Journal: Molecular Medicine

Article Title: Exploring the mechanism of endothelial Pim-1 upregulation of tissue factor to initiate the hypercoagulable state in sepsis

doi: 10.1186/s10020-026-01433-4

Figure Lengend Snippet: Inhibition of Pim-1 activity increased the survival rate of CLP-induced septic mice (WT) and alleviated the coagulation response. a Immunohistochemical staining (the staining agent was horseradish peroxidase, HRP) and Pim-1 + positive cells statistical plot of WT mice. b Pim-1 protein bands and statistical results in lung/liver of CLP WT mice ( n ≥ 3/group). c Dual immunofluorescence staining images of Pim-1 and CD31 in lung tissues were obtained using Cy3-conjugated goat anti-rabbit IgG (red) and Alexa Fluor 488-conjugated goat anti-mouse IgG (green). d Pim-1 protein bands and statistical results after LPS stimulation of HUVECs. e Survival of experimental mice. WT mice aged 6–8 weeks were given CLP, and six hours later SMI-4a (15 mg/kg or 30 mg/kg) was injected via the tail vein ( n = 10/group; Kaplan-Meier survival analysis). f Plasma TM, TAT, FIB and D-dimer contents of WT mice ( n ≥ 3/group), CLP + SMI-4a (L) : 15 mg/kg, CLP + SMI-4a (H) : 30 mg/kg. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns represent no statistical significance

Article Snippet: We contracted Cyagen Biosciences Inc. to construct a mouse model of specific endothelial cell Pim-1 knockout (KO).

Techniques: Inhibition, Activity Assay, Coagulation, Immunohistochemical staining, Staining, Immunofluorescence, Injection, Clinical Proteomics

Journal: Molecular Medicine

Article Title: Exploring the mechanism of endothelial Pim-1 upregulation of tissue factor to initiate the hypercoagulable state in sepsis

doi: 10.1186/s10020-026-01433-4

Figure Lengend Snippet: Pim-1 relies on the transcription factor Sp1 to regulate TF in HUVECs stimulated with LPS. a Protein bands and statistical plots of p-Sp1 and TF after Pim-1 kinase inhibition in HUVECs (SMI-4a 80 µM). b Protein bands and statistical plots of p-Sp1 with increasing degree of Pim-1 kinase inhibition in HUVECs (SMI-4a 30, 60, 90, 120 µM). c Protein bands and statistical plots of p-Sp1 and TF after LPS stimulation in HUVEs transfected with Pim-1 overexpression plasmid. d Protein bands of TF after HUVECs treated with Pim-1 overexpression plasmid and 200 nM Mithramycin A (Sp1 inhibitor). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns represent no statistical significance

Article Snippet: We contracted Cyagen Biosciences Inc. to construct a mouse model of specific endothelial cell Pim-1 knockout (KO).

Techniques: Inhibition, Transfection, Over Expression, Plasmid Preparation

Journal: Molecular Medicine

Article Title: Exploring the mechanism of endothelial Pim-1 upregulation of tissue factor to initiate the hypercoagulable state in sepsis

doi: 10.1186/s10020-026-01433-4

Figure Lengend Snippet: Pim-1 activates the mTOR/Sp1/TF axis to initiate SIC in LPS-induced HUVECs. a Protein bands and statistical plots of p-mTOR after Pim-1 overexpression in HUVECs. b Protein bands and statistical plots of p-Sp1 and TF after mTOR inhibition in HUVECs (RAPA 5 µM). c - d Protein bands and statistical plots of p-Sp1 and TF after transfection of HUVECs with mTOR overexpression plasmid. e Protein bands of p-Sp1 and TF after HUVEC treated with mTOR overexpression plasmid and 80 µM SMI-4a. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns represent no statistical significance

Article Snippet: We contracted Cyagen Biosciences Inc. to construct a mouse model of specific endothelial cell Pim-1 knockout (KO).

Techniques: Over Expression, Inhibition, Transfection, Plasmid Preparation

Journal: Molecular Medicine

Article Title: Exploring the mechanism of endothelial Pim-1 upregulation of tissue factor to initiate the hypercoagulable state in sepsis

doi: 10.1186/s10020-026-01433-4

Figure Lengend Snippet: Pim-1 endothelial cell-specific knockout affects coagulation function after CLP-induced sepsis in mice. a and b Images of lung/liver tissues from Pim-1 fl/fl and Pim-1 fl/fl Cre + group subjected to H&E staining and fibrin immunohistochemical staining ( n ≥ 3/group; The staining agents were Hematoxylin-Eosin and HRP; magnification 200×, Scale bar = 10 μm, black arrow pointing to microvascular thrombus). c The blood perfusion volume of the right kidney and mesentery in four groups of mice ( n ≥ 3/group). d Plasma TM, TAT, FIB and D-D dimer levels were measured in Pim-1 fl/fl and Pim-1 fl/fl Cre + group ( n ≥ 3/group). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns represent no statistical significance

Article Snippet: We contracted Cyagen Biosciences Inc. to construct a mouse model of specific endothelial cell Pim-1 knockout (KO).

Techniques: Knock-Out, Coagulation, Staining, Immunohistochemical staining, Clinical Proteomics

Journal: Molecular Medicine

Article Title: Exploring the mechanism of endothelial Pim-1 upregulation of tissue factor to initiate the hypercoagulable state in sepsis

doi: 10.1186/s10020-026-01433-4

Figure Lengend Snippet: Pim-1 endothelial cell-specific knockout affects coagulation function and signaling pathways after CLP-induced sepsis in mice. a and b Protein was extracted from lung tissue of Pim-1 fl/fl and Pim-1 fl/fl Cre + group for immunofluorescence to observe TF and Thrombin protein expression ( n ≥ 3/group), Scale bar = 50 μm. c Protein was extracted from lung tissue of Pim-1 fl/fl and Pim-1 fl/fl Cre + group for western blot to observe TF and Thrombin protein expression ( n ≥ 3/group). d and e Bands and statistical plots of mouse p-mTOR and p-Sp1 proteins in Pim-1 fl/fl and Pim-1 fl/fl Cre + group ( n ≥ 3/group). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns represent no statistical significance

Article Snippet: We contracted Cyagen Biosciences Inc. to construct a mouse model of specific endothelial cell Pim-1 knockout (KO).

Techniques: Knock-Out, Coagulation, Protein-Protein interactions, Immunofluorescence, Expressing, Western Blot

Journal: Molecular Medicine

Article Title: Exploring the mechanism of endothelial Pim-1 upregulation of tissue factor to initiate the hypercoagulable state in sepsis

doi: 10.1186/s10020-026-01433-4

Figure Lengend Snippet: TLR4 affects the expression of Pim-1 and TF in CLP/LPS-induced sepsis. a Immunofluorescence images and statistical graphs of Pim-1 protein in lung tissues of WT group and TLR4 mut group ( n ≥ 3/group), Scale bar = 50 μm. b Immunofluorescence images and statistical graphs of TF protein in lung tissues of WT group and TLR4 mut group ( n ≥ 3/group), Scale bar = 50 μm. c Bands and statistics of Pim-1 protein in lung tissues of WT and TLR4 mut groups ( n ≥ 3/group). d Bands and statistics of TF protein in lung tissues of WT and TLR4 mut groups ( n ≥ 3/group). e Bands and statistical analysis of Pim-1 and TF proteins after inhibition of TLR4 in HUVECs (5 nM TAK-242). f Protein expression and statistical graph of Pim-1 and TF after HUVECs were stimulated with different concentrations of LPS (2.5, 5, 10 μg/ml; LPS is a TLR4 activator). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, ns represent no statistical significance

Article Snippet: We contracted Cyagen Biosciences Inc. to construct a mouse model of specific endothelial cell Pim-1 knockout (KO).

Techniques: Expressing, Immunofluorescence, Inhibition

Journal: bioRxiv

Article Title: Macrophage PIM1 Drives Atherosclerosis by Enhancing Foam Cell Formation Via CD36

doi: 10.1101/2025.10.31.685966

Figure Lengend Snippet: (A) Violin plots based on scRNA-seq show that Pim1 mRNA was enriched and upregulated in plaque from Ldlr -null mice fed HFD. Each black dot represents an individual lesional Mφ. (B) Comparison of surface CD36 expression between vehicle-treated Mφs and AZD1208-treated Mφs. Examples of histograms of CD36 signals were shown. Flow cytometry data were quantified (n = 3 mice for each group). (C) Comparison of surface CD36 expression between peritoneal Mφs from Pim1 -/- mice and WT mice. Examples of histograms of CD36 signals were shown. Flow cytometry data were quantified (n = 3 mice for each group). (D) CD36 expression in WT and Pim1 -/- Mφ was evaluated by Western blot analysis. A representative blot image was shown on the left. Protein expressions were quantified by densitometry and shown in the bar graphs on the right (n = 3 mice for each group). (E) WT murine peritoneal Mφs were treated with increasing doses of oxLDL (0, 5, 20, and 50 µg/mL) for 24 hours and PIM1 protein expression was then evaluated using flow cytometry. Examples of histograms of PIM1 signals were shown. Mean fluorescence intensity (MFI) normalized to control is shown in the bar graph (n = 3 mice for each group).

Article Snippet: For intracellular staining, cells were fixed and permeabilized using the eBioscienceTM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions, then stained with FITC-conjugated anti-PIM1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX) for flow cytometry assays.

Techniques: Comparison, Expressing, Flow Cytometry, Western Blot, Fluorescence, Control

Journal: bioRxiv

Article Title: Macrophage PIM1 Drives Atherosclerosis by Enhancing Foam Cell Formation Via CD36

doi: 10.1101/2025.10.31.685966

Figure Lengend Snippet: WT peritoneal Mφ were treated with 10µM of AZD1208 or vehicle for 72 hours. (A) DiI-oxLDL-binding and -uptake assays by Mφ were evaluated by flow cytometry, respectively. Examples of histograms of DiI signals were shown. Flow cytometry data were quantified (n = 3 mice for each group). (B) Mφ from WT or Pim1 -/- cells were either treated with PBS or with 50µg/ml oxLDL for 24 hours. Oil Red O (ORO) staining was performed and representative images after staining were shown. (C) Foam cell rates were quantified (n = 3 mice for each group). More than 300 cells were counted for each condition. (D) Cholesterol was measured in peritoneal Mφs from WT and Pim1 -/- mice treated with PBS or oxLDL. Cholesterol concentrations were adjusted with protein content. Data in the bar graph were combined from three independent experiments.

Article Snippet: For intracellular staining, cells were fixed and permeabilized using the eBioscienceTM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions, then stained with FITC-conjugated anti-PIM1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX) for flow cytometry assays.

Techniques: Binding Assay, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Macrophage PIM1 Drives Atherosclerosis by Enhancing Foam Cell Formation Via CD36

doi: 10.1101/2025.10.31.685966

Figure Lengend Snippet: Mφ from WT or Pim1 -/- mice were injected intraperitoneally into Apoe -/- mice fed on a HFD for 6 weeks to induce hyperlipidemia and a proatherogenic environment. (A) Schematic diagram showing the experimental design. (B) ORO staining was performed on peritoneal Mφs from HFD-treated Apoe -/- mice transplanted with WT mice-or Pim1 -/- mice-derived Mφs. Foam cell formation was quantified using ORO staining (n = 6 mice for each group). More than 300 cells were counted for each condition. (C) Cholesterol was measured from cells in (B). Cholesterol concentrations were adjusted to protein content. Data in the bar graph were combined from four independent experiments. (D) Apoe -/- mice on HFD for 6 weeks were injected with WT Mφ along with oral gavage of 30mg/g body weight of PIM inhibitor (AZD1208) or just vehicle. A schematic diagram showing the experimental design. (E) ORO staining was performed on peritoneal Mφs isolated from HFD-treated Apoe -/- mice intraperitoneally injected with or without AZD1208. Foam cell formation was quantified by ORO staining (n = 11 mice for each group). More than 300 cells were counted for each condition. (F) Cholesterol was measured from cells in (E). Cholesterol concentrations were adjusted to protein content. Data in the bar graph were combined from 10-12 independent experiments.

Article Snippet: For intracellular staining, cells were fixed and permeabilized using the eBioscienceTM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions, then stained with FITC-conjugated anti-PIM1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX) for flow cytometry assays.

Techniques: Injection, Staining, Derivative Assay, Isolation

Journal: bioRxiv

Article Title: Macrophage PIM1 Drives Atherosclerosis by Enhancing Foam Cell Formation Via CD36

doi: 10.1101/2025.10.31.685966

Figure Lengend Snippet: (A) A schematic diagram showing the experimental design. (B) The levels of plasma cholesterol (n = 7-10 mice for each group). (C) The levels of plasma MCP-1 (n = 7-10 mice for each group). (D) Aortic arches from four mice with Apoe -/- Pim1 fl/fl and Apoe -/- Lyz2 Cre/+ Pim1 fl/fl fed a HFD for 12 weeks were dissected, pinned open, and stained en-face for lipid-rich plaque with ORO. The yellow arrow indicates plaque. Representative images from four mice in each group are shown. Percent of plaque area was shown at bottom left corner in each image. (E) Left panels show ORO staining of histological sections obtained at the level of the aortic sinus from these mice with. Immunofluorescence images with antibodies to PPARψ, and CD68 are shown in the 3 panels to the right. (F-G) The quantifications of positive ORO, PPARψ, CD68, and αSMA areas in the aortic sinus were measured (n = 7-10 mice for each group).

Article Snippet: For intracellular staining, cells were fixed and permeabilized using the eBioscienceTM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions, then stained with FITC-conjugated anti-PIM1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX) for flow cytometry assays.

Techniques: Clinical Proteomics, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Macrophage PIM1 Drives Atherosclerosis by Enhancing Foam Cell Formation Via CD36

doi: 10.1101/2025.10.31.685966

Figure Lengend Snippet: Peritoneal Mφs from WT and Pim1 -/- mice were isolated and cultured in vitro . Their RNA was then extracted and analyzed by bulk RNA sequencing. (A) A heatmap presents differentially expressed genes related to lipids, atherosclerosis, and PPAR signaling between WT and Pim1 -/- Mφ (n = 3 mice for each group). (B) Expression of PIM1, PPARγ, and CD36 in WT and Pim1 -/- Mφ were evaluated by Western blots. Representative blot images were shown (n = 3 mice for each group). (C) The effect of si Pim1 on the expression of PIM1, PPARγ, and CD36 in Mφs was assessed by Western blots. Representative blot images were shown (n = 4 mice for each group). (D) The effect of rosiglitazone (a PPARγ agonist) on CD36 expression in Mφ was assessed by Western blots. Representative blot images were shown (n = 3 mice for each group).

Article Snippet: For intracellular staining, cells were fixed and permeabilized using the eBioscienceTM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer’s instructions, then stained with FITC-conjugated anti-PIM1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX) for flow cytometry assays.

Techniques: Isolation, Cell Culture, In Vitro, RNA Sequencing, Expressing, Western Blot