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

Journal: Materials Today Bio

Article Title: 4D-printed Fe3O4-PLLA scaffolds modulate osteoimmune microenvironment for oral bone repair

doi: 10.1016/j.mtbio.2025.102691

Figure Lengend Snippet: In vitro immunomodulatory properties of PLLA, Fe 3 O 4 -PLLA, CSMA/Fe 3 O 4 -PLLA, NG-CSMA/Fe 3 O 4 -PLLA scaffolds. a-b) 3 days of co-culture of RAW 264.7 with PLLA, Fe 3 O 4 -PLLA, CSMA/Fe 3 O 4 -PLLA, and NG-CSMA/Fe 3 O 4 -PLLA scaffolds, flow cytometry was performed to analyze M1, M2 markers CD86 and CD206. c) mRNA levels of TNF-α, IL-1β, Arg-1, and CD206 by qRT-PCR; d) Immunofluorescence images of M1 (iNOS) and M2 (Arg-1) macrophage markers; and e) quantitative analysis of fluorescence intensity. n = 3. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Article Snippet: After 3 days, cells were harvested, blocked, and stained with PE-anti-CD206 and CL647- anti -CD86 antibodies (Proteintech, China).

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Quantitative RT-PCR, Immunofluorescence, Fluorescence