Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Apoptosis induction and change in HO activity after treatment with ZnPP IX of AH136B solid. Apoptosis ( A, B ) and HO activity ( C ) were assessed on day 14 after tumour implantation. Each specimen was analysed via TUNEL staining 24 h after treatment with vehicle (the control) ( A, a ), with ZnPP IX (500 μ g kg −1 i.a.) ( A, b ), or with CuPP IX (500 μ g kg −1 i.a.) ( A, c ). Quantitative analysis of TUNEL-positive cells in each specimen is shown ( B ). TUNEL-positive cells were counted in four different fields of magnification at × 100 per sample, and then the number of positive cells per mm 2 was calculated. Haem oxygenase activity of the solid tumour was measured after treatment in the same manner as in the TUNEL analysis ( C ). ** P <0.05, ** P <0.01 vs control ( n =3 for each group). Data are means±s.e. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Activity Assay, TUNEL Assay, Staining, Control

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Zinc protoporphyrin IX-induced apoptosis of AH136B cells in vitro . AH136B cells were incubated for 24 h with indicated concentrations of ZnPP IX, with or without caspase-3 inhibitor ( A ) or SnPP IX ( B ), or with 100 μ M CuPP IX. ( A, B ) TUNEL-positive cells were counted in four different fields of magnification at × 100 per sample, and then the percentage of positive cells was calculated. ** P <0.05 vs control ( n =3 for each group). Cell viability after the same treatment is shown in the inset. ** P < 0.05, ** P <0.01 vs control ( n =4 for each group). Data are means±s.e. ( C ) Representative TUNEL staining of control and ZnPP IX-treated tumour cells. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: In Vitro, Incubation, TUNEL Assay, Control, Staining

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Effect of ZnPP IX on HO activity and caspase-3 activity of AH136B cells cultured in vitro . Haem oxygenase activity was measured 24 h after incubation with vehicle (control), ZnPP IX, or CuPP IX ( A ). Similarly, cells were treated with 100 μ M ZnPP with or without caspase-3 inhibitor (1 or 10 μ M ) for 24 h ( B ). Caspase-3 activity was measured fluorome-trically in cell extracts by using a fluorescent substrate. ** P <0.05, ** P <0.01 vs control ( n =3 for each group). Data are means±s.e. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Activity Assay, Cell Culture, In Vitro, Incubation, Control

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Protective effect of bilirubin against ZnPP IX-induced apoptosis of AH136B cells. AH136B cells were incubated for 24 h with 100 μ M ZnPP IX in the presence or absence of indicated concentrations of bilirubin. TUNEL-positive cells were counted in four different fields of magnification at × 100 per sample, and then the percentage of positive cells was calculated. ** P <0.05 vs ZnPP IX alone ( n =3 for each group). Data are means±s.e. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Incubation, TUNEL Assay

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Tumour blood flow after ZnPP IX or L -NAME treatment. Tumour blood flow was measured in AH136B solid tumour on day 14 after tumour implantation. At the indicated times after injection of 500 μ g kg −1 ZnPP IX (i.a.) or 6 mg kg −1 L -NAME (i.p.), a laser Doppler flowmeter and a probe needle were used to measure blood flow. Relative changes in tumour blood flow after injection of ZnPP IX or L -NAME are shown. Inset, change in tumour blood flow during early period after treatment (within 25 min). ** P <0.01 by ANOVA between the groups ( n =3 for each time point). Data are means±s.e. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Injection

