Journal: Endocrine-Related Cancer

Article Title: Diet-induced macrophage inhibitory cytokine 1 promotes prostate cancer progression

doi: 10.1530/erc-13-0227

Figure Lengend Snippet: Figure 3 Administration of MIC1 stimulates cell proliferation and ERK1/2 activation in PCa cells. (A) LNCaP and C4-2 cells were cultured with DMEM containing 5% FBS or 5% mouse serum in the presence or absence of the anti-MIC1 neutralizing antibody MAB957 or recombinant MIC1 at the indicated concentrations for 24 h. MTT assay was performed and cell viability was compared with that of cells cultured with FBS. MeanGS.D., *P!0.05. (B) LNCaP cells were cultured with DMEM containing 5% FBS or 5% HFD mouse serum in the presence or absence of MAB957 or MAB004 or 20 ng/ml rMIC1 for 15 min. In the same experiments, the DMEM containing 5% HFD mouse serum had been previously incubated with 10 mg/ml MAB97 or MAB004 at 4 8C overnight. Western blot analysis was performed using anti- ERK1/2, anti-phospho-ERK1/2 (p-ERK1/2), and anti-b actin antibodies.

Article Snippet: The sections of formalin-fixed paraffin-embedded xenograft tumors were stained with rabbit anti-human MIC1 polyclonal antibody (Cell Signaling) used at a 1:100 dilution.

Techniques: Activation Assay, Cell Culture, Recombinant, MTT Assay, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: LPS challenge induces time-dependent mitochondrial dysfunction and metabolic stress with compensatory GDF15 upregulation in liver injury (A) Temporal liver histopathology (H&E) post-LPS. Scale bar: 50 μm. (Time-resolved hepatic damage progression.) (B) Serum ALT/AST kinetics (n = 3). Data: mean ± SD. Study groups and individual replicates are identified in the figure key. ***p < 0.001. (Biomarker-confirmed hepatocyte injury.) (C) Progressive loss of hepatic UQCRC1 (mitochondrial complex III core subunit). β-actin: loading control.(Impaired mitochondrial electron transport.) (D) Time-elevated serum TNF-α, IL-6, and lactate in mice (n = 5). Study groups and individual replicates are identified in the figure key. ***p < 0.001. (Concomitant systemic inflammation and metabolic stress.) (E) Time-elevated cell culture supernatant TNF-α, IL-6, and lactate (n = 5). ***p < 0.001. (Concomitant inflammation and metabolic stress in cell culture system.) (F) Spatiotemporal GDF15-F4/80 co-localization in liver. GDF15 (orange), macrophages (F4/80, green), nuclei (DAPI, blue). Scale bar: 20 μm. Representative image showing focal GDF15 induction. Note that the signal is localized to discrete macrophage-enriched inflammatory niches, consistent with the physiological distribution of immune cells in hepatic tissue. (G) Hepatic GDF15 accumulation after LPS challenge. β-actin served as a loading control. Note: the detected band at ~34 kDa corresponds to the glycosylated pro-form of GDF15. (Liver-wide stress adaptation response.) (H) Persistent UQCRC1 suppression in hepatocytes (validating in vivo impairment in C).(Cell-level mitochondrial dysfunction.) (I) GDF15 induction in RAW264.7 cells. β-actin: loading control. (Cell-autonomous GDF15 upregulation.).

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Histopathology, Biomarker Discovery, Control, Cell Culture, In Vivo

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: Hepatoprotective effects of GDF15 overexpression against LPS-induced injury via mitochondrial function restoration. (A) Robust GDF15 expression in mouse liver following rAAV8-mGdf15 delivery. β-actin: loading control.(rAAV8-mediated hepatic GDF15 overexpression. β-actin: loading control.) (B) H&E-stained liver sections: Untreated (NC), LPS-challenged (LPS), and LPS + rAAV8-mGdf15 (LPS+GDF15). Scale bar: 50 μm. (Histopathological rescue by GDF15.) (C) TUNEL assay (green) showing apoptotic cell death. DAPI (blue): nuclei. GDF15 significantly reduces apoptosis. Scale bars: 20 μm.(GDF15-mediated suppression of apoptosis. TUNEL cells (green), nuclei (DAPI, blue).) (D) Macrophage infiltration (F4/80+, green) attenuated by GDF15 overexpression. DAPI (blue): nuclei. Scale bars: 100 μm.(Inhibition of macrophage recruitment. F4/80+ cells (green), nuclei (DAPI, blue).) (E) Hepatic UQCRC1 recovery with GDF15. β-actin: loading control.(Mitochondrial complex III rescue.) (F) Serum TNF-α, IL-6, and lactate levels (n = 5). Study groups and individual replicates are identified in the figure key. ***p < 0.001. (Systemic inflammation and metabolic stress reversal.) (G) rAAV8-mGdf15 elevates GDF15 in RAW264.7. β-actin: loading control.(Macrophage-targeted GDF15 overexpression.) (H) Effective GDF15 knockdown (si-GDF15) in RAW264.7. β-actin: loading control.(GDF15 knockdown efficiency.) (I) UQCRC1 expression in RAW264.7: Loss of GDF15 (si-GDF15) exacerbates LPS-induced UQCRC1 suppression, while GDF15 restores it.(GDF15-dependent mitochondrial protection in macrophages.) (J) Inflammatory (TNF-α, IL-6) and metabolic (lactate) markers in RAW264.7 supernatant (n = 5 independent experiments). GDF15 inhibits LPS-induced release; si-GDF15 amplifies it. Study groups and individual replicates are identified in the figure key.(GDF15-modulated macrophage inflammatory output.).

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Over Expression, Expressing, Control, Staining, TUNEL Assay, Inhibition, Knockdown

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: GDF15 preserves mitochondrial homeostasis in LPS-stimulated macrophages through dual regulation of SMAD7 and PKM2 pathways. (A) HIF-1α and SMAD7 expression in RAW264.7 macrophages across conditions: Untreated, LPS, LPS with rAAV8-mGdf15 overexpression (LPS+GDF15), and LPS with GDF15 knockdown (si-GDF15). β-actin: loading control.(HIF-1α suppression and SMAD7 induction by GDF15.) (B) Cytosolic and nuclear PKM2 protein levels. Lamin B1 (nuclear) and α-tubulin (cytosolic) markers validate fractionation efficiency. Study groups and individual replicates are identified in the figure key.(PKM2 subcellular redistribution modulated by GDF15.) (C) Immunofluorescence of PKM2 (red) and nuclei (DAPI, blue). Arrows indicate nuclear PKM2 accumulation. Scale bar: 15 μm.(Nuclear PKM2 enrichment upon LPS challenge mitigated by GDF15 and exacerbated by GDF15 knockdown.).

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Expressing, Over Expression, Knockdown, Control, Fractionation, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: HIF-1α and PKM2 are critical effectors of GDF15-driven mitochondrial protection and anti-inflammatory responses. (A) HIF-1α inhibition by BAY 87-2243 (5 μM, 24 h). β-actin: loading control.(Pharmacological HIF-1α blockade.) (B) PKM2 inhibition by Shikonin (2 μM, 24 h). β-actin: loading control.(PKM2 activity suppression.) (C) UQCRC1 recovery in LPS-injured macrophages treated with: GDF15 overexpression, HIF-1α inhibitor (BAY), or PKM2 inhibitor (Shikonin). β-actin: loading control.(Mitochondrial complex III rescue via HIF-1α/PKM2 inhibition mirrors GDF15 effects.) (D) Inflammatory (TNF-α, IL-6) and metabolic (lactate) markers in cell supernatant (n = 5). Study groups and individual replicates are identified in the figure key. ***p < 0.001.(HIF-1α/PKM2 targeting replicates GDF15-mediated anti-inflammatory and metabolic homeostasis.) (E) UQCRC1 expression under GDF15 loss-of-function: si-GDF15 alone vs. combined with BAY 87–2243 or Shikonin. β-actin: loading control. Study groups and individual replicates are identified in the figure key.(Mitochondrial rescue in GDF15-deficient macrophages requires HIF-1α/PKM2 inhibition.) (F) Supernatant cytokines and lactate in si-GDF15 macrophages with/without inhibitors (n = 5). ***p < 0.001. (Inflammation reversal in GDF15-knockdown macrophages depends on HIF-1α/PKM2 blockade.).

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Inhibition, Control, Activity Assay, Over Expression, Expressing, Knockdown

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: SMAD7 activation suppresses HIF-1α to mediate GDF15-dependent mitochondrial protection in LPS-challenged macrophages. (A) Pharmacological SMAD7 activation by Asiaticoside (20 μM, 48 h). β-actin: loading control. (B) HIF-1α expression under LPS challenge: LPS alone, LPS + AVV-GDF15, or LPS + SMAD7 activation (Asiaticoside). β-actin: loading control. (C) HIF-1α modulation across conditions: LPS, LPS + si-GDF15, LPS + Asiaticoside, or LPS + si-GDF15 + Asiaticoside. β-actin: loading control.

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Activation Assay, Control, Expressing

Journal: Frontiers in Immunology

Article Title: GDF15 orchestrates mitochondrial-immune crosstalk via SMAD7-HIF-1α-PKM2 cascade to attenuate septic liver injury

doi: 10.3389/fimmu.2025.1712741

Figure Lengend Snippet: GDF15 correlates with clinical severity in sepsis. (A) Circulating GDF15 levels: Healthy controls (HC, n=91) vs. Sepsis patients (SP, n=119). (B–N) Correlation network of GDF15 with clinical parameters: WBC, CRP, SOFA, SAA, PCT, Glu, Lactate, PLT, SOFA, ALT, AST, TBIL, IL-6 and TNF-α (n=210). Solid lines represent linear regression fits for visual trend estimation, while r and P values are derived from Spearman’s rank correlation analysis.

Article Snippet: Proteins (30 μg/lane) were separated on 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, IPVH00010), and probed with the following primary antibodies: rabbit anti-mouse UQCRC1 (Proteintech, Cat. No. 21705-1-AP, 1:1000), rabbit anti-mouse GDF15 (Proteintech, Cat. No. 27455-1-AP, 1:1000), rabbit anti-mouse HIF-1α (Proteintech, Cat. No. 20960-1-AP, 1:1000), rabbit anti-mouse SMAD7 (Proteintech, Cat. No. 725840-1-AP, 1:1000), mouse anti-mouse PKM2 (Proteintech, Cat. No. 60268-1-Ig, 1:1000), rabbit anti-mouse H3 (Proteintech, Cat. No. 17168-1-AP, 1:5000) and mouse anti-mouse β-actin (Proteintech, Cat. No. 66009-1-Ig, 1:5000).

Techniques: Derivative Assay