Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: Ubiquitin ligase–specific and whole-genome CRISPR-Cas9 screens identify STUB1 and CHIC2 as regulators of CSF2RB protein stability in TF1 and 32D cell lines. A , diagram of CSF2RB reporter. B , bar graph showing normalized GFP/mCherry ratio of CSF2RB reporter in TF1 Cas9 cells treated with DMSO control, 1 μM MLN7243 (E1 inhibitor), 5 μM MLN4924 (neddylation inhibitor), or 10 μM MG132 (proteasomal inhibitor) for 4 h in 5 ng/ml GM-CSF as measured by flow cytometry. Bars are the mean ± SD normalized to the DMSO sample from n = 3 replicates. p -values calculated by unpaired Student’s t test between DMSO and other conditions. C , bar graph showing normalized GFP/mCherry ratio of CSF2RB reporter in TF1 Cas9 cells treated with 10 μM Chloroquine, 100 nM Bafilomycin A1, or a DMSO control for 4 h as measured by flow cytometry. Bars are the mean ± SD normalized to the DMSO sample from three biological replicates. p -values calculated by unpaired Student’s t test between DMSO and other conditions. D , volcano plot showing gene level analysis of CSF2RB reporter-based ubiquitin ligase–specific CRISPR screen in TF1 cells cultured in 5 ng/ml GM-CSF. Guide counts were collapsed to gene level (n = 4 guides/gene; two-sided empirical rank sum test statistics). E , volcano plot showing gene level analysis of CSF2RB reporter-based ubiquitin ligase–specific CRISPR screen in TF1 cells starved (0 min) and then stimulated with 5 ng/ml GM-CSF for 10 and 120 min. Guide counts were collapsed to gene level (n = 4 guides/gene; two-sided empirical rank-sum test statistics). F , volcano plot showing gene level analysis of Csf2rb reporter-based whole-genome CRISPR screen in 32D cells cultured in 0.01 ng/ml IL-3. Guide counts were collapsed to gene level (n = 4 guides/gene; two-sided empirical rank-sum test statistics). G , volcano plot of Pearson correlation coefficient and -log10( p -value) for linear correlation analysis of all gene CERES scores with STUB1 CERES scores in the Cancer Dependency Map (21Q4 public dataset). Inset shows scatterplot of CERES scores for STUB1 (horizontal axis) and CHIC2 (vertical axis).DMSO, dimethyl sulfoxide.

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Ubiquitin Proteomics, CRISPR, Control, Flow Cytometry, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: Stub1 and Chic2 KO lead to increased endogenous total and cell surface Csf2rb. A , Western blots of 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 for STUB1 and Vinculin. B , Western blots of 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 for Chic2 and Vinculin. C , Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 cultured in 5, 0.1, or 0.01 ng/ml IL-3 (S.E. = short exposure, L.E. = long exposure). Representative of three independent biological replicates with similar results. D , Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 cultured in 5, 0.1, or 0.01 ng/ml IL-3. Representative of three independent biological replicates with similar results. E , Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 treated with 10 μM cycloheximide (CHX) for 0, 2, 4, or 8 h. F , Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 treated with 10 μM cycloheximide (CHX) for 0, 2, 4, or 8 h. G , Western blots of Csf2rb, Stub1, Chic2, and Vinculin in 32D Cas9 cells with sgNT/sgNT, sgStub1-1/sgNT, sgNT/sgChic2-1, or sgStub1-1/sgChic2-2. H , bar graph showing ratio of mean GFP to mean mCherry signal of Csf2rb reporter in 32D cells with sgNT, sgChic2-1/2, or sgStub1-1/2 in 0.01 ng/ml IL-3 as measured by flow cytometry. Bars show GFP/mCherry ± SD from n = 3 replicates. I , bar graph showing median fluorescence intensity for anti-Csf2rb-PE (CD131) in 32D Cas9 cells with sgNT, sgChic2-1/2, or sgStub1-1/2 in 0.01 ng/m IL-3 as measured by flow cytometry. Bars show mean ± SD from n = 3 replicates. p -values calculated by unpaired Student’s t test between sgNT and other conditions at each cytokine concentration.

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Western Blot, Cell Culture, Flow Cytometry, Fluorescence, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: The Stub1/Chic2 complex interacts with Csf2rb and Chic2 and Stub1 KO reduce Csf2rb ubiquitination. A , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Chic2, Csf2rb, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. B , Western blots of anti-V5 immunoprecipitation of V5-CHIC2 and whole cell lysate for V5, Stub1, Csf2rb, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. C , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Csf2rb, and Vinculin from 32D Cas9 cells cultured in 5, 0.1, or 0.01 ng/ml IL-3. D , Western blots of anti-V5 immunoprecipitation of STUB1-V5 or STUB1 ΔTPR-V5 and whole cell lysate for V5, Csf2rb, Chic2, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. E , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Csf2rb, Chic2, and Vinculin in 32D Cas9 cells with sgNT or sgChic2-1/2 cultured in 0.01 ng/ml IL-3. F , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Csf2rb, and Vinculin from 32D Cas9 cells cultured in 5, 0.1, or 0.01 ng/ml IL-3. G , Western blots of anti-V5 immunoprecipitation of STUB1-V5 and whole cell lysate for V5, Csf2rb, Chic2, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3 and treated with or without Ruxolitinib (100 nM) for 2 h.

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: Stub1 and Chic2 KO lead to deceased ubiquitination of Csf2rb and blocks the effect of a lysosomal acidification inhibitor on Csf2rb stability. A , Western blots of TUBE immunoprecipitation and whole cell lysate for Csf2rb, Chic2, Stub1, and Vinculin as a loading control in 32D Cas9 cells with sgNT, sgChic2-1/2, or sgStub1-1/2 cultured in 0.01 ng/ml IL-3 and treated with 1 μM Bafilomycin A1 and 10 μM MG132 for 4 h. B , Western blots of Csf2rb and Vinculin in 32D cas9 cells with sgNT, sgStub1-1, or sgChic2-1 treated with DMSO or bafilomycin A1 (100 nM) for 16 h. DMSO, dimethyl sulfoxide.

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Control, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: Primary antibodies:

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common β chain (CSF2RB) protein stability

doi: 10.1016/j.jbc.2022.102484

Figure Lengend Snippet: RT-qPCR Primers

Article Snippet: Mm00655745_m1 , Csf2rb , Mouse , FAM-MGB , Thermo Fisher Scientific , 4331182.

Techniques:

Journal: bioRxiv

Article Title: The emerging fungal pathogen Candida auris induces IFNγ to colonize mammalian hair follicles

doi: 10.1101/2025.01.15.632653

Figure Lengend Snippet: (A-C) scRNAseq dot plot of epithelial cells separated by (A) fungal colonization type or (B) epithelial cell type or (C) IFNγ -/- , WT, IL-17 -/- , and IFNγ OE genotype. Mice were treated with Candida spp indicated on days 0,2,4,6 and back skin was harvested on day 14. (D) Representative 3D confocal microscopy of Stat1 expression in the upper hair follicle in WT mice treated with either mock colonization or C. auris colonization on day 14. (E) Correlation between Stat1 expression, assessed via mean fluorescence index (MFI), and CD8 T cells per hair follicle. (F) Quantitation of Stat1 mean fluorescence intensity (MFI) per hair follicle in mice indicated. WT C. auris colonized mice were subdivided based on having less than or greater than 15 CD8 T-cells per hair follicle. (G) Stat1 MFI in the epithelial surface (CD49f) compared to all other skin structures (non-CD49f). (H) Stat1 MFI between the interfollicular epithelium (IFE), upper hair follicle, and lower hair follicle in mock colonized (white) and C. auris colonized (red) mice. (I,J) Schematics showing models used to test colonization and epicutaneous infection. For epicutaneous infection, intact back skin was pre-colonized with C. auris on day 0,2,4,6; on day 14, hair was removed, and the skin was abraded with sandpaper. C. auris was reapplied on day 15 and back skin was harvested 2 days later for fungal burden. For ‘Re-Colonization’, intact back skin was pre-colonized with C. auris on days 0,2,4,6 and intact back skin was re-colonized on day 14. Back skin was harvested for fungal CFU 5 days after final colonization. Graph showing fungal burden (CFU) after C. auris (I) epicutaneous infection and (J) recolonization of B6 wild type (WT), IFNγ -/- , IL-17 -/- mice, IFNγ OE mice (Yeti/+), and Ker14 ΔIFNγR1 (Ker14 Cre ;IFNγR1 Flox ). (K) IFNγ overexpressing mice ( IFNγ OE ) colonized with C. auris , at day 14 with numerous yeast forms at the hair follicle orifice. *P<0.05, ****P<0.0001 for one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test (F) . In (E), statistics shown comparing C. auris <15 CD8 per hair follicle to all other groups. **P<0.01, ***P<0.001 for Kruskal-Wallis test with Dunn’s multiple comparison test (I,J) . *P<0.05, **P<0.01 student T-Test (G,H). *P<0.05, **P<0.01 Mann-Whitney Test (I,J) .

Article Snippet: To generate keratinocyte specific knock out of IL-17 and IFNγ signaling, we crossed Ker14 Cre mice (B6N.Cg-Tg(KRT14-cre)1Amc/J) from Jackson with Il17Ra floxed mice (B6.Cg-Il17ratm2.1Koll/J; MGI:J:237978) and IFNγR1 floxed mice (C57BL/6N-Ifngr1tm1.1Rds/J, MGI:J:206257) from Jackson Labs. To track type 1 lymphocytes we used Tbet (Tbx21)-zsGreen transgenic mice (generously provided by Jinfang Zhu, Lab of Immune System Biology, NIH).

Techniques: Confocal Microscopy, Expressing, Fluorescence, Quantitation Assay, Infection, Comparison, MANN-WHITNEY

Journal: The FASEB Journal

Article Title: Vascular Bed‐Specific Endothelial Dysfunction and Age‐Dependent Circadian Hypertension in Mice Lacking the Resolvin D2 Receptor GPR18

doi: 10.1096/fj.202503386R

Figure Lengend Snippet: Upper panels show In vivo MRI examinations of endothelial function in the femoral artery (A) and thoracic aorta (B) in response to acetylcholine (ACh, 16.6 mg/kg i.p.) administered to female wildtype (WT, open symbols, n = 6 for the femoral artery and n = 4 for the thoracic aorta) and GPR18 knockout (KO, green symbols, n = 5 for the femoral artery and n = 44 for the thoracic aorta) mice. The unaltered endothelium‐independent responses to infusion of the NO‐donor to sodium nitroprusside (SNP, 1 mg/kg i.v.; WT: N = 5 for the femoral artery and n = 3 for the thoracic aorta; KO: N = 5 for the femoral artery and n = 4 for the thoracic aorta) are shown as insets. Results are shown as changes in vessel volume. The horizontal line represents the mean. p ‐value from Student's t‐test vs. WT. (C) Ex vivo analysis of endothelium‐dependent relaxations in response to ACh administered to 10 μM PE‐precontracted arterial rings of the femoral artery derived from female mice (WT, n = 7; KO, n = 9) in the presence of 10 μM indomethacin. Inset shows endothelial‐independent relaxations in response to DEANO measured in 10 μM PE‐precontracted arterial rings of the femoral artery (WT, n = 8; KO, n = 8), in the presence of 10 μM indomethacin and 300 μM L‐NAME, to inhibit any possible confounding effects of endogenous NOS or COX activity, respectively. The overall P of significant two‐way ANOVA comparison of KO vs. WT is indicated in each concentration‐response graph. * p < 0.05 (Holm‐Sidak post hoc test vs. WT). (D) Representative images and analysis of immunofluorescent staining for endothelial nitric oxide synthase (eNOS) in femoral arteries derived from female GPR18 KO (green circles, n = 3) compared with female WT mice (open circles, n = 4). The horizontal line represents the mean; P‐value from Student's t‐test vs. WT. The micrographs show representative immunofluorescence stainings. Size bar represents 50 μm. (E) Concentration‐response relaxations to acetylcholine (ACh) in the absence (circles; WT, n = 7; KO, n = 10) and presence (squares; WT, n = 7; KO, n = 9) of 10 μM indomethacin in 10 μM PE‐precontracted isolated femoral arteries derived from female WT (left panel with open symbols) and GPR18 KO (left panel with green symbols). The overall p of two‐way ANOVA comparison of KO vs. WT is indicated in each concentration‐response graph. * p < 0.05 (Holm‐Sidak post hoc test vs. WT).

Article Snippet: The breeding and experimental protocols in GPR18 KO (MGI:107859; NCBI ID: 110168; C57BL/6J‐Gpr18 em1 cyagen) mice and WT littermates were approved by the local ethics committee and the Ministry of National Education, Higher Education, and Research and Innovation in France (APAFIS#22187‐2 019 093 011 424 973 v5).

Techniques: In Vivo, Knock-Out, Ex Vivo, Derivative Assay, Activity Assay, Comparison, Concentration Assay, Staining, Immunofluorescence, Isolation

Journal: The FASEB Journal

Article Title: Vascular Bed‐Specific Endothelial Dysfunction and Age‐Dependent Circadian Hypertension in Mice Lacking the Resolvin D2 Receptor GPR18

doi: 10.1096/fj.202503386R

Figure Lengend Snippet: Telemetry mean arterial blood pressures (MAP) in young (A–C) and old (D) WT (open symbols) and GPR18 KO (green symbols) mice. Each datapoint represents the MAP recorded during 1 h for all mice of each group during 3 days and nights. (A, B): The comparison between males (WT, n = 3; KO, n = 3) and females (WT, n = 4; KO, n = 3) in sex‐disaggregated analysis did not reveal any significant sex differences in either WT or GPR18 KO young mice. (C): In the sex aggregated analysis, with young KO ( n = 7; n = 3 males [4.64 ± 0.51 months] and n = 4 females [4.13 ± 0.19 months]) did not exhibit any significant differences compared with young WT ( n = 7; n = 3 males [4.49 ± 0.45 months] and n = 4 females [4.13 ± 0.08 months]) during either day or night. (D): Old KO ( n = 4 [3 females and 1 male; 20.7 ± 0.63 months]) exhibited a significantly increased MAP during daytime and a moderate increase during active night‐time compared with old WT mice ( n = 4 [2 females and 2 males; 21.3 ± 0.48 months]).

Article Snippet: The breeding and experimental protocols in GPR18 KO (MGI:107859; NCBI ID: 110168; C57BL/6J‐Gpr18 em1 cyagen) mice and WT littermates were approved by the local ethics committee and the Ministry of National Education, Higher Education, and Research and Innovation in France (APAFIS#22187‐2 019 093 011 424 973 v5).

Techniques: Comparison