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