Journal: Cell

Article Title: Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.

doi: 10.1016/j.cell.2019.01.041

Figure Lengend Snippet: Figure 2. Cross-Reactive Tmem Recognize Antigens from C. albicans and A. fumigatus (A and B) Re-stimulation of expanded CD154+ T cells. (A) Percentage of CD154+TNF-a+ within CD4+ is indicated. (B) Statistical summary (n = 45). (C) Percentage of cross-reactivity in relation to total reactivity after re-stimulation with the specific fungal lysate (n = 45). (D) Experimental setup for expansion of cross-reactive and non-cross-reactive clones. A. fumigatus-reactive cell lines were stimulated with C. albicans, cross- reactive CD154+, and non-cross-reactive CD154 cells were fluorescence-activated cell sorting (FACS) sorted as single cells and expanded. (E) Reactivity of expanded single cell clones from (D) (both n = 37) against A. fumigatus and C. albicans was assessed by re-expression of CD154. (F–H) Re-stimulation of expanded A. fumigatus-reactive CD154+ memory or naive T cells. (F) Percentage of CD154+TNF-a+ within CD4+ is indicated. (G) Statistical summary (n = 4). (H) Percentage of cross-reactivity to C. albicans in relation to total reactivity after re-stimulation with A. fumigatus lysate (n = 10). (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human IL-4 Miltenyi Biotec Cat#130-093-922 Human GM-CSF Miltenyi Biotec Cat#130-093-866 Brefeldin A Sigma Aldrich Cat#B7651 Critical Commercial Assays MS Columns Miltenyi Biotec Cat#130-042-201 Viobility 405/520 Fixable Dye Miltenyi Biotec Cat#130-110-206 Inside Stain Kit Miltenyi Biotec Cat#130-090-477 Eppendorf twin.tec PCR Plates Eppendorf Cat#0030128680 Super Script III One-Step RT PCR system Invitrogen Cat#12574018 SUPERase-In RNase inhibitor Invitrogen Cat#AM2694 AllPrep DNA/RNA Micro Kit QIAGEN Cat#80284 Oligonucleotides TaqMan Assay CXCR6 Thermo Fisher Hs00174843_m1 TaqMan Assay ITGA1 (CD49a) Thermo Fisher Hs01673848_m1 TaqMan Assay ITGAE (CD103) Thermo Fisher Hs01025366_m1 TaqMan Assay CCR2 Thermo Fisher Hs01560352_m1 TaqMan Assay CCR8 Thermo Fisher Hs00174764_m1 TaqMan Assay CCR6 Thermo Fisher Hs00171121_m1 TaqMan Assay ITGAL (CD11a, LFA-1) Thermo Fisher Hs01035627_m1 TaqMan Assay ITGB7 Thermo Fisher Hs01565750_m1 TaqMan Assay CCR10 Thermo Fisher Hs00706455_s1 TaqMan Assay CX3CR1 Thermo Fisher Hs00365842_m1 TaqMan Assay ITGA4 (CD49d) Thermo Fisher Hs01047118_m1 TaqMan Assay CCR7 Thermo Fisher Hs01013469_m1 TaqMan Assay CXCR5 Thermo Fisher Hs00173527_m1 TaqMan Assay CXCR4 Thermo Fisher Hs00976734_m1 TaqMan Assay CXCR3 Thermo Fisher Hs00171041_m1 TaqMan Assay CCR4 Thermo Fisher Hs00747615_s1 TaqMan Assay CCR5 Thermo Fisher Hs00152917_m1 TaqMan Assay CCR9 Thermo Fisher Hs01011469_m1 TaqMan Assay ITGB1 (CD29) Thermo Fisher Hs01127536_m1 TaqMan Assay SELL (CD62L) Thermo Fisher Hs01053460_m1 TaqMan Assay S1PR1 Thermo Fisher Hs00173499_m1 Software and Algorithms FlowJo Treestar RRID:SCR_000410 MACSQuantify Miltenyi Biotec Cat#130-094-556 Genesis N/A Sturn et al., 2002 R implementation of Yue-Clayton similarity index N/A Bacher et al., 2016 Other Cytomegalovirus Siemens Healthcare Diagnostics Cat#OREA05 C. albicans MP65 Miltenyi Biotec Cat#130-096-776 A. fumigatus Aspf22 Miltenyi Biotec Cat#130-099-776 A. fumigatus Gel1 Miltenyi Biotec Cat#130-097-289 A. fumigatus Crf1 Miltenyi Biotec Cat#130-096-775 A. fumigatus Aspf3 (pmp20) Miltenyi Biotec Cat#130-096-772 A. fumigatus Catalase B Miltenyi Biotec Cat#130-097-291 A. fumigatus Sod3 (SOD) Miltenyi Biotec Cat#130-097-288 (Continued on next page) Cell 176, 1–16.e1–e7, March 7, 2019 e3 Please cite this article in press as: Bacher et al., Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans, Cell (2019), https://doi.org/10.1016/j.cell.2019.01.041

