Journal: Cell Death & Disease

Article Title: SKA3-mediated hypoxia tolerance and metabolic reprogramming promote liver metastasis in lung adenocarcinoma

doi: 10.1038/s41419-025-08270-z

Figure Lengend Snippet: A Pathway enrichment analysis indicated that the HIF-1α pathway was enriched in A549-LMs. B GSEA analyses suggested that the HIF-1α pathway may be activated in A549-LMs. C Immunoprecipitation (IP) was performed with SKA3 antibody, and total lysate from A549 cells (input) was used as a positive control, while IgG antibody was used as a negative control. Representative images of silver-stained PAGE gel showed separated proteins. D The protein interaction between SKA3 and PHD2 was analyzed using HDOCK software. E Immunofluorescence co-localization analysis showed SKA3 (Red) co-localized with PHD2 (Green) in A549 and A549-LMs cells, assessed by Pearson’s coefficient (Pr), nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. F , G Co-IP experiments verified the interaction between SKA3 and PHD2 in A549 and A549-LMs cells. H qRT-PCR results revealed no impact of SKA3 knockdown on PHD2 and HIF-1α mRNA expression in A549 and A549-LMs cells. I WB analysis indicated increased OH-HIF-1α and decreased HIF-1α levels, with no significant change in PHD2 and VHL protein levels following SKA3 knockdown in A549 cells. J WB analysis indicated decreased OH-HIF-1α and increased HIF-1α levels after SKA3 overexpression, without significant changes in PHD2 and VHL protein levels in A549 cells. K Knockdown of SKA3 did not affect HIF-1α protein levels in A549 cells treated with the proteasome inhibitor MG132 (25 μM), the numbers represented relative HIF-1α expression. L Knockdown of SKA3 accelerated degradation of HIF-1α protein in A549 cells treated with the transcription inhibitor CHX (200 μg/mL), the numbers represented relative HIF-1α expression. M IP assays demonstrated that the interaction of SKA3 and PHD2 was weakened in A549 cells after being transfected with SKA3 siRNA. N IP assays demonstrated that the interaction of HIF-1α and PHD2 was strengthened in A549 cells after transfected with SKA3 siRNA. MG132 (25 μM) was added to inhibit HIF-1α degradation. O Proximity ligation assay (PLA) showing protein interactions in A549 cells. The upper panel displays the interaction between SKA3 and PHD2 (red puncta), while the lower panel shows the interaction between PHD2 and HIF-1α (red puncta) in both control (si-Ctrl) and SKA3 knockdown (si-SKA3) cells. Nuclei are stained with DAPI (blue); Scale bars: 100 µm. P IP assays revealed that HIF-1α ubiquitination levels were increased in A549 cells after SKA3 siRNA transfection. MG132 (25 μM) was added to inhibit HIF-1α degradation. Q IP assays followed by immunoblotting (IB) with an antibody specific for K48-linked ubiquitin chains (Ub-K48), SKA3 knockdown increased Ub-K48 of HIF-1α in A549 cells. * p < 0.05; ** p < 0.01; *** p < 0.001; ns no significance.

Article Snippet: For immunoprecipitation, antibodies against PHD2 (1:200; 4835, CST), SKA3 (1:200; 186003, Abcam) were added to the lysates and incubated overnight at 4 °C, with rabbit IgG (1–5 μg) as the control antibody.

Techniques: Immunoprecipitation, Positive Control, Negative Control, Staining, Software, Immunofluorescence, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Knockdown, Expressing, Over Expression, Transfection, Proximity Ligation Assay, Control, Ubiquitin Proteomics, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside CK targets PHD2 to prevent platelet adhesion and enhance blood circulation by modifying the three-dimensional arrangement of collagen

doi: 10.1016/j.apsb.2024.12.038

Figure Lengend Snippet: Untargeted plasma metabolomics analysis and evaluation on rat soft tissue injury. (A) Cluster analysis of significantly differential metabolites affected by CK. (B) Enrichment analysis of metabolic pathways based on significantly differential metabolites. (C) The impact of CK on the key metabolites 2-OG and Vc in HIF-1 signaling pathway. (D) Schematic diagram of the role of PHD2 and its associated metabolites in the HIF-1 α signaling pathway. (E) Western blot detection of the effect of CK on the expression of PHD2, HIF-1 α and VEGF. ∗ P < 0.05, ∗∗ P < 0.01 vs . mod group, ( n = 6).

