Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Deep spatial profiling of human palatine tonsil tissues. (A) Hematoxylin and eosin (H&E) staining after 92 MICS cycles, including the marked epithelium, germinal center (GC), and T cell zone of the lymphoid follicle. MICS DAPI and stroma staining depicting the composition and structure of the tonsil. Markers: Collagen III, collagen IV, fibronectin (all extracellular matrix (ECM), cytokeratin (epithelium), podoplanin (lymphatic vessels), CD105/SM Actin (blood vessels). (B) Immune cell content of a human palatine tonsil comprising T cells (CD3), B cells (CD19/CD20), plasma cells (PCs) (CD38/CD138), NK cells (CD56), granulocytes (CD15/CD66b), mast cells (CD117), macrophages (MΦ) (CD163/CD169/CD206), myeloid dendritic cells (mDCs) (CD11c), and plasmacytoid dendritic cells (pDCs) (CD123). (C) Detailed view on the T cell zone, mainly composed of CD4 + helper T cells (T h ) and CD8 + cytotoxic T cells (T c ), mDCs (CD11c), and PCs (CD38/CD138). (D) Detailed view on the GC-mantle zone border, showing different B cells (CD11b, CD21, CD22), mDCs (CD11c), and PCs (CD38/CD138). (E) Cell annotations of three different tonsil samples plus respective bar graphs of gated cell populations, comparing the cell content between the three tonsil samples. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: 976 x 640 µm, zoomed-in subregions in (C, D) : 334 µm x 219 µm. Scale bar: 100 µm.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Staining, Clinical Proteomics

Journal: iScience

Article Title: Aurkb deficiency disrupts microglial development, homeostasis and hinders remyelination following cuprizone-induced demyelination

doi: 10.1016/j.isci.2026.114718

Figure Lengend Snippet: Aurkb loss transiently elevates CD68 in homeostatic microglia but compromises its upregulation in an LPS-induced inflammation model (A–D) Adult Aurkb fl/fl and Cx3cr1 CreERT2/+ Aurkb fl/fl littermates (8-week-old) were i.p. injected with TAM for 5 consecutive days, followed by tissue collection at 1-month and 3-month post TAM induction. Representative immunofluorescence and quantification of CD68 in microglia at (A and B) 1-month and (C and D) 3-month post-TAM induction ( n = 5 mice per genotype per time point, Scale bars: 50 μm). (E–H) Neonatal Aurkb fl/fl and Cx3cr1 CreERT2/+ Aurkb fl/fl littermates were i.p. injected with TAM for 3 consecutive days at P1-P3, followed by tissue collection at P13. Representative immunofluorescence and quantification of (E and F) CD68 and (G and H) CD206 in microglia at P13 ( n = 6 mice per genotype, Scale bars: 50 μm). (I and J) Representative immunofluorescence and quantification of CD68 in microglia from adult Aurkb fl/fl and Cx3cr1 Cre/+ Aurkb fl/fl littermates ( n = 5 mice per genotype, Scale bars: 50 μm). The representative immunofluorescence image of the Cx3cr1 Cre/+ Aurkb fl/fl group is shared in C. (K and L) Adult Aurkb fl/fl and Cx3cr1 Cre/+ Aurkb fl/fl littermates were i.p. injected with LPS (1 mg/kg) and sacrificed at 48 h post LPS administration ( n = 5 mice per genotype, Scale bars: 50 μm). Data are presented as the mean ± SD. Two-tailed unpaired t-tests in (B, D, J, and I); two-way ANOVA with Bonferroni multiple comparisons test in (F, H); ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant compared with the Aurkb fl/fl group. See also and .

Article Snippet: The following primary antibodies were used: rabbit anti-Iba-1 antibody (1:500, Wako, Cat: 019–19741), mouse anti-Iba-1 antibody (1:400, Abcam, Cat: ab283319), rat anti-CD206 antibody (1:200, Bio-Rad, Cat: MCA2235), rat anti-BrdU antibody (1:400, Abcam, Cat: ab6326), mouse anti-phospho-Histone H3 (Ser10) antibody (1:200, Cell Signaling Technology, Cat: 9706S), mouse anti-APC (1:100, CC-1, Merck, Cat: OP80), rat anti-myelin basic protein (Mbp) monoclonal antibody (1:500, Abcam, Cat: ab7349), rabbit anti-degraded myelin basic protein (dMbp) antibody (1:2000, Millipore Sigma, Cat: AB5864), rabbit anti-Olig2 antibody (1:500, Proteintech, Cat: 13999-1-AP) and rat anti-CD68 antibody (1:500, Abcam, Cat: ab53444).

Techniques: Injection, Immunofluorescence, Two Tailed Test

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: ZXDB is required for pro‐inflammatory macrophage activation and metabolic reprogramming. (A, B) Flow cytometric analysis of M1‐like (CD86 + INOS + ), M2a‐like (CD206 + ARG1 + ), and M2b‐like (CD86 + IL‐10 + ) surface markers on RAW264.7 (A) and THP‐1 (B) macrophages following stimulation with LPS (100 ng/mL) for 6 h, with or without Zxdb knockdown (shZxdb). Representative plots and quantification are shown. (C, D) Western blot analysis of key M1‐like (iNOS, CD40, CD86, CD80) and M2‐like (CD206, CD163, Arg1) protein markers in RAW264.7 (C) and THP‐1 (D) cells under the same conditions. GAPDH served as the loading control. (E, F) ELISA quantification of pro‐inflammatory (TNF‐α, IFN‐γ, IL‐1β) and anti‐inflammatory (IL‐10, IL‐4, IL‐13) cytokines secreted into the supernatant of RAW264.7 (E) and THP‐1 (F) cells. (G‐J) Assessment of metabolic state via relative lactate production (G, H) and intracellular ATP levels (I, J) in RAW264.7 and THP‐1 cells. p < 0.05, * p < 0.01, ** p < 0.001.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Activation Assay, Knockdown, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: ACACA mediates the pro‐inflammatory action of ZXDB on macrophages. (A) Overlap of ZXDB‐regulated genes/proteins and ACACA‐interacting proteins. (B) Protein–protein interaction network showing ACACA's association with the ZXDB network. (C) ACACA mRNA expression in THP‐1 cells, treated with LPS (100 ng/mL) +/− shZxdb for 6 h. (D) ACACA protein expression in THP‐1 cells, treated as in (C). (E–H) Cell proliferation (E, G) and apoptosis (F, H) in RAW264.7 (E, F) and THP‐1 cells (G, H) subjected to rescue experiments (LPS +/− shZxdb +/− Acaca overexpression). (I‐J) M1‐like (CD86 + INOS + ), M2a‐like (CD206 + ARG1 + ), and M2b‐like (CD86 + IL‐10 + ) macrophage populations in RAW264.7 (I) and THP‐1 cells (J) from the rescue experiments. (K‐L) Cytokine secretion (TNF‐α, IFN‐γ, IL‐1β, IL‐10, IL‐4, IL‐13) in RAW264.7 (K) and THP‐1 cell supernatants from the rescue experiments. (M‐P) Relative ATP levels (M, N) and lactate production (O, P) in RAW264.7 (M, O) and THP‐1 cells (N, P) from the rescue experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Expressing, Over Expression

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: The ZXDB‐EIF4A3 Interaction Is required for Its Pro‐inflammatory Functions. (A) Co‐IP of HA‐EIF4A3 with Flag‐ZXDB or ZXDB‐MUT in THP‐1 cells. (B) ACACA protein expression in THP‐1 cells transfected with the indicated plasmids and treated with LPS for 6 h. (C) Polysome profiling of ACACA mRNA in THP‐1 cells with Flag‐ZXDB or ZXDB‐MUT, stimulated with LPS. (D) RIP‐qPCR analysis of ACACA mRNA associated with ZXDB or ZXDB‐MUT. (E‐F) Cell proliferation (E) and apoptosis (F) in THP‐1 cells transfected and treated with LPS for 6 h. (G) M1‐like (CD86 + INOS + ), M2a‐like (CD206 + ARG1 + ) and M2b‐like (CD86 + IL‐10 + ) populations in THP‐1 cells. (H) M1‐like (iNOS, CD40, CD86, CD80) and M2‐like (CD206, CD163, ARG1) protein expression in THP‐1 cells. (I) Cytokine secretion (IFN‐γ, TNF‐α, IL‐6, IL‐1β, IL‐4, IL‐13, IL‐10) in THP‐1 cell supernatants. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Co-Immunoprecipitation Assay, Expressing, Transfection

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: Macrophage‐Specific Deletion of Zxdb Protects Against Sepsis‐Induced Acute Kidney Injury. (A) Generation strategy for myeloid‐specific Zxdb knockout (Mac‐Zxdb‐KO) mice. (B) H&E staining of kidney sections from Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice. (C‐D) Serum creatinine (Scr) (C) and blood urea nitrogen (BUN) (D) levels in Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice at 24 h post‐CLP. (E) KIM‐1 immunohistochemistry of kidney sections from Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice. (F) TUNEL staining of kidney sections from Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice. (G) Immunofluorescence of kidney sections stained for F480, CD86, and CD206. (H) Serum IL‐1β, IL‐10, and TNF‐α levels in Sham, WT SI‐AKI, and Mac‐Zxdb‐KO SI‐AKI mice at 24 h post‐CLP. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Knock-Out, Staining, Immunohistochemistry, TUNEL Assay, Immunofluorescence

Journal: The FASEB Journal

Article Title: ZXDB Drives Macrophage Inflammatory Programming in Sepsis‐Induced Acute Kidney Injury by Recruiting EIF4A3 to Enhance ACACA Translation

doi: 10.1096/fj.202502962RR

Figure Lengend Snippet: Schematic diagram of the proposed mechanism by which ZXDB promotes M1‐like macrophage polarization and exacerbates SI‐AKI. In macrophage, ZXDB interacts with EIF4A3, promoting the translation of the ACACA gene. The resulting increase in ACACA protein expression enhances glycolysis and lactate production. This metabolic reprogramming shifts macrophage polarization toward M1‐like macrophage activation (characterized by increased Cd86, Cd80, Cd40, and iNOS) and away from an anti‐inflammatory M2‐like phenotype (characterized by Cd206, Cd163, and Arg1). The dominance of M1‐like macrophages leads to an elevated secretion of pro‐inflammatory cytokines (TNF‐α, IFN‐γ, IL‐1β) and reduced anti‐inflammatory cytokines (IL‐10, IL‐4, IL‐13), which collectively drive the pathogenesis of acute kidney injury.

Article Snippet: Membranes were then incubated overnight at 4°C with primary antibodies against: ZXDB (A303‐656A; Invitrogen, 1:1000), ACACA (21923‐1‐AP; Proteintech, RRID:AB_11042445, 1:1000), EIF4A3 (17 504‐1‐AP; Proteintech, RRID:AB_2097393, 1:1000), iNOS (22226‐1‐AP; Proteintech, RRID:AB_2879038, 1:1000), CD86 (13395‐1‐AP; Proteintech–, RRID:AB_2074882, 1:1000), ARG1 (16001‐1‐AP; Proteintech–, RRID:AB_2289842, 1:1000), CD206 (18704‐1‐AP; Proteintech, RRID:AB_10597232, 1:1000), GAPDH (60004‐1‐Ig; Proteintech, RRID:AB_2107436, 1:10000).

Techniques: Expressing, Activation Assay