Techniques: Clone Assay, FACS, Expressing

Journal: Cell

Article Title: Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.

doi: 10.1016/j.cell.2019.01.041

Figure Lengend Snippet: Figure 7. Cross-Reactive Th17 Cells Selectively Contribute to Acute ABPA (A) Ex vivo IL-4 production of A. fumigatus-reactive Tmem in PBMCs (healthy, n = 130; asthma and/or COPD, n = 75; CF, n = 188). (B) Direct comparison of A. fumigatus-reactive IL-4 and IL-17A production in individual CF patients (n = 188). Classification of sensitized CF patients: acute ABPA before the onset of treatment (red), acute ABPA after the onset of treatment (light blue), chronic recurrent ABPA with prophylactic treatment (dark blue), and patients without ABPA at the time point of measurement (dark gray). (C and D) Ex vivo IL-17A or IL-4 production of A. fumigatus-reactive cells of sensitized CF patients (C) within CD154+ Tmem; (D) within total CD4+ T cells. Acute ABPA, n = 13; acute ABPA with >7 days therapy, n = 7; chronic ABPA with prophylactic therapy, n = 6; ABPA history, n = 6; and no ABPA, n = 15. (E) A. fumigatus-specific IgE levels in serum of sensitized CF patients.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Human IL-4 Miltenyi Biotec Cat#130-093-922 Human GM-CSF Miltenyi Biotec Cat#130-093-866 Brefeldin A Sigma Aldrich Cat#B7651 Critical Commercial Assays MS Columns Miltenyi Biotec Cat#130-042-201 Viobility 405/520 Fixable Dye Miltenyi Biotec Cat#130-110-206 Inside Stain Kit Miltenyi Biotec Cat#130-090-477 Eppendorf twin.tec PCR Plates Eppendorf Cat#0030128680 Super Script III One-Step RT PCR system Invitrogen Cat#12574018 SUPERase-In RNase inhibitor Invitrogen Cat#AM2694 AllPrep DNA/RNA Micro Kit QIAGEN Cat#80284 Oligonucleotides TaqMan Assay CXCR6 Thermo Fisher Hs00174843_m1 TaqMan Assay ITGA1 (CD49a) Thermo Fisher Hs01673848_m1 TaqMan Assay ITGAE (CD103) Thermo Fisher Hs01025366_m1 TaqMan Assay CCR2 Thermo Fisher Hs01560352_m1 TaqMan Assay CCR8 Thermo Fisher Hs00174764_m1 TaqMan Assay CCR6 Thermo Fisher Hs00171121_m1 TaqMan Assay ITGAL (CD11a, LFA-1) Thermo Fisher Hs01035627_m1 TaqMan Assay ITGB7 Thermo Fisher Hs01565750_m1 TaqMan Assay CCR10 Thermo Fisher Hs00706455_s1 TaqMan Assay CX3CR1 Thermo Fisher Hs00365842_m1 TaqMan Assay ITGA4 (CD49d) Thermo Fisher Hs01047118_m1 TaqMan Assay CCR7 Thermo Fisher Hs01013469_m1 TaqMan Assay CXCR5 Thermo Fisher Hs00173527_m1 TaqMan Assay CXCR4 Thermo Fisher Hs00976734_m1 TaqMan Assay CXCR3 Thermo Fisher Hs00171041_m1 TaqMan Assay CCR4 Thermo Fisher Hs00747615_s1 TaqMan Assay CCR5 Thermo Fisher Hs00152917_m1 TaqMan Assay CCR9 Thermo Fisher Hs01011469_m1 TaqMan Assay ITGB1 (CD29) Thermo Fisher Hs01127536_m1 TaqMan Assay SELL (CD62L) Thermo Fisher Hs01053460_m1 TaqMan Assay S1PR1 Thermo Fisher Hs00173499_m1 Software and Algorithms FlowJo Treestar RRID:SCR_000410 MACSQuantify Miltenyi Biotec Cat#130-094-556 Genesis N/A Sturn et al., 2002 R implementation of Yue-Clayton similarity index N/A Bacher et al., 2016 Other Cytomegalovirus Siemens Healthcare Diagnostics Cat#OREA05 C. albicans MP65 Miltenyi Biotec Cat#130-096-776 A. fumigatus Aspf22 Miltenyi Biotec Cat#130-099-776 A. fumigatus Gel1 Miltenyi Biotec Cat#130-097-289 A. fumigatus Crf1 Miltenyi Biotec Cat#130-096-775 A. fumigatus Aspf3 (pmp20) Miltenyi Biotec Cat#130-096-772 A. fumigatus Catalase B Miltenyi Biotec Cat#130-097-291 A. fumigatus Sod3 (SOD) Miltenyi Biotec Cat#130-097-288 (Continued on next page) Cell 176, 1–16.e1–e7, March 7, 2019 e3 Please cite this article in press as: Bacher et al., Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans, Cell (2019), https://doi.org/10.1016/j.cell.2019.01.041

Techniques: Ex Vivo, Comparison

Journal: Redox biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Fig. 8. siRNA-mediated knockdown of FoxO1 or FoxO3a increased basal Runx2 levels and H2O2-induced increases in Runx2 in VSMC. A, Western blot showed successful knockdown of FoxO1 by FoxO1-specific siRNA. Addition of H2O2 into the medium induced the inhibition of FoxO1 (indicated by the increase in pFoxO1 (S256)). In these studies, FoxO1 siRNA (P < 0.05), but not H2O2 (P = 0.07), affected levels of pFoxO1(S253) in VSMC, but there was an interaction effect (P < 0.05) between H2O2 and FoxO1 siRNA on pFoxO1(S256). (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA) B, Knockdown of FoxO1 increased basal expression of Runx2 and increased H2O2-mediated increases in Runx2. H2O2 (P < 0.05) and FoxO1 siRNA (P < 0.05) affected levels of Runx2 in VSMC and an interaction effect (P < 0.05) between H2O2 and FoxO1-specific siRNA on levels of pFoxO1(S256) was observed. (n = 4 samples in each group; *P < 0.05; Two-way ANOVA) C, Western blot showed successful knockdown of FoxO3a by FoxO3a-specific siRNA. Addition of H2O2 into the medium induced the inhibition of FoxO3a (indicated by the increase in pFoxO3a(S253)). In these studies, H2O2 (P < 0.05) and FoxO3a siRNA (P < 0.05) affected levels of pFoxO3a(S253) in VSMC and an interaction effect (P < 0.05) between H2O2 and FoxO3a siRNA on pFoxO3a(S253) was observed (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA). D, Knockdown of FoxO3a increased basal expression of Runx2 and increased H2O2-mediated increases in Runx2. H2O2 (P < 0.05) and FoxO3a siRNA (P < 0.05) affected levels of Runx2 in VSMC, but an interaction effect (P > 0.05) between H2O2 and FoxO3a siRNA on levels of pFoxO3a(S253) was not observed (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA).

Article Snippet: RNA interference was accomplished using Catalase Human siRNA Oligo Duplex (Locus ID 847, OriGene Technologies Inc., Rockvile, MD), SignalSilence® FoxO1 siRNA I (Cat# 6242, Cell Signaling Technology, Danvers, MA), and SignalSilence® FoxO3a siRNA I (Cat# 6302, Cell Signaling Technology, Danvers, MA); Trilencer-27 universal scrambled negative siRNA (Cat# SR30004, OriGene Technologies Inc.) served as a control in these experiments.

Techniques: Knockdown, Western Blot, Inhibition, Expressing

Journal: Redox biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Fig. 9. Effect of siRNA-mediated knockdown of catalase on catalase protein, activity, and bioavailable H2O2. in VSMC. A, Western blotting demonstrated successful knockdown of catalase protein by catalase-specific siRNA. Addition of H2O2 into the culture medium decreased (P < 0.05) levels of catalase in VSMC that received nontargeting siRNA. H2O2 (P < 0.05) and catalase siRNA (P < 0.05) affected catalase protein levels in VSMC and an interaction effect (P < 0.05) between H2O2 and catalase siRNA on catalase was observed. (n = 4 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA) B, Catalase activity followed a similar pattern, with H2O2 (P < 0.05) and catalase siRNA (P < 0.05) affecting catalase activity in VSMC and an interaction effect (P < 0.05) between H2O2 and catalase siRNA on catalase was observed. (n = 8 samples in each group; ns, not significant; *P < 0.05; Two-way ANOVA) C, H2O2 accumulation during vehicle treatment of VSMC pre-treated with siRNA that targeted catalase and VSMC that received nontargeting siRNA did not differ (*P > 0.05). In contrast, incubation of VSMC that received nontargeting siRNA in medium containing H2O2, 0.4 mM, showed significant (**P < 0.05) increases in H2O2 at each time point, compared with the vehicle-treated groups. Incubation in medium containing H2O2, 0.4 mM, of VSMC pre-treated with siRNA that targeted catalase produced significantly higher (***P < 0.05) amounts of H2O2 at each time point, compared with the other three groups in the study. (n = 8 samples in each group; one-way ANOVA).

Article Snippet: RNA interference was accomplished using Catalase Human siRNA Oligo Duplex (Locus ID 847, OriGene Technologies Inc., Rockvile, MD), SignalSilence® FoxO1 siRNA I (Cat# 6242, Cell Signaling Technology, Danvers, MA), and SignalSilence® FoxO3a siRNA I (Cat# 6302, Cell Signaling Technology, Danvers, MA); Trilencer-27 universal scrambled negative siRNA (Cat# SR30004, OriGene Technologies Inc.) served as a control in these experiments.

Techniques: Knockdown, Activity Assay, Western Blot, Incubation, Produced

Journal: Redox biology

Article Title: Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4.

doi: 10.1016/j.redox.2022.102296

Figure Lengend Snippet: Fig. 10. Effect of siRNA-mediated knockdown of catalase on H2O2-induced decreases in PP2a activity and increases in Runx2 in VSMC. A and B, Immu noprecipitation of the active subunit of PP2a and subsequent analysis of PP2a activity in the immunoprecipitants showed that loss of catalase enhanced the effects of H2O2 on PP2a activity, but neither catalase knockdown nor H2O2 altered the amount of PP2a C subunit. In these experiments, H2O2 (P < 0.05) and catalase siRNA (P < 0.05) affected PP2a activity in VSMC but there was no interaction (P > 0.05) between H2O2 and catalase siRNA on PP2a activity. (n = 8 samples in each group; *P < 0.05; Two-way ANOVA) C, Knockdown of catalase enhanced the effects of H2O2 on Runx2 in VSMC. H2O2 (P < 0.05) and catalase siRNA (P < 0.05) affected Runx2 levels in VSMC and an interaction effect (P < 0.05) between H2O2 and catalase siRNA on Runx2 was observed. (n = 4 samples in each group; *P < 0.05; Two- way ANOVA).

Article Snippet: RNA interference was accomplished using Catalase Human siRNA Oligo Duplex (Locus ID 847, OriGene Technologies Inc., Rockvile, MD), SignalSilence® FoxO1 siRNA I (Cat# 6242, Cell Signaling Technology, Danvers, MA), and SignalSilence® FoxO3a siRNA I (Cat# 6302, Cell Signaling Technology, Danvers, MA); Trilencer-27 universal scrambled negative siRNA (Cat# SR30004, OriGene Technologies Inc.) served as a control in these experiments.

Techniques: Knockdown, Activity Assay