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Western blot analysis of HSP70 and HO-1 proteins in AH136B cells and solid tumours. ( A ) Cells were incubated with SNAP (10 or 100 μ M ), P-NONOate (10 or 100 μ M ), or ZnPP IX (100 μ M ) for 6 h or, as a positive control, were treated by heat at 42°C for 30 min, followed by incubation at 37°C for 6 h. ( B ) AH136B solid tumours were treated with 0.9% NaCl solution (control), ZnPP IX (500 μ g kg −1 i.a.), L -NAME (6 mg kg −1 day −1 for 5 days i.p.), or SMT (6 mg kg −1 day −1 for 5 days i.p.). ( C ) Solid tumours were resected at 24 or 48 h after initiation of surgical occlusion of the common iliac artery, which serves the tumour-feeding artery of the tumour-implanted side. Control rats were treated by sham operation. Then, expression of HSP70 and HO-1 proteins was detected by Western blotting by using a monoclonal antibody to mammalian inducible HSP70 or a polyclonal antibody to rat HO-1. Each data point shown represents three independent experiments. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Western Blot, Incubation, Positive Control, Control, Expressing

Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Time line depicting in vitro protocols for resistant oculomotor neurons (OMN) and vulnerable spinal motor neuron (SC MN) generation. ESC column reports the four cell lines used in this study. Patterning was performed for 5 days after ESC expansion and EB formation. mRNA-seq coupled with FACS was performed on EB dissociation day (day 9 of the protocol). Survival assay was performed after five days from EB dissociation. (B-C) SC MNs generated from E14.1 ESCs express Hb9, Islet1 and NF200, OMNs generated from E14.1 ESCs over-expressing the transcription factor Phox2a under the Nestin enhancer co-express Islet1 and NF200 in absence of Hb9. Scale bar in c 60 μm. (D) Percentage of Hb9+/Islet1+ cells in SC MN (64.1±5.2%, mean±SEM, n=4) and OMN (6.3±1.3%, mean±SEM) cultures respectively. (For quantification, experiments were run in quadruplicates including two technical replicates per experiment, with at least 120 Islet1+ cells counted per condition). (E) Microphotographs showing Islet1+/Tuj1+ cells in OMN cultures, (F) OMNs also express the specific marker Phox2a as indicated by asterisks. Scale bars in e and g 100 μm. (G) Quantification of Islet1+ over Tuj1+ cells demonstrates that half neuronal population appears to be Islet1+ (47.5±5.9%, mean±SEM, experiments performed in quadruplicates with two technical replicates each, total number of Tuj1+ cells counted: 1325). Quantification of Phox2A+ over Islet1+ cells (experiments performed in quadruplicates with two technical replicates each, total number Islet1+ cells counted: 746) indicates that 62±5.2% (mean±SEM) of the Islet1+ population is also Phox2a+. All quantifications were performed 5 days after EB dissociation (mean ± SEM). (H) PCA of OMN and SC MN samples based on all genes expressed confirmed cell differential identities. mRNA-seq analysis of OMNs and SC MNs isolated by FACS was performed after 5 days of patterning (day 9 of the protocol). (I) 1,017 differentially expressed genes were found between the two different cell types (adjusted P<0.05). Heatmap shows the top 500 most significant differentially expressed genes by adjusted p-value. (J) Heatmap of expected progenitors and motor neuron transcripts but also OMN and SC MN specific transcripts obtained in the two generated motor neuron populations. (K) Using the top 100 DEGs obtained from our RNAseq analysis we could separate datasets originating from in vivo microarray studies on early postnatal (J) and adult (L) rodent OMNs and SC MNs. (M) Venn diagram showing gene sets enriched either in brain stem cultures or OMN-specific as revealed by PAGODA analysis, (N) gene sets preferentially found in spinal cord cultures or MN-specific.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: In Vitro, Clonogenic Cell Survival Assay, Generated, Expressing, Marker, Isolation, In Vivo, Microarray

Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Oculomotor neurons (OMNs) and spinal motor neurons (SC MNs) express similar mRNA levels of glutamate ionotropic receptor AMPA, NMDA and kainate type subunits. The heatmap shows log2 RPKM values of these subunits and no separate clustering of the two cell types is observed. (B-C) Immunohistochemistry performed on generated OMNs and SC MNs at D1 in vitro in control conditions; similar levels of glutamate ionotropic receptor kainate type subunit 5 (Grik5) are found in both cell types. Scale bar in d 60 μm. (D-E) Microphotographs presenting SC MN and OMN response to kainic acid induced toxicity (20 μM) for a week. Scale bars in f = 100 μm. (F) Curves represent percentages of MN survival over time in OMN and SC MN cultures. OMNs were visualized as NF200+Islet1+Hb9- clls, while SC MNs as NF200+Islet1+Hb9+ cells. OMNs show increased survival to KA toxicity at D7 (mean ± SEM, 2way ANOVA and Tukey’s multiple comparison test, F(9, 56)=2.333, *P=0.0261, SC MNs n=4; OMNs n=5) when compared to SC MNs (experiments were performed at least in quadruplicates, with technical replicates and with at least 130 motor neurons counted per condition in each experiment). Analysis of the length of neuronal processes in both oculomotor and spinal motor neuron cultures exposed to kainic acid for seven days. showed that oculomotor neurons were unaffected by kainic acid while spinal motor neurons displayed a shortening of neurites (G). (H-K) Sholl analysis was performed on OMN at D7 survival assay in control and KA20 conditions to further assess individual MN arborization complexity during toxicity. Scale bar = 100 μm. (I) Sholl mask was applied to individual OMNs after specifying the radius from the center of the soma of the neuron and created concentric circles every 25 μm of increasing radius. (J) Comparison of average number of neurite intersections of OMN in control and KA20 toxicity conditions with radial step size of 25 μm. OMNs did not show reduction in arborization (multiple t test, n = 10 per condition). (K) Schematic depicting identification of neurite segments by Sholl analysis. Color code is assigned depending on arbor localization from the soma in an inside-out manner following the given radius. Multiple intersections within the same segment display the same colour.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: Immunohistochemistry, Generated, In Vitro, Clonogenic Cell Survival Assay

Journal: The EMBO Journal

Article Title: DNGR-1 signalling limits dendritic cell activation for optimal antigen cross-presentation

doi: 10.1038/s44318-025-00620-z

Figure Lengend Snippet: ( A ) ELISA for IFN-γ release from OT-I effector T (T E ) cells co-cultured with wild-type (WT) or DNGR-1 deficient Clec9a knock-in Cre (C9 KI-Cre ) bone marrow-FLT3L cultured (BM-FLT3L) cDC1s incubated with ovalbumin (OVA)-dead cells (left) or SIINFEKL peptide (right). Mean ± SD from biological duplicates is plotted. ( B ) Uptake of Cell Tracker-Deep Red (CT-DR)-labelled dead cell debris by WT or C9 KI-Cre BM-FLT3L cDC1s assessed by flow cytometry. Plotted as phagocytic index (% CT-DR + cells x CT-DR MFI of CT-DR + cells/arbitrary unit) mean ± SD from n = 3. ( C ) Schematic of WT (C9), W155A-W250A (C9(2WA)), or Y7F (KO/C9(Y7F)) DNGR-1 transduced into DNGR-1 knockout (KO) splenic cDC1 line MuTuDC1940 (MuTuDCs). Intracellular cytoplasmic domain (ICD); transmembrane domain (TM); extracellular domain (ECD). ( D ) ELISA for IFN-γ release from OT-I T E cells co-cultured with C9 KO, KO/C9, KO/C9(2WA), or KO/C9(Y7F) MuTuDCs incubated with OVA-dead cells (left), SIINFEKL peptide (right), or ( E ) DNGR-1 ligand (DNGR-1L)-OVA coupled beads. Mean ± SD from biological ( D ) quadruplet or ( E ) duplicates is plotted. All lines are plotted even when they cannot be seen because of superimposition. ( F ) Uptake of CT-DR-labelled dead cell debris as in ( B ) by C9 KO or KO/C9 MuTuDCs. Cytochalasin D (CD) co-culture was included as a negative control. ( G , H ) C9 KO or KO/C9 MuTuDCs transduced with lysenin-mCherry fusion protein were co-cultured with α-DNGR-1 IgG coupled beads and assessed by confocal microscopy. ( G ) Representative images, scale bar = 5 µm. Beads not internalised marked by α-rat IgG staining. ( H ) Quantification of lysenin-mCherry + phagosomes per cells in field of view (Lysenin index), bars indicate mean ± SEM. ( I ) Absorbance of β-galactosidase activity from B3Z-DNGR-1-SYK reporter cells stimulated ± DNGR-1L (left) or plate-bound α-DNGR-1 IgG (right). Mean ± SEM of four replicates. ( J ) Confocal microscopy of KO/C9 MuTuDCs treated as in ( G ). ( K , L ) WT, C9 KI-Cre BM-FLT3L cDC1s or ( M , N ) C9 KO MuTuDCs or cells reconstituted with indicated receptors were cultured overnight ± indicated stimuli and assessed in triplicate by flow cytometry for ( K , M ) surface or ( N ) intracellular protein expression, or ( L ) release of IL-12 p40 by ELISA in cultured supernatants. Cells treated with 200 U/mL IFN-α or 20 nM DNGR-1L ( K ) or 10 µg/mL Poly(I:C) or 20 nM DNGR-1L ( L ). Mean ± SEM of each group from biological triplicates ( K , L ) or pooled duplicates from two independent experiments ( M , N ) is plotted. MFI mean fluorescence intensity. Data are representative of two ( A , B , F – H , J – N ) or ≥ three ( D , E , I ) independent experiments. Data were analysed using Tukey-corrected two-way ANOVA ( A – F , K – N ) or unpaired t test ( H ). Significant values comparing against C9 KI-Cre BM-FLT3L cDC1 ( A ), C9 KO MuTuDCs ( D , E , H ), or untreated samples ( K – N ) are plotted. ( A , D , E ) **** P < 0.0001, ( H ) *** P = 0.0002, ( K ) **** P < 0.0001, ( L ) ** P = 0.0020, **** P < 0.0001, ( M ) *** P = 0.0004, **** P < 0.0001, ( N ) **** P < 0.0001. See also Fig. . .

Article Snippet: Unless otherwise stated, cultured supernatant analytes were quantified using mouse CCL17/TARC, CCL22/MDC, IL-12 p40, TNF-α DuoSet ELISA kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Knock-In, Incubation, Flow Cytometry, Knock-Out, Co-Culture Assay, Negative Control, Transduction, Confocal Microscopy, Staining, Activity Assay, Expressing, Fluorescence

Journal: The EMBO Journal

Article Title: DNGR-1 signalling limits dendritic cell activation for optimal antigen cross-presentation

doi: 10.1038/s44318-025-00620-z

Figure Lengend Snippet: ( A ) Analysis of the indicated surface marker expression (top, middle; flow cytometry) or CCL22 released into cultured supernatants (bottom; ELISA) from C9 KO MuTuDCs reconstituted or not with the indicated receptors and stimulated overnight ± DNGR-1L. Mean ± SEM from biological replicates pooled from two independent experiments (left) and representative flow cytometry profiles (right) are plotted. Data here are partly represented in Fig. and are relative to average of untreated controls to better emphasise the response to DNGR-1L. Dotted line represents 1. ( B ) Flow cytometric analysis of surface DNGR-1 (C9) expression in C9 KO MuTuDCs or those reconstituted or not with the indicated receptors. Cell lines were established after sorting for equal expression of DNGR-1. ( C ) Flow cytometric analysis of surface Dectin-1 (C7) expression by parental RAW 264.7 cells or cells ectopically expressing the indicated receptors or transduced with empty vector (EV). Cell lines were established after sorting for equal expression of Dectin-1. ( B , C ) Representative histograms (left) and mean MFI ± SEM (right) are plotted from biological ( B ) quintuplets or ( C ) triplicates. ( D ) Analysis of the indicated surface marker expression (top, middle; flow cytometry) or TNF-α released into cultured supernatants (bottom; ELISA) from RAW 264.7 cells ectopically expressing the indicated receptors or transduced with EV and stimulated overnight ± Zym-D. Mean ± SEM from biological replicates pooled from two independent experiments (left) and representative flow cytometry profiles (right) are plotted. Data here are partly represented in Fig. and are relative to average of untreated controls to better emphasise the response to Zym-D. Dotted line represents 1. Data are representative of two ( D ), or three ( A – C ) independent experiments. Data were analysed using Tukey-corrected two-way ANOVA with significant values comparing against untreated samples plotted ( A , D ). ( A ) I-A/E hi CD86 hi * P = 0.0117, **** P < 0.0001; CCR7 * P = 0.0279 (KO/C7::C9), P = 0.0134 (KO/C9(I6G)), **** P < 0.0001; CCL22 **** P < 0.0001, ( D ) H2-D d ** P = 0.0011, **** P < 0.0001; CCR7 * P = 0.0319, ** P = 0.0040, **** P < 0.0001; TNF-α ** P = 0.0033, *** P = 0.0004, **** P < 0.0001.

Article Snippet: Unless otherwise stated, cultured supernatant analytes were quantified using mouse CCL17/TARC, CCL22/MDC, IL-12 p40, TNF-α DuoSet ELISA kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Marker, Expressing, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation

Journal: The EMBO Journal

Article Title: DNGR-1 signalling limits dendritic cell activation for optimal antigen cross-presentation

doi: 10.1038/s44318-025-00620-z

Figure Lengend Snippet: ( A – C ) ELISA for IFN-γ release from OT-I T E cells co-cultured with C9 KO MuTuDCs or those reconstituted with indicated receptors incubated with ( A ) DNGR-1L-OVA-coupled beads, ( B ) OVA-dead cells, or ( C ) SIINFEKL peptide. Mean ± SD from biological ( B , C ) duplicates or ( A ) triplicates are plotted. ( D , E ) Uptake of DNGR-1L-coupled beads by C9 KO MuTuDCs or those reconstituted with indicated receptors assessed by flow cytometry. ( D ) Representative plot depicting the use of post-uptake streptavidin staining to distinguish MuTuDCs that have internalised (in) biotinylated-DNGR-1L coupled beads from those attached to surface DNGR-1 (out). ( E ) Representative histogram of cells treated 20:1 with beads (left) and phagocytic index (% internalised beads x bead MFI/arbitrary unit) mean ± SD from biological duplicates (right). ( F – H ) Confocal microscopic analysis of lysenin-mCherry fusion protein-expressing C9 KO MuTuDCs or those reconstituted with indicated receptors co-cultured with α-DNGR-1 IgG-coupled or isotype IgG-coupled beads. Beads not internalised marked by α-rat IgG staining. ( F ) Violin plot quantification of internalised beads per cell per field of view ( n = 20). ( G ) Violin plot of lysenin-mCherry + phagosomes per cells in field of view (Lysenin index). ( H ) Representative images of lysenin-mCherry + phagosomes from indicated MuTuDCs, scale bar = 5 µm. ( I – K ) ELISA for IFN-γ release from OT-I T E cells co-cultured with KO/C9(I6G) or SHIP1 sufficient (guide control; gControl) or deficient KO/C9 MuTuDCs incubated with ( I ) DNGR-1L-OVA-coupled beads, ( J ) OVA-dead cells, or ( K ) SIINFEKL peptide. Mean ± SD from ( I – K ) biological duplicates or quadruplets (KO/C9 SHIP1 KO) are plotted. ( L ) Uptake of DNGR-1L-coupled beads by KO/C9 SHIP1 sufficient or deficient MuTuDCs assessed by flow cytometry. Phagocytic index plotted with mean ± SD from biological triplicates. Data are representative of two ( F – L ) or ≥ three ( A – C ) independent experiments. Data were analysed using Tukey-corrected two-way ANOVA with only significant values observed between KO/C9 and KO/C9(I6G) or KO/C7::C9 MuTuDCs plotted in ( A – C , E – G ) or between KO/C9 gControl and KO/C9(I6G) or KO/C9 SHIP1 KO MuTuDCs in ( I – K ). ( A , B , G , I , J ) **** P < 0.0001. See also Fig. . .

Article Snippet: Unless otherwise stated, cultured supernatant analytes were quantified using mouse CCL17/TARC, CCL22/MDC, IL-12 p40, TNF-α DuoSet ELISA kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Flow Cytometry, Staining, Expressing, Control

Journal: The EMBO Journal

Article Title: DNGR-1 signalling limits dendritic cell activation for optimal antigen cross-presentation

doi: 10.1038/s44318-025-00620-z

Figure Lengend Snippet: ( A , B ) Flow cytometric analyses of surface proteins and ELISA of indicated proteins released in culture supernatants from ( A ) RAW 264.7 cells ectopically expressing indicated receptors or transduced with EV stimulated overnight ± Zym-D or ( B ) C9 KO MuTuDCs reconstituted with indicated receptors stimulated overnight ± DNGR-1L. Data shown as mean ± SEM from biological triplicates. Note that the TNF-α data in ( A ) are plotted in two different ways (absolute versus relative concentration to untreated controls) to emphasise the similarity between C7 and C9(I6G)::C7. ( C ) Uptake of DNGR-1L-coupled beads by C9 KO MuTuDCs or those reconstituted with indicated receptors assessed by flow cytometry. Plotted as phagocytic index (% internalised beads x bead MFI of bead + cells/arbitrary unit) mean ± SD from biological duplicates. ( D , E ) C9 KO MuTuDCs reconstituted with C7::C9 or C7(G14I)::C9 receptors co-cultured with DNGR-1L-OVA coupled beads. Single internalised bead + cells were subsequently sorted and co-cultured with OT-I T E cells. ( D ) Schematic of experiment (left) and representative flow plots from pre- and post-sort enrichment (right). ( E ) ELISA for IFN-γ released from OT-I T E cells co-cultured with MuTuDCs from ( D ) or MuTuDCs loaded with exogenous SIINFEKL peptide. Mean ± SD from biological duplicates is plotted. Data are representative of two ( D , E ) or ≥ three ( A – C ) independent experiments. Data were analysed using Tukey-corrected two-way ANOVA ( A – C , E ). Significant values comparing against untreated controls ( A , B ) or between KO/C7(G14I)::C9 ( E ) are plotted. ( A ) H2-D d ** P = 0.0043, *** P = 0.0006, **** P < 0.0001; CD83 * P = 0.0374, *** P = 0.0002, **** P < 0.0001; TNF-α ** P = 0.0017 (C7), P = 0.0067 (C9(Y7F)::C7, *** P = 0.0004 (C7(G14I)), P = 0.0006 (C9::C7), **** P < 0.0001, ( B ) CD86 * P = 0.0469, **** P < 0.0001; CCR7 ** P = 0.0097, *** P = 0.0004, **** P < 0.0001; CCL17 **** P < 0.0001; CCL22 * P = 0.0217, ** P = 0.0018, **** P < 0.0001, ( C , E ) **** P < 0.0001. See also Fig. . .

Article Snippet: Unless otherwise stated, cultured supernatant analytes were quantified using mouse CCL17/TARC, CCL22/MDC, IL-12 p40, TNF-α DuoSet ELISA kits (R&D Systems) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transduction, Concentration Assay, Flow Cytometry, Cell Culture