Journal: Bioactive Materials

Article Title: Magnesium ion implantation enhances the osseointegration and vascularization of 3D-Printed CoCrMo alloy scaffolds for load-bearing orthopedic applications

doi: 10.1016/j.bioactmat.2025.11.012

Figure Lengend Snippet: Evaluation of angiogenic, and osteogenic abilities in vitro . (A) Immunofluorescence staining of HUVECs for VEGF. (B) Fluorescence intensity quantification of VEGF. (C and D) The expression levels of angiogenesis-related genes VEGF and HIF-1α in HUVECs. (E) ELISA analysis of VEGF secretion by HUVECs. (F) Western blot analysis of angiogenesis-related proteins in HUVECs. (G and H) Quantitative analysis of Western blot in HUVECs normalized to β-actin. (I) Images of ALP staining on day 7 and 14. (J) Images of ARS staining on day 21 and 28. (K) Quantitative analysis of ALP detected by the ALP assay. (L) Quantitative analysis of calcium nodule elution. (M) Immunofluorescence staining of BMSCs for OPN. (N) Fluorescence intensity quantification of OPN. (O–Q) The expression levels of osteogenic-related genes OPN, COL-I and ALP in BMSCs. (R) Western blot analysis of osteogenesis-related proteins in BMSCs. (S–U) Quantitative analysis of Western blot in BMSCs normalized to β-actin. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Western blotting was then performed using the same method as described above, with primary antibody against p-VEGFR2 (Affinity Biosciences, China) and HIF-1α (Proteintech, China).

Techniques: In Vitro, Immunofluorescence, Staining, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, ALP Assay

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: Pharmacological agents included: HIF-1α inhibitor BAY 87-2243 [MedChemExpress, CAS 1227158-85-1; a potent and selective inhibitor of mitochondrial complex I that effectively suppresses HIF-1α protein accumulation under normoxic and hypoxic conditions ( )], PKM2 inhibitor Shikonin [MedChemExpress, CAS 54952-43-1; a specific inhibitor that binds to the PKM2 subunit, suppressing its enzymatic activity and nuclear translocation, with well-documented anti-inflammatory effects ( )], and SMAD7 activator Asiaticoside (MedChemExpress, 16830-15-2; a triterpenoid compound known to upregulate SMAD7 expression and ameliorate inflammation in macrophage models ( )).

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: Pharmacological agents included: HIF-1α inhibitor BAY 87-2243 [MedChemExpress, CAS 1227158-85-1; a potent and selective inhibitor of mitochondrial complex I that effectively suppresses HIF-1α protein accumulation under normoxic and hypoxic conditions ( )], PKM2 inhibitor Shikonin [MedChemExpress, CAS 54952-43-1; a specific inhibitor that binds to the PKM2 subunit, suppressing its enzymatic activity and nuclear translocation, with well-documented anti-inflammatory effects ( )], and SMAD7 activator Asiaticoside (MedChemExpress, 16830-15-2; a triterpenoid compound known to upregulate SMAD7 expression and ameliorate inflammation in macrophage models ( )).

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: Pharmacological agents included: HIF-1α inhibitor BAY 87-2243 [MedChemExpress, CAS 1227158-85-1; a potent and selective inhibitor of mitochondrial complex I that effectively suppresses HIF-1α protein accumulation under normoxic and hypoxic conditions ( )], PKM2 inhibitor Shikonin [MedChemExpress, CAS 54952-43-1; a specific inhibitor that binds to the PKM2 subunit, suppressing its enzymatic activity and nuclear translocation, with well-documented anti-inflammatory effects ( )], and SMAD7 activator Asiaticoside (MedChemExpress, 16830-15-2; a triterpenoid compound known to upregulate SMAD7 expression and ameliorate inflammation in macrophage models ( )).

Techniques: Activation Assay, Control, Expressing

Journal: Cell Death Discovery

Article Title: Mitochondrial retrograde signaling initiates HIF-1α/BNIP3/NIX-mediated mitophagy in Tibetan high-altitude adaptation

doi: 10.1038/s41420-025-02933-8

Figure Lengend Snippet: A , B Western blot analysis of HIF-1α, BNIP3, NIX, MFN1, DRP1, LC3B, and p62 protein levels in M7/8+shCTR ( n = 4), M9a+shCTR ( n = 4), and M9a+sh EPAS1 ( n = 4) cells under hypoxia. Grayscale analysis of target protein levels was normalized to β-Tubulin. C Autophagosomes were analyzed in M7/8+shCTR ( n = 2), M9a+shCTR ( n = 2), and M9a+sh EPAS1 ( n = 2) cells using TEM under hypoxia. Scale bar: 1 μm. D Autophagic flux was assessed by quantifying the colocalization of LC3B (green) and Mitotracker (red) (marking mitophagosomes) using immunofluorescence in M7/8+shCTR ( n = 4), M9a+shCTR ( n = 4), and M9a+sh EPAS1 ( n = 4) cells under hypoxia. DAPI (blue) stains the nucleus. Scale bar: 20 μm. E Cytoplasmic free mtDNA was determined by staining the double-stranded DNA (dsDNA) (green) and Mitotracker (red) using immunofluorescence in M7/8+shCTR ( n = 4), M9a+shCTR ( n = 4), and M9a+sh EPAS1 ( n = 4) cells under hypoxia. DAPI (blue) stains the nucleus. dsDNA that does not colocalize with either the nucleus or mitochondria is considered cytoplasmic free mtDNA. Scale bar: 20 μm. F Western blot analysis of NLRP3, IL1β, cGAS, STING, P65, and P-P65 protein levels in M7/8+shCTR ( n = 4), M9a+shCTR ( n = 4), and M9a+sh EPAS1 ( n = 4) cells under hypoxia. Grayscale analysis of target protein levels was normalized to β-Tubulin. G Inflammatory cytokines IL6, IL1β, and TNF-α in the culture medium were measured in M7/8+shCTR ( n = 4), M9a+shCTR ( n = 4), and M9a+sh EPAS1 ( n = 4) cells under normoxic and hypoxic conditions using an ELISA kit. All cells were cultured under normoxia (21% O₂) or hypoxia (1% O₂) for 48 h.#1–4 represent individual samples. Data are presented as the mean ± SD of three independent experiments. One-way ANOVA followed by Tukey’s test; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Inhibitors were used as follows: N-acetylcysteine (NAC) was dissolved in the medium to a final concentration of 10 mM (diluted in ddH2O, Sigma, USA), the HIF-1α inhibitor PX-478 was dissolved in ddH2O to a final concentration of 30 μM (MCE, USA), and Mdivi-1 was used at 50 μM (diluted in DMSO, MCE, USA).

Techniques: Western Blot, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cell Death Discovery

Article Title: Mitochondrial retrograde signaling initiates HIF-1α/BNIP3/NIX-mediated mitophagy in Tibetan high-altitude adaptation

doi: 10.1038/s41420-025-02933-8

Figure Lengend Snippet: A Total ROS and mtROS contents were determined in the M9a+shCTR ( n = 4), M9a+sh EPAS1 ( n = 4) and M9a+sh EPAS1 -NAC ( n = 4). The mtROS and total ROS contents in the M9a+sh EPAS1 and M9a+sh EPAS1 -NAC cells were normalized to those in the M9a+shCTR cells. B Western blot analysis of HIF-1α, BNIP3, NIX, LC3B, and p62 protein levels in the three cell lines under hypoxia. Grayscale analysis of target protein levels was normalized to β-Tubulin. C Autophagic flux was quantified by colocalizing LC3B (green) and Mitotracker (red) (mitophagosomes) using immunofluorescence in the three cell lines under hypoxia. DAPI (blue) stains the nucleus. Scale bar: 20 μm. D – F Mitochondrial OCR (basal, ATP-linked, maximal), MMP, and ATP content were measured in the three cell lines under hypoxia. MMP and ATP levels in M9a+sh EPAS1 and M9a+sh EPAS1 -NAC cells were normalized to those in M9a+shCTR cells. G CCK-8 assay showing cell viability in the three cell lines under hypoxia. H Cell apoptosis was determined using Annexin V-FITC staining in the three cell lines under hypoxia. I Western blot analysis of C-Caspase-3 and BAX protein levels in the three cell lines under hypoxia. Grayscale analysis of target protein levels was normalized to β-Tubulin. J Cytoplasmic free mtDNA was determined by staining dsDNA (green) and Mitotracker (red) using immunofluorescence in the three cell lines under hypoxia. DAPI (blue) stains the nucleus. dsDNA that does not colocalize with either the nucleus or mitochondria is considered cytoplasmic free mtDNA. Scale bar: 20 μm. K Western blot analysis of NLRP3, IL1β, cGAS, and STING protein levels in the three cell lines under hypoxia. Grayscale analysis of target protein levels was normalized to β-Tubulin. L Inflammatory cytokines IL6, IL1β, and TNF-α in the culture medium supernatant were measured in the three cell lines under hypoxia using an ELISA kit. The three cell lines, M9a+shCTR ( n = 4), M9a+sh EPAS1 ( n = 4), and M9a+sh EPAS1 -NAC ( n = 4), were treated with hypoxia (1% O₂) for 48 h, with NAC treatment (10 mM) administered during the last 24 h.#1–4 represent individual samples. Data are presented as the mean ± SD of three independent experiments. One-way ANOVA followed by Dunnett’s test; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Inhibitors were used as follows: N-acetylcysteine (NAC) was dissolved in the medium to a final concentration of 10 mM (diluted in ddH2O, Sigma, USA), the HIF-1α inhibitor PX-478 was dissolved in ddH2O to a final concentration of 30 μM (MCE, USA), and Mdivi-1 was used at 50 μM (diluted in DMSO, MCE, USA).

Techniques: Western Blot, Immunofluorescence, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell Death Discovery

Article Title: Mitochondrial retrograde signaling initiates HIF-1α/BNIP3/NIX-mediated mitophagy in Tibetan high-altitude adaptation

doi: 10.1038/s41420-025-02933-8

Figure Lengend Snippet: A Schematic diagram of the mechanism of action of PX-478. B Western blot analysis of HIF-1α, BNIP3, NIX, DRP1, LC3B, and p62 protein levels in M9a+sh EPAS1 cells following PX-478 treatment under hypoxia. Grayscale analysis of protein levels was normalized to β-Tubulin. C Autophagic flux was quantified by colocalizing LC3B (green) and Mitotracker (red) (mitophagosomes) using immunofluorescence in M9a+sh EPAS1 cells following PX-478 treatment under hypoxia. DAPI (blue) stains the nucleus. Scale bar: 20 μm. D – F Mitochondrial OCR (basal, ATP-linked, maximal), MMP, and ATP levels were determined in M9a+sh EPAS1 cells after PX-478 treatment under hypoxia. G CCK-8 assay showing cell viability in M9a+sh EPAS1 cells after PX-478 treatment under hypoxia. H Cell apoptosis was measured in M9a+sh EPAS1 cells after PX-478 treatment under hypoxia using Annexin V-FITC staining. I Western blot analysis of C-Caspase-3 and BAX protein levels in M9a+sh EPAS1 cells following PX-478 treatment under hypoxia. Grayscale analysis of protein levels was normalized to β-Tubulin. J Cytoplasmic free mtDNA was determined by staining dsDNA (green) and Mitotracker (red) in M9a+sh EPAS1 cells following PX-478 treatment under hypoxia. DAPI (blue) stains the nucleus. dsDNA that does not colocalize with either the nucleus or mitochondria is considered cytoplasmic free mtDNA. Scale bar: 20 μm. K Western blot analysis of NLRP3, IL1β, cGAS, and STING protein levels in M9a+sh EPAS1 cells after PX-478 treatment under hypoxia. Grayscale analysis of protein levels was normalized to β-Tubulin. L Inflammatory cytokines IL6, IL1β, and TNF-α in the culture medium were measured in M9a+sh EPAS1 cells after PX-478 treatment under hypoxia using an ELISA kit. M9a+sh EPAS1 cells ( n = 4) were treated with 30 μM PX-478 (for the last 22 h) under hypoxia (1% O₂) for 48 h. #1–4 represent individual samples. Data are presented as the mean ± SD of three independent experiments. Two-tailed, paired Student’s t tests; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Inhibitors were used as follows: N-acetylcysteine (NAC) was dissolved in the medium to a final concentration of 10 mM (diluted in ddH2O, Sigma, USA), the HIF-1α inhibitor PX-478 was dissolved in ddH2O to a final concentration of 30 μM (MCE, USA), and Mdivi-1 was used at 50 μM (diluted in DMSO, MCE, USA).

Techniques: Western Blot, Immunofluorescence, CCK-8 Assay, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test