acc1  (Proteintech)

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: ZXDB interacts through its RRM domain with EIF4A3 to stimulate translation of ACACA mRNA. (A) ZXDB IP from THP‐1 cell lysates, followed by SDS‐PAGE and Coomassie staining. (B) Overlap of ZXDB interactors identified by IP‐MS and EIF4A3 interactors. (C) Co‐IP of endogenous ZXDB and EIF4A3 from THP‐1 cells. (D) Co‐IP of Flag‐ZXDB and HA‐EIF4A3, ectopically expressed in THP‐1 cells. (E) Co‐localization of endogenous ZXDB (red) and EIF4A3 (green) in THP‐1 cells by immunofluorescence. (F, G) Effect of EIF4A3 knockdown (shEIF4A) on ACACA mRNA (F) and protein (G) expression. THP‐1 cells transfected with indicated plasmids were analyzed. (H) Polysome profiling of ACACA mRNA in THP‐1 cells transfected with indicated constructs and stimulated with LPS. (I) ACACA 5′UTR‐luciferase reporter assay in THP‐1 cells, treated with indicated plasmids. (J, K) RIP‐qPCR analysis of ACACA mRNA associated with endogenous ZXDB (J) or EIF4A3 (K) in THP‐1 cells. (L) RNA pull‐down assay: EIF4A3 binding to ACACA 5′UTR probe. (M) Schematic of ZXDB and its truncation mutants. (N) Co‐IP of HA‐EIF4A3 with Flag‐tagged ZXDB truncations in HEK293T cells. (O) GST pull‐down assay: Interaction of GST‐ZXDB truncation mutants with His‐EIF4A3. * 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: SDS Page, Staining, Protein-Protein interactions, Co-Immunoprecipitation Assay, Immunofluorescence, Knockdown, Expressing, Transfection, Construct, Luciferase, Reporter Assay, Pull Down Assay, Binding 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: 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: 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