Journal: Frontiers in Cell and Developmental Biology

Article Title: Human placenta-derived mesenchymal stem cells stimulate neuronal regeneration by promoting axon growth and restoring neuronal activity

doi: 10.3389/fcell.2023.1328261

Figure Lengend Snippet: Characterization of hPMSCs. (A) Morphology at passage 3. Both normoxic and hypoxic cultures show highly homogeneous spindle-like shape. Scale bar: 500 µm (B) Proliferation capacity. Cumulative population doublings of hPMSCs grown under hypoxia (square) are higher than cultures grown in normoxia (dot). (C) Flow cytometry shows specific hMSCs marker expression pattern: positive for CD105, CD44, CD90, CD73 and negative for CD14, CD19, CD34, CD45 and HLA-DR. (D) Osteogenic differentiation was confirmed by Alizarin red staining of calcium deposits. (E) Adipogenesis differentiation was followed by Oil Red O staining of lipids vacuoles (black arrows indicate lipid droplets). (F) Alcian Blue staining of proteoglycans demonstrated chondrogenesis differentiation. Scale bar: 100 μm. Two-way ANOVA and post hoc Sidak test for multiple comparisons between means (** p ≤ 0.005; * p ≤ 0.05).

Article Snippet: Antibodies against the following human antigens were used: CD105-FITC (Miltenyi Biotect, Bergisch Gladbach, Germany, cat# 130-112-327, 1:50), CD90-FITC (Miltenyi Biotec, cat# 130-114-901, 1:50), CD44-VioBlue (Miltenyi Biotec, cat# 130-113-906, 1:50), CD73-APC (Miltenyi Biotec, cat# 130-111-909, 1:50), MSC Phenotyping Cocktail-PE (CD34, CD14, CD19, CD45, Miltenyi Biotec cat# 130-125-285, dilution according to the manufacturer’s instructions).

Techniques: Flow Cytometry, Marker, Expressing, Staining

Journal: Materials Today Bio

Article Title: Developing immune-regulatory materials using immobilized monosaccharides with immune-instructive properties

doi: 10.1016/j.mtbio.2020.100080

Figure Lengend Snippet: Flow cytometry data are shown as a heat map summarizing the expression profile of three surface markers (CD86, CD40, and CD274) on DCs after incubation on immobilized monosaccharide libraries in the presence of a TLR4 ligand (LPS). Unstimulated (immature) DCs were used as a negative control. All changes are expressed as a percentage of expression levels in mature (LPS-stimulated) DCs. A reduction in CD40 expression was observed in most conditions with the highest reduction in fucose-contained libraries compared to LPS alone. CD86 expression was higher in a number of conditions (particularly mixers of Gal1 and Gal2); however, these did not reach statistical significance. CD274 (PD-L1) expression showed a significant increase in Man1 and Gal2 combinations. Data are shown as mean ± SD of three independent donors where ∗ p <0.05, ∗∗ p <0.01, ∗∗∗ p <0.001, and ∗∗∗∗ p <0.0001.

Article Snippet: DCs were then incubated with labeled antibodies CD274 (APC clone REA1197), CD40 PE (clone HB14), and CD86 FITC (clone FM95) and isotype-matched mouse antibody controls (all from Miltenyi Biotec) for 20 min in the dark at 4 °C.

Techniques: Flow Cytometry, Expressing, Incubation, Negative Control

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: