Journal: The Journal of Cell Biology

Article Title: Galectin-9 regulates dendritic cell polarity and uropod contraction by modulating RhoA activity

doi: 10.1083/jcb.202404079

Figure Lengend Snippet: cDC2 cell purity and supplementary phenotype analysis. (A) Gating strategy to determine cDC2 purity after isolation from PBMCs. cDC2s were stained for CD1c, CD11c, CD14, and CD20. A first gate is set based on the physical FCS-A/SSC-A parameters followed by selecting on negative cells for CD14 and CD19. cDC2s are then identified as positive cells for CD11c and CD1c. (B) Surface galectin-9 and CCR7 expression in galectin-9 and CCR7-positive cDC2s. Graph shows average ± SEM gMFI of five individual donors. One-way ANOVA followed by Dunnett’s test for multiple comparisons was performed. *P < 0.05; **P < 0.01; ***P < 0.001. gMFI, geometric mean fluorescence intensity.

Article Snippet: Human cDC2s were isolated from peripheral blood mononuclear cells derived from healthy individuals (Sanquin, Nijmegen, The Netherlands) using the MACS CD1c + isolation kit (130-119-475; Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Isolation, Staining, Expressing, Fluorescence

Journal: Frontiers in immunology

Article Title: Genomic Multiple Sclerosis Risk Variants Modulate the Expression of the ANKRD55 - IL6ST Gene Region in Immature Dendritic Cells.

doi: 10.3389/fimmu.2021.816930

Figure Lengend Snippet: FIGURE 5 | Digital droplet PCR (ddPCR) quantification of gene transcript copy numbers in pDC, mDC, CD14+ monocytes, PBMC fraction minus monocytes (F-), and moDC. Analysis was performed in 350 pg cDNA (A, E, F) or 30 ng of cDNA (B–D). (C) ddPCR quantification of ANKRD55 transcript copy numbers in moDC treated with AM-580. (D) ANKRD55 copy numbers in moDC matured or not with IFN-g/LPS measured at 6 h after addition of stimulus. (F) Transcript levels of markers specific for mDC (CD1c) and pDC (IL3RA, CLEC4C).

Article Snippet: To verify the purity of each subpopulation, cells were analyzed by flow cytometry (MACSQuant® flow cytometer, Miltenyi Biotec) using antihuman CD1c (BCDA-1)-APC antibody (Miltenyi Biotec, Ref. 130-110-595) for mDC and CD303 (BDCA-2)-APC antibody (Miltenyi Biotec, Ref. 130-114-177) for pDC, as shown in Supplementary Figure S1.

Techniques:

Journal: Cell Death & Disease

Article Title: Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c + dendritic cells

doi: 10.1038/s41419-022-05191-z

Figure Lengend Snippet: Representative serial section immunohistochemical (IHC) staining of control/non-fibrotic (left panel) and fibrotic (middle panel) kidney tissue probed for ( A ) aquaporin-1, ( B ) GPX4, ( C ) 4-HNE and ( D ) CD1c. Regions of CD1c + DC co-localisation with aqupaporin-1 + PTEC displaying evidence of ferroptotic cell death (↓ GPX4 and ↑ 4-HNE) are highlighted with black arrows. Scale bars represent 60 µm. Quantitative analysis (positive pixel intensity/µm 2 total area) of IHC staining in control and fibrotic tissue is presented (right panels). Symbols represent values for individual donor tissue. Results represent mean ± SEM of values from four randomly selected areas for each tissue sample. * P < 0.05, *** P < 0.001, Welch’s t test.

Article Snippet: Serial sections were probed with antibodies against AQP-1 (Cat. No. sc-20810; Santa Cruz Biotechnology, Dallas, TX, USA), GPX4 (Cat. No. ab41787; Abcam, Cambridge, MA, USA), 4-HNE (Cat. No. ab46544; Abcam) or CD1c (Cat. No. AF5910; R&D Systems, Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Immunohistochemistry, Control

Journal: Cell Death & Disease

Article Title: Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c + dendritic cells

doi: 10.1038/s41419-022-05191-z

Figure Lengend Snippet: A Heatmap representing secreted cytokine levels following 24-h co-culture (DC & PTEC) of flow cytometry sorted CD1c + DC with pre-conditioned normoxic (N; 21% O 2 ) or hypoxic (H; 1% O 2 ) PTEC in the absence (Nil) or presence of poly I:C. PTEC alone and DC alone cultures are included as controls; n = 4. The colour bar represents the z-score. Yellow indicates low expression; purple indicates high expression. IL-12p70, IL-17, interferon (IFN)-α and IFN-γ were not detectable in any culture conditions (data not shown). B , C Secreted IL-1β ( B ) and IL-18 ( C ) protein levels (measured by LEGENDplex™ assay; pg/ml) following 24-h co-culture (DC & PTEC) of flow cytometry sorted CD1c + DC with pre-conditioned normoxic (white bars) or hypoxic (grey bars) PTEC in the absence (Nil) or presence of poly I:C. PTEC alone and DC alone cultures are included as controls; N.D. not detectable. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 4. **** P < 0.0001, one-way ANOVA with Tukey’s multiple-comparison test. D Flow cytometric detection of intracellular IL-1β protein following 4-h co-culture of magnetic bead-enriched CD1c + DC with pre-conditioned hypoxic PTEC in the presence of poly I:C. Co-cultures were treated prior to the 4-h culture period with either brefeldin A or VX-765 to enable intracellular IL-1β accumulation/detection. Representative flow cytometric histograms of IL-1β staining (black unfilled) compared with unstained control (grey filled) for PTEC (gated on live, single, CD45 − cells) and CD1c + DC (gated on live, single, CD45 + lineage − HLA-DR + CD1c + cells) are presented. One representative of three individual donor PTEC experiments is shown. E Fold changes (relative to –Fer-1) in secreted IL-1β levels (measured by ELISA; pg/ml) following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c + DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (-Fer-1) or presence of ferrostatin-1 (+Fer-1). Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 7. ns not significant, paired t test.

Article Snippet: Serial sections were probed with antibodies against AQP-1 (Cat. No. sc-20810; Santa Cruz Biotechnology, Dallas, TX, USA), GPX4 (Cat. No. ab41787; Abcam, Cambridge, MA, USA), 4-HNE (Cat. No. ab46544; Abcam) or CD1c (Cat. No. AF5910; R&D Systems, Minneapolis, MN, USA).

Techniques: Co-Culture Assay, Flow Cytometry, Expressing, Comparison, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c + dendritic cells

doi: 10.1038/s41419-022-05191-z

Figure Lengend Snippet: A NLRP3 mRNA expression relative to housekeeping gene β-2-microglobulin ( B2M) in flow cytometry sorted CD1c + DC freshly isolated (0 h) or following 24-h co-culture (DC & PTEC) with pre-conditioned normoxic (white bar) or hypoxic (grey bar) PTEC in the presence of poly I:C. DC alone cultures are included as controls. Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 7. * P < 0.05, one-way ANOVA with Tukey’s multiple-comparison test. B Heatmap representing secreted cytokine levels following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c + DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (NIL) or presence of inflammasome inhibitors (VX-765, MCC950) or DAMP inhibitors (apyrase, isotype control or HMGB1 antibody); n = 6. The colour bar represents the z-score. Yellow indicates low expression; purple indicates high expression. C , D Fold changes (relative to NIL inhibitor) in secreted IL-1β ( C ) and IL-18 ( D ) protein levels (measured by LEGENDplex™ assay) following 24-h co-culture (DC & PTEC) of magnetic bead-enriched CD1c + DC with pre-conditioned hypoxic PTEC and poly I:C in the absence (NIL) or presence of inflammasome inhibitors (VX-765, MCC950) or DAMP inhibitors (apyrase, isotype control or HMGB1 antibody). Bar graphs represent mean ± SEM. Symbols represent individual donor PTEC experiments; n = 6. * P < 0.05; ** P < 0.01; **** P < 0.0001, one-way ANOVA with Tukey’s multiple-comparison test.

Article Snippet: Serial sections were probed with antibodies against AQP-1 (Cat. No. sc-20810; Santa Cruz Biotechnology, Dallas, TX, USA), GPX4 (Cat. No. ab41787; Abcam, Cambridge, MA, USA), 4-HNE (Cat. No. ab46544; Abcam) or CD1c (Cat. No. AF5910; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Flow Cytometry, Isolation, Co-Culture Assay, Comparison, Control

Journal: Cell Death & Disease

Article Title: Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c + dendritic cells

doi: 10.1038/s41419-022-05191-z

Figure Lengend Snippet: A , B Immunofluorescent labelling of frozen sections from control/non-fibrotic ( A ) and fibrotic kidney tissue ( B ) stained for ASC (green), CD1c (red), aquaporin-1 (AQP-1) (white) and DAPI (blue). Scale bars represent 50 µm for small frames (left panels) and 20 µm for large frames (right panels). ASC-speck-positive CD1c + DC are highlighted with white arrows. C Quantification (mean cells/mm 2 ) of ASC-speck-negative CD1c + DC (white bars) and ASC-speck-positive CD1c + DC (grey bars) in control/non-fibrotic kidney tissue ( n = 4) and fibrotic kidney tissue ( n = 4). Symbols represent values for individual donor tissue. Results represent mean ± SEM of values from five randomly selected areas for each tissue sample. * P < 0.05, ** P < 0.01, Welch’s t test.

Article Snippet: Serial sections were probed with antibodies against AQP-1 (Cat. No. sc-20810; Santa Cruz Biotechnology, Dallas, TX, USA), GPX4 (Cat. No. ab41787; Abcam, Cambridge, MA, USA), 4-HNE (Cat. No. ab46544; Abcam) or CD1c (Cat. No. AF5910; R&D Systems, Minneapolis, MN, USA).

Techniques: Control, Staining