enzo Life Science) after 2, 6, and 15 h. Red asterisks indicate the position of apoptotic cell. Scale bars are 50 µm. C Apoptosis dynamics analysis of CD95L-IZ apoptosis induction at different stages: post-purification, post-His-tag cleavage, post-biotinylation, and comparison with CD95L-Enzo. D The efficiency of CD95L-IZ-His apoptosis induction is increased by anti-His-tag antibody crosslinking. E The presence of CD95-His (in 10x, 50x, 100x, and 200x molar excess in comparison to CD95L-IZ-biotin) as competitive binders to the natural CD95 on the cell membrane, successively inhibits CD95L-IZ-biotin apoptosis induction. In panels C, D, and E data points are marked with dots. Hill equation fit curves are represented by solid lines, and standard deviations are indicated with shaded regions.

Article Snippet: Protein samples of 100 ng CD95-His incubated with 100 ng CD95L-IZ-biotin at 37 °C for 1 h, or 100 ng CD95-His alone (Sino Biological, Beijing, China) were loaded with 10 % glycerol into 10 µl loading volume.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Comparison, Purification, Membrane