Article Snippet: Antibodies targeting PHD2 (4835S), HIF-1 α (48085S), VEGF (9698S), collagen I (72026S), and GAPDH (5174S) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: Clinical Proteomics, Western Blot, Expressing

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside CK targets PHD2 to prevent platelet adhesion and enhance blood circulation by modifying the three-dimensional arrangement of collagen

doi: 10.1016/j.apsb.2024.12.038

Figure Lengend Snippet: PHD2 protein is identified as a target of CK. (A) DARTS assay of CK's effect on damaged soft tissue proteins on SDS-PAGE. (B) Identification of potential target proteins of CK by HPLC–MS/MS analysis. (C) Western blot of PHD2 target proteins on DARTS samples. ∗ P < 0.05 vs . mod group ( n = 3). (D) Molecular docking of CK molecules and PHD2 protein. (E) FTSA analysis of PHD2 protein treated with or without 50 μmol/L CK. (F) SPR analysis of CK and PHD2 ( K D = 18.0 μmol/L). (G) MST analysis of CK and PHD2 ( K D = 17.6 μmol/L).

Article Snippet: Antibodies targeting PHD2 (4835S), HIF-1 α (48085S), VEGF (9698S), collagen I (72026S), and GAPDH (5174S) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: SDS Page, Tandem Mass Spectroscopy, Western Blot

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside CK targets PHD2 to prevent platelet adhesion and enhance blood circulation by modifying the three-dimensional arrangement of collagen

doi: 10.1016/j.apsb.2024.12.038

Figure Lengend Snippet: CK affects collagen structure by inhibiting prolyl hydroxylase. (A) Schematic diagram of hydroxylation modification of collagen structure by PHD2. (B) MST analysis of CK on PHD2–collage interaction. (C) Effect of CK on HYP content in collagen. # P < 0.05 vs . collagen group; ∗∗∗ P < 0.001 vs . PHD2 group ( n = 5). (D) Protein profile identification flow chart of collagen α 1 chain. The effect of CK on the hydroxylation ratio (E) or hydroxylation number (F) of representative collagen α 1 peptides. (G) SEM imaging analysis of architecture for collagen with or without PHD2 or CK administration.

Article Snippet: Antibodies targeting PHD2 (4835S), HIF-1 α (48085S), VEGF (9698S), collagen I (72026S), and GAPDH (5174S) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: Modification, Imaging

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside CK targets PHD2 to prevent platelet adhesion and enhance blood circulation by modifying the three-dimensional arrangement of collagen

doi: 10.1016/j.apsb.2024.12.038

Figure Lengend Snippet: CK reduces platelet adhesion and disrupts the binding between VWF and collagen. (A) Effect of CK on fluorescence imaging of platelet adhesion with collagen protein which coated on microporous plates. ### P < 0.001 vs . con group; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . mod group ( n = 3). (B) VWF-collagen interaction diagram. CK affects PHD2 mediated (C) collagen–platelet adhesion (D) collagen–VWF binding, and (E) the adhesion of collagen–VWF-platelet. ### P < 0.001 vs . con group; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . mod group ( n = 6). (F) MST analysis of VWF and collagen. (G) The impact of PHD2 on VWF–collagen interaction. (H) The effect of CK on the interaction between VWF and collagen.

