Journal: Advanced Science

Article Title: SIRT5–RAC2 Axis Drives Monocyte‐to‐Macrophage Differentiation to Promote Inflammatory Injury in Premature Ovarian Insufficiency

doi: 10.1002/advs.202518417

Figure Lengend Snippet: Ubiquitination‐mediated degradation of RAC2 impairs macrophage differentiation in the Sirt5 ‐deficient ovarian environment. (A) Identification of SIRT5‐interacting proteins in M‐CSF