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: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). Heat map illustrating differential expression data from RNA-Seq analysis. Heatmap of FASLG gene expression, represented as fold changes in mRNA levels, was generated for the ARHL (n = 12) and NH (n = 12); (B). Expression of FASLG was analyzed using RT-qPCR and confirmed through WB (NH: n = 12, ARHL: n = 14). (C). Expression of miR-5195 was detected using RT-qPCR in WB (NH: n = 6, ARHL: n = 5). (D). Expression of miR-3941 was detected using RT-qPCR in WB (NH: n = 14, ARHL: n = 12). U6 snRNA was used as an internal control (** p < 0.01). Abbreviations used are as follows: Normal hearing group (NH), Age-related hearing loss (ARHL), Whole blood (WB), Fas ligand ( FASLG ).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Quantitative Proteomics, RNA Sequencing, Gene Expression, Generated, Expressing, Quantitative RT-PCR, Control

Journal: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). Identification of 3′-UTR sequences in the FASLG gene predicted to include binding sites for miR-3941; (B). Identification of 3′-UTR sequences in the FASLG gene predicted to harbor binding sites for miR-5195-5p; (C). Dual luciferase reporter assay results for miR-3941, miR-5195, and their corresponding target 3’-UTR of FASLG in HEI-OC-1 cells; (D). Dual luciferase assay results of miR-3941, miR-5195 and their corresponding target 3’-UTR of FASLG in HeLa cell; The significant differences between FASLG and the miRNA target group are represented as ** p < 0.01. Abbreviations used are as follows: Fas ligand ( FASLG ), House Ear Institute-Organ of Corti 1 (HEI-OC-1).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Binding Assay, Luciferase, Reporter Assay

Journal: PLOS One

Article Title: Differential expression and regulation of FASLG by miR-5195/miR-3941 in age-related hearing loss

doi: 10.1371/journal.pone.0331661

Figure Lengend Snippet: (A). The light blue rectangular node represents the FASLG gene, while the light blue circular nodes represent the 35 selected miRNAs. The nodes with red text indicate the two miRNAs of interest, miR-3941 and miR-5195-5p. The arrows connecting FASLG to the miRNAs indicate that the thickness of the arrow corresponds to the context++ score of the miRNA, with thicker arrows representing lower scores. (B). We utilized blood samples from individuals with ARHL and found that A notable rise in FASLG expression was observed. Additionally, we identified miR-3941 and miR-5195 as potential regulators of FASLG expression. These miRNAs may bind to the 3’ UTR of the elevated FASLG , leading to down-regulation of gene expression. Abbreviations used are as follows: Fas ligand ( FASLG ), Age-related hearing loss (ARHL), Fas receptor (FAS-R), 3’untranslated region (3’UTR).

Article Snippet: For the transfection experiments, the FASLG Human 3’ UTR Clone (#SC210991, OriGene, Rockville, Maryland, USA), miR-5195 (HmiR1468 – MR04, Genecopoeia, Rockville, Maryland, USA), and miR-3941 (HmiR1052 – MR04, Genecopoeia, Rockville, Maryland, USA) were used.

Techniques: Expressing, Gene Expression

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Zinc modulates cytokine-induced lung epithelial cell barrier permeability.

doi: 10.1152/ajplung.00207.2006

Figure Lengend Snippet: Fig. 1. Zinc depletion enhances extrinsic apoptosis and barrier rupture. Fully differentiated cultures of primary human upper airway epithelia were placed in serum-free, zinc-deprived, culture medium for 48 h and than exposed to interferon (IFN)-, tumor necrosis factor (TNF)-, a Fas cross-linking anti- body (FasAb), or a combination of the three (ITF) for an additional 48 h. The zinc chelator N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) was added during the last 4 h of the cytokine treatment period. Apoptosis was determined by M30 staining (A) and compared across individual cytokines and TPEN. Under similar conditions, a combination of ITF increasing concen- trations of TPEN was also evaluated for cellular apoptosis (B). Results are representative of experiments conducted on 3 separate donors.

Article Snippet: In preparation of all experiments, unless indicated, the cells were cultured under serum-free conditions for 48 h followed by the addition of 250 U/ml interferon (IFN)(catalog no. PHC 4031, BioSource, Camarillo, CA), 100 ng/ml tumor necrosis factor (TNF)- (Knoll Pharmaceuticals, Whippany, NY), and/or 200 ng/ml anti-human CD95 (FasAb) (catalog no. MAB 142, R&D Systems, Minneapolis, MN) for an additional 48 h. To achieve zinc deprivation, cultures were grown in nonzinc-supplemented DMEM 48 h before cytokine addition.

Techniques: Staining