Journal: Disease Models & Mechanisms

Article Title: C-C motif receptor 2 is a core profibrotic factor in uremic cardiomyopathy

doi: 10.1242/dmm.052395

Figure Lengend Snippet: CCR-2 blockade suppresses left ventricular (LV) fibrosis in MNx-induced UC. (A) Schedule of INCB3344 administration. w, weeks. (B) CCR-2 inhibition significantly alleviated LV fibrosis (scale bars: 50 μm), and slightly reduced myocyte cross-section area. 2-week CCR-2 treatment obtained similar effects but without causing reduced myocyte cross-section area (scale bars: 50 μm, n =360 cells per group). (C) UC model with INCB3344 treatment demonstrated larger heart (Gross, scale bar: 2 mm), slightly reduced LV wall thickness, increased LV diameter [B-mode, scale bar: 2 mm; M-mode, scale bars: 1 mm (longitudinal) and 100 ms (transverse); WGA, scale bar: 50 μm] and compromised LV systolic function. However, 2-week CCR-2 treatment did not induce LV dilation and compromised LV systolic function. Sham (sham group), n =6; MNx, n =6; MNx+5w INCB3344 (modified nephrectomy group with 5-week INCB3344 injection), n =6; MNx+2w INCB3344 (modified nephrectomy group with 2-week INCB3344 injection), n =6. One-way ANOVA followed by Tukey's multiple comparisons test or Kruskal–Wallis test followed by Dunn's multiple comparisons test was used. * P <0.05, ** P <0.01.

Article Snippet: For CCR-2 analysis, LV tissue slides were fixed in EDTA pH 9.0 solution at 95°C for 10 min after rehydration, followed by incubation with peroxidase blocking buffer (ZSGB-BIO Co.; ZLI-9311) for 15 min. After PBS washing, slides were incubated with QuickBlock Blocking Buffer for Immunol Staining (Beyotime Biotechnology Co., Ltd.) for 30 min. Antibodies against CCR-2 (1:100; Proteintech Group, Inc.; 16153-1-AP) were applied overnight at 4°C, followed by incubation with goat anti-rabbit IgG (ZSGB-BIO Co.; PV-6000) for 30 min.

Techniques: Inhibition, Modification, Injection

Journal: Disease Models & Mechanisms

Article Title: C-C motif receptor 2 is a core profibrotic factor in uremic cardiomyopathy

doi: 10.1242/dmm.052395

Figure Lengend Snippet: INCB3344 reduces LV inflammation and may increase LV residual macrophages. (A) Inhibition of CCR-2 downregulated the expression of markers of inflammation. (B) Inhibition of CCR-2 upregulated CCR-2 − residual macrophage markers in LV tissues. (C) Immunofluorescence showed increased infiltration of F4/80 + , TIMD-4 + and CD163 + cells in LV tissues after INCB3344 injection, and some F4/80 + cells were co-stained with CD163 and TIMD-4. White arrows indicate F4/80 + TIMD-4 + CD163 + cells (scale bar: 100 μm). (D) Flow cytometry showed that INCB3344 increased the proportion of CD163 + TIMD-4 + cells in F4/80 + cells derived from LV tissues. Sham, n =6; MNx, n =6; MNx+5w INCB3344, n =6; MNx+2w INCB3344, n =6. One-way ANOVA followed by Tukey's multiple comparisons test or Kruskal–Wallis test followed by Dunn's multiple comparisons test was used. * P <0.05, ** P <0.01.

Article Snippet: For CCR-2 analysis, LV tissue slides were fixed in EDTA pH 9.0 solution at 95°C for 10 min after rehydration, followed by incubation with peroxidase blocking buffer (ZSGB-BIO Co.; ZLI-9311) for 15 min. After PBS washing, slides were incubated with QuickBlock Blocking Buffer for Immunol Staining (Beyotime Biotechnology Co., Ltd.) for 30 min. Antibodies against CCR-2 (1:100; Proteintech Group, Inc.; 16153-1-AP) were applied overnight at 4°C, followed by incubation with goat anti-rabbit IgG (ZSGB-BIO Co.; PV-6000) for 30 min.

Techniques: Inhibition, Expressing, Immunofluorescence, Injection, Staining, Flow Cytometry, Derivative Assay

Journal: Cell stem cell

Article Title: Oligodendrocyte Death in Pelizaeus-Merzbacher Disease Is Rescued by Iron Chelation.

doi: 10.1016/j.stem.2019.09.003

Figure Lengend Snippet: Figure 7. Iron Chelation Rescues Phenotypes of Multiple PLP1 Mutations In Vitro and In Vivo (A) Treatment strategy to assess effects of DFO in additional human PMD mutations. (B–G) Differentiation phenotype of additional human PMD mutations after treatment with DFO (E–G) compared to control culture condition (B–D) (n = 3–4, biological replicates). B&E, T75P point mutation; C&F, Exon6 deletion; D&G, duplication. (H) Jimpy mouse OPCs isolated at postnatal days 5–8 were tested for differentiation phenotype and transiently treated with iron chelation. (I–L) Jimpy OPCs (I) completely failed to differentiate upon removal of growth factors that was associated with increased cell death. Transient (4 day) DFO treatment (J) rescued both differentiation and cell death as measured by caspase-3 and MBP expression (n = 10 fields/treatment from 2 biological replicates) (K and L). (M) In vivo iron chelation strategy in Jimpy mice. Subcutaneous injections with the brain-permeable iron chelator deferiprone (DFP) were given from postnatal days 7–14 and 21–death. (N and O) Corpus callosum of vehicle-treated Jimpy mice (N) at postnatal day 28 showed caspase-3+ apoptotic cells compared to the DFP-treated Jimpy mice (O). (P–Q0) Electron microscopic analysis of corpus callosum in DFP- (Q) and vehicle-treated (P) Jimpy mice showed surviving oligodendrocytes in the DFP-treated mice. Higher magnification of (P) and (Q) are depicted in (P0) and (Q0), respectively. (R–W) Fluorescent in situ hybridization (RNA scope) showed a significant reduction in Atf4 (R, S, and V) and Chop (T, U, and W) gene expressions in Mbp+ mature oligodendrocytes after a 7-day in vivo DFP treatment from P7 in Jimpy mice (n = 3–4). (legend continued on next page) 538 Cell Stem Cell 25, 531–541, October 3, 2019

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Olig2 Courtesy of Charles Stiles N/A NKX2.2 Developmental Studies Hybridoma Bank Cat# 74.5A5 SOX1 R&D Cat# AF3369 PAX6 Developmental Studies Hybridoma Bank Cat# Pax6 O4 Mouse Hybridoma N/A PLP1 Abcam Cat# 28486 Tra1-60 Cell Signaling 4746T Oct3/4 Santa Cruz sc-5279 MBP AbD Serotec Cat# MCA409S Cleaved Caspase-3 Cell Signaling Cat# 9664S KDEL Abcam Cat# ab50601 GFP Aveslab Cat# GFP-1020 Human cytoplasmic marker Clontech Cat# STEM121 NeuN EMD Millipore Cat# MAB377 PDGFRa BD Bioscience Cat# 556001 Transferrin Abcam Cat# ab137744 Ferritin Novus Biologicals Cat# NB600-920 IRP1 Cell Signaling Cat# 20272 IRP2 Cell Signaling Cat# 37135S Transferrin receptor BD Bioscience Cat# 561939 Ferroportin Novus Biologicals Cat# NBP1-21502 beta Actin Sigma Cat# A5441 GFAP Fisher Cat# 13-0300 Iba1 Wako Cat# 019-19741 EIf2a Cell Signaling Cat# 9722 Phospho-EIf2a Fisher Cat# MA5-15133 ATF4 Aviva Systems Biology Cat# ARP37017 CHOP Fisher Cat# MA1-250 O4-APC Miltenyi Biotec Inc 130-099-211 TFRC-GFP BD Biosciences 561939 Bacterial and Virus Strains AAV serotype DJ This paper Melo et al., 2014 Lenti-GFP Addgene Tet-O-FUW-EGFP Biological Samples Dermal fibroblasts from PMD patients This paper N/A Human brain tissues from fetal gestational week 20 to 23 This paper N/A Chemicals, Peptides, and Recombinant Proteins T3 Sigma Cat# T6397 SB431542 Tocris Cat# 1614 LDN193189 Stemgent Cat# 04-0074 SAG Shenzhen Shengjie Chemical Cat# 912545-86-9 Thiazovivin Santa Cruz Cat# sc-361380 basic FGF Thermo Fisher Cat# PHG0024 PDGF-A Peprotech Cat# 100-13A (Continued on next page) e1 Cell Stem Cell 25, 531–541.e1–e6, October 3, 2019

Techniques: In Vitro, In Vivo, Control, Mutagenesis, Isolation, Expressing, In Situ Hybridization, RNAscope

Journal: Clinical and Translational Medicine

Article Title: LRH‐1/NR5A2 targets mitochondrial dynamics to reprogram type 1 diabetes macrophages and dendritic cells into an immune tolerance phenotype

doi: 10.1002/ctm2.70134

Figure Lengend Snippet: Reagents and resources used in this study.

Article Snippet: CD194 , Miltenyi Biotec , Cat#130‐117‐376.

Techniques: Recombinant, Cell Isolation, TUNEL Assay, Extraction, Modification, Multiplex Assay, Software, Selection, Control

Journal: Clinical and Translational Medicine

Article Title: LRH‐1/NR5A2 targets mitochondrial dynamics to reprogram type 1 diabetes macrophages and dendritic cells into an immune tolerance phenotype

doi: 10.1002/ctm2.70134

Figure Lengend Snippet: LRH‐1/NR5A2 agonism alters CD4 + and CD8 + T‐cell subpopulations in T1D individuals. PBMCs were purified from individuals with T1D and exposed to 10 µM BL001 every 24 h for a total duration of 48 h with a final dose given 30 min prior to analysis. Flow cytometry immunophenotyping of (A) CD4 + CD25 + and (B) Tregs; CD4 + CD25 + FoxP3 + cell subpopulations. The initial gating defining the CD4 population was always set at 10 000 cells. n = 8 independent individuals with T1D. Relative transcript levels of (C) FoxP3, (D) CTLA4 and (E) GATA3. Data were normalised to the housekeeping gene RSP9. n = 5 T1D independent donors. Flow cytometry immunophenotyping of (F) Th1; CD4 + CD196 − CD183 + CD194 − and (G) Th2; CD4 + CD196 − CD183 − CD194 + . The initial gating defining the CD4 population was always set at 10000 cells. n = 8 independent individuals with T1D. Relative (H) transcript and (I) secreted levels of IFNγ as well as of (J) IL‐4. Transcript levels of IFNγ were normalised to the housekeeping gene RSP9. n = 4–6 T1D independent donors. (K) Th17/22; CD4 + CD196 + CD183 − CD194 + immunophenotyping and (L) IL17 transcript levels normalised to the housekeeping gene RSP9. n = 6–8 T1D independent donors. (M) CD8 + immunophenotyping. n = 8 independent individuals with T1D. (O) CD4 + cells were isolated from PBMCs and treated with BL001 as described above. Cells were then analysed by flow cytometry for the cell surface markers CD25 + FoxP3 + . Relative (P) LRH‐1/NR5A2 and (Q) FoxP3 transcript levels in either siScrambled (siSc) or siNR5A2‐treated PBMCs. Data were normalised to the housekeeping gene RSP9. n = 3 T1D independent donors. CD14 − PBMCs were labelled with CFSE and stimulated/expanded using antiCD3/CD28 before the addition of CD4 + T‐cells (at a 1:2 ratio, respectively, from the same donor). Proliferation of (R) CD4 + /CFSE + and (S) CD8 + /CFSE + subpopulations was assessed by flow cytometry 4 days post co‐culturing. n = 6 T1D independent donors. Each donor is colour coded. Data are presented as means ± SEM. Paired Student t ‐test * p < 0.05 and ** p < 0.01 as compared with untreated cells.

Article Snippet: CD194 , Miltenyi Biotec , Cat#130‐117‐376.

Techniques: Purification, Flow Cytometry, Isolation

Journal: iScience

Article Title: Single-cell RNA-sequencing of circulating eosinophils from asthma patients reveals an inflammatory signature

doi: 10.1016/j.isci.2025.112609

Figure Lengend Snippet: Asthmatic eosinophils have distinct gene expression profiles from healthy eosinophils (A) UMAP plot of down sampled eosinophils from healthy and asthma patients colored by condition (left) and cluster (right). (B) Bar plot of fraction of cells per condition for each cluster. (C) Volcano plot of significant DEGs in healthy vs. asthmatic eosinophils (P adj < 0.05, log2FC > 0.25). The top 5 most significant genes per group are labeled. (D) Violin plots of expression of class I HLA genes, antigen presentation genes, granule genes and CCR3. Adjusted p value from Wilcoxon rank-sum test.

Article Snippet: CD193 (CCR3) FITC , REA574 , 2 , Eosinophil isolation buffer , 130-116-662 , Miltenyi.

Techniques: Gene Expression, Labeling, Expressing, Immunopeptidomics