Article Snippet: Antibodies targeting PHD2 (4835S), HIF-1 α (48085S), VEGF (9698S), collagen I (72026S), and GAPDH (5174S) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: Binding Assay, Fluorescence, Imaging

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside CK targets PHD2 to prevent platelet adhesion and enhance blood circulation by modifying the three-dimensional arrangement of collagen

doi: 10.1016/j.apsb.2024.12.038

Figure Lengend Snippet: Construction of sh- Phd2 mice confirms that PHD2 is a potential target for inhibiting platelet adhesion and thrombus. (A) Molecular docking of PHD2 inhibitors Rox, Vad, Dap and IOX2 with PHD2 protein, and dashed lines indicate catalytic pocket. (B) Effect of PHD2 inhibitors on platelet adhesion; ns, multiple comparisons test no significant ( n = 3). (C) Determination and analysis of the enzymatic activity of PHD2 in the presence of PHD2 inhibitors. ## P < 0.01, ### P < 0.001 vs . con group; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs . mod group; Δ P < 0.05, Dap vs . Rox group ( n = 3). (D) Construction scheme of sh- Phd2 knockdown mice. (E) Western blot analysis for assessing the knockdown efficiency of PHD2 in the sh- Phd2 group. ∗∗ P < 0.01, vs . sh-con, ( n = 3). (F, G) Measurement of tail bleeding time and volume of DIC model mice. (H) Masson staining for thrombus analysis of lung tissue. (I, J) Fibrinogen and thrombin immunofluorescence analysis and statistics. (K) Fluorescence co-localization analysis of VWF and collagen in lung tissue. ## P < 0.01, ### P < 0.001, vs . con group; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . mod group; Δ P < 0.05, ΔΔ P < 0.01, ΔΔΔ P < 0.001, vs . mod group in sh- Phd2 group ( n = 6).

Article Snippet: Antibodies targeting PHD2 (4835S), HIF-1 α (48085S), VEGF (9698S), collagen I (72026S), and GAPDH (5174S) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: Activity Assay, Knockdown, Western Blot, Staining, Immunofluorescence, Fluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside CK targets PHD2 to prevent platelet adhesion and enhance blood circulation by modifying the three-dimensional arrangement of collagen

doi: 10.1016/j.apsb.2024.12.038

Figure Lengend Snippet: Effect of PHD2 inhibitor combined with ASP on FeCl 3 -induced arterial thrombosis. (A) Occlusive carotid artery thrombus images. (B) Effect of PHD2 inhibitors in combination with ASP on thrombus wet weight and (C) thrombus length. (D–G) Analysis of key coagulation parameters in plasma including PT, APTT, TT, and FIB. (H) Analysis of arterial thrombus histopathology by Masson staining. ### P < 0.001, vs . ctrl group; ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 vs . mod group; Δ P < 0.05, ΔΔ P < 0.01, ΔΔΔ P < 0.001, vs . respective monotherapy group ( n = 6).

Article Snippet: Antibodies targeting PHD2 (4835S), HIF-1 α (48085S), VEGF (9698S), collagen I (72026S), and GAPDH (5174S) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: Coagulation, Clinical Proteomics, Histopathology, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Ginsenoside CK targets PHD2 to prevent platelet adhesion and enhance blood circulation by modifying the three-dimensional arrangement of collagen

doi: 10.1016/j.apsb.2024.12.038

Figure Lengend Snippet: The schematic diagram for antiplatelet adhesion by regulating the three-dimensional structure of collagen. (A) Targets for antiplatelet thrombosis with current research. Light red shading indicates clinically approval drug targets, dark red highlighting denotes newly emerging tactics, and blue representing the collagen target illuminated in this paper. (B) The mechanism diagram targets the PHD2 protein to modulate the three-dimensional structure of collagen for inhibiting platelet adhesion.

Article Snippet: Antibodies targeting PHD2 (4835S), HIF-1 α (48085S), VEGF (9698S), collagen I (72026S), and GAPDH (5174S) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: