Journal: Nature communications

Article Title: Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy.

doi: 10.1038/s41467-018-05763-8

Figure Lengend Snippet: Fig. 5 Paclitaxel increases IRE1-dependent cytokine secretion. a MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times, and cell lysates were immunoblotted for XBP1s and Actin. b MDA-MB-231 cells were treated with 10 nM paclitaxel in the presence of 20 μM MKC8866 or vehicle (DMSO) for 72 h, cell lysates were harvested and immunoblotted for XBP1s and Actin. c MDA-MB-231 cells were treated with 10 nM paclitaxel in combination with DMSO or 20 μM MKC8866 for 72 h in the presence of Boc-D-fmk (40 μM). Following treatment conditioned medium was collected and analyzed by ELISA for secretion of IL-6, IL-8, CXCL1, and GM-CSF. Cells were lysed and protein quantified (n = 3). Results shown for a and b are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, based on a Student’s t test. Error bars represent s.e.m.

Article Snippet: Neutralizing antibodies against IL-6 (R&D Systems, MAB206), IL-8 (R&D Systems, MAB208), CXCL1 (R&D Systems, MAB275), and GM-CSF (R&D Systems, MAB215) were used at 0.5 μg ml−1, while TGFβ2 neutralizing antibody (R&D Systems, AF302) was used at 1.12 μg ml−1.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

doi: 10.1038/mi.2016.10

Figure Lengend Snippet: Figure 3 Role of decidual CD3 CD56 þ-derived cytokines on neutrophil phenotype and survival. (a) Concentration of GM-CSF, IFNg, and TNF (pg ml 1)±s.e.m. in spt obtained from p- (white bars) or dCD3 CD56 þ (black bars) cells cultured, for 48 h, in the absence or in the presence of IL2 or IL15 (n ¼ 2–13). Statistical analysis was performed using Mann–Whitney test. (*) Indicates comparison between the two different cell subsets (p- vs. d-CD3 CD56 þ) activated using the same stimulus, while (y) indicate comparison between stimulated and unstimulated cells within the same cell subset. When not indicated, data were not statistically significant. (b) Percentages of annexin V-negative±s.e.m. of pNs cultured, for 48 h, with unstimulated dCD3 CD56 þ spt in the absence (black bars; n ¼ 10) or in the presence of neutralizing a-GM-CSF Ab (stripped bars; n ¼ 7). Statistical analysis was performed using Mann–Whitney test. (c) Expression of CD11b and CD62L on pNs cultured, for 48 h, with unstimulated p- (filled gray profile) or dCD3 CD56 þ spt (black line). One representative experiment out of four performed. (d) Statistical analysis of mean±s.e.m. of CD11b and CD62L on pNs cultured with unstimulated p- (white bars; n ¼ 4–6) or dCD3 CD56 þ spt (black bars; n ¼ 4–6) either in the absence or in the presence of neutralizing a-GM-CSF Ab (stripped bars; n ¼ 4). Statistical analysis was performed using Mann–Whitney test. When not indicated, data were not statistically significant.

Article Snippet: All experiments were performed in the absence or in the presence of polyclonal neutralizing a-human GM-CSF Ab (10 mg ml 1, R&D System).

Techniques: Derivative Assay, Concentration Assay, Cell Culture, MANN-WHITNEY, Comparison, Expressing

Journal: Mucosal immunology

Article Title: Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

doi: 10.1038/mi.2016.10

Figure Lengend Snippet: Figure 4 GM-CSF-dependent HB-EGF expression in decidual neutrophils. (a, b) Immunohistochemical analysis of HB-EGF. (a) DB, arrows indicate examples of HB-EGF þ polymorphonuclear neutrophils (upper panel). The lower panel shows DB tissue section stained with secondary antibody, as negative control. (b) Spleen (upper panel) and gastritis (lower panel) tissue, arrows indicate HB-EGF-negative neutrophils. (c, d) Levels of HB-EGF mRNA expression by (c) freshly purified pNs (white; n ¼ 4) or dNs (black; n ¼ 7) or by (d) pNs incubated for 2.5 h with medium alone or with spt derived from IL15-stimulated pCD3 CD56 þ (as positive control) or unstimulated dCD3 CD56 þ cells, in the absence (white bars) or in the presence of a-GM- CSF Ab (black bars). (e, f) Levels of IL1ra mRNA expression by (e) freshly purified pNs (white; n ¼ 4) or dNs (black; n ¼ 7) or by (f) pNs incubated for 2.5 h with medium alone or with spt derived from IL15-stimulated pCD3 CD56 þ (as positive control) or unstimulated dCD3 CD56 þ cells, in the absence (white bars) or in the presence of a-GM-CSF Ab (black bars). (d, f) Bars indicate the MNE±s.e.m. from three independent experiments. When not indicated, data were not statistically significant. MNE, mean normalized expression.

Article Snippet: All experiments were performed in the absence or in the presence of polyclonal neutralizing a-human GM-CSF Ab (10 mg ml 1, R&D System).

Techniques: Expressing, Immunohistochemical staining, Staining, Negative Control, Purification, Incubation, Derivative Assay, Positive Control

Journal: Mucosal immunology

Article Title: Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

doi: 10.1038/mi.2016.10

Figure Lengend Snippet: Figure 5 NCR þILC3 promote neutrophil migration and survival via production of CXCL8 and GM-CSF. (a) Frequency±s.e.m. of dNs and NCR þ

Article Snippet: All experiments were performed in the absence or in the presence of polyclonal neutralizing a-human GM-CSF Ab (10 mg ml 1, R&D System).

Techniques: Migration

Journal: Mucosal immunology

Article Title: Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

doi: 10.1038/mi.2016.10

Figure Lengend Snippet: Figure 6 NCR þILC3, but not dNK cells, induce HB-EGF and IL1ra expression in neutrophils via GM-CSF production. (a, b) Expression of HB-EGF (a) or IL1ra (b) mRNA in pNs incubated 2.5 h with 10 ng ml 1

Article Snippet: All experiments were performed in the absence or in the presence of polyclonal neutralizing a-human GM-CSF Ab (10 mg ml 1, R&D System).

Techniques: Expressing, Incubation

Journal: iScience

Article Title: Multimodal cartography of human lymphopoiesis reveals B and T/NK/ILC lineages are subjected to differential regulation

doi: 10.1016/j.isci.2023.107890

Figure Lengend Snippet: Dynamics of CD127 - and CD127 + ELPs in the BM of HSC-engrafted mice (A) Experimental design: neonatal CD34 hi CD45RA − CD38 lo/− Lin - progenitors (Lin: CD3, CD4, CD10, CD19, CD24, CD56, CD115, CD116, CD123, CD127, ITGB7; thereafter referred to as HSCs) pooled from different donors were transduced with egfp -reporter lentiviruses (1.5 × 10 5 cells/mouse) before mixing with non-transduced bulk CD34 + HSPCs (1.5 × 10 5 cells/mouse) and intravenous injection to irradiated NSG mice. Note that non-transduced bulk CD34 + HSPCs were used as carriers to ensure full comparability with mice reconstituted with LMDPs (see also Figures S1 E and S1F). To follow multi-lineage reconstitution, hu-CD45 + egfp + cells harvested from the BM of mice sacrificed at weekly endpoints were analyzed by multiparameter flow cytometry; percentages of corresponding cell subsets are indicated; results from individual mice are representative of 3 independent experiments. (B) Density plots showing the kinetics of monocytes (Mo; CD116 + CD123 - ) (upper panels; blue rectangles), dendritic cells (DC; CD116 + CD123 + (medium panels; blue rectangles) and B lymphocytes (BL CD19 + ) (lower panels; blue rectangles) in the BM of xenografted mice. (C) Asynchronous in vivo emergence of CD127 - and CD127 + ELPs. Upper density plots show the kinetics of CD34 hi CD45RA hi Lin − HSPCs (red circles) in xenografted mice; gates are set on Lin − negative cells (Lin: CD115, CD116, CD123, CD10, CD19). Lower contour plots show the dynamics of CD127 - (green) and CD127 + (red) ELPs; blue lines correspond to LMDPs; red arrows show the gating strategy. (D) Asynchronous in vitro emergence of CD127 - and CD127 + ELPs. LMDPs cultured under standard scf condition were analyzed by flow cytometry at the indicated time points; gates are set on CD45RA + My − CD19 − cells (My − : CD15 − , CD115 - , CD116 - , CD123 - ; see also Figure S1 C). Bidimensional density plots show the kinetics of differentiation of CD127 - and CD127 + ELPs. Data are from ≥ 10 concatenated wells; percentages of the corresponding populations are indicated. Results are from 1 representative experiment. (E–G) Time-dependent changes in the lymphoid potential of cultured LMDPs. (E) Experimental design: LMDPs sorted from NSG mice at week-3 post-grafting were cultured by 100 cell-pools for 7 days onto OP9 stromal cells before analysis by flow cytometry (see also Figure S1 C for the gating procedure). Condition-1 (d0-LMDPs; left panel ): LMDPs are cultured for 7-day under the standard scf condition. Condition-2 (d2-LMDPs; medium panel ): LMDPs sorted from cultures at day-2 are seeded for 5 more days in secondary cultures under the standard scf condition; Condition-3 (d5-LMDPs; right panel ): LMDPs sorted from cultures at day-5 are seeded for 2 more days in secondary cultures under the standard scf condition. (F) Shown are absolute cell yields (upper panel) and median percentages of ELPs (lower panel); circles correspond to individual wells. (G) Stacked bar plot shows normalized ratios of CD127 - (green) vs. CD127 + (red) ELPs; median numbers of ELPs per well are indicated (upper row); positivity threshold for ELP detection is set arbitrarily at ≥ 10 ELPs/well. Results are expressed as medians of 4–9 replicates from a representative experiment. (H and I) CD127 - and CD127 + ELPs emerge independently from LMDPs. (H) Density plot shows the gating strategy used for fractionation of CD34 hi CD45RA hi CD127 − Lin - HPCs (Lin: CD10, CD19, CD115, CD116, CD123) from HSC-xenografted mice (week-3). (I) The indicated ITGB7 neg; int; hi cellular fractions were sorted from the BM of mice at week-3 after grafting and then cultured for 3 or 7 days under the under standard scf condition before analysis by flow cytometry. Density plots show the percentages of in vitro-generated CD127 + ELPs (blue rectangles); gates are set as aforementioned; results from one representative experiment are pooled from 10 concatenated wells. ITGB7 neg cells correspond to LMDPs.

Article Snippet: anti-human CD116 APC-vio770 , Miltenyi , Cat# 130-100-992; RRID: AB_2654571.

Techniques: Transduction, Injection, Irradiation, Flow Cytometry, In Vivo, In Vitro, Cell Culture, Fractionation, Generated

Journal: iScience

Article Title: Multimodal cartography of human lymphopoiesis reveals B and T/NK/ILC lineages are subjected to differential regulation

doi: 10.1016/j.isci.2023.107890

Figure Lengend Snippet:

Article Snippet: anti-human CD116 APC-vio770 , Miltenyi , Cat# 130-100-992; RRID: AB_2654571.

Techniques: Recombinant, Reverse Transcription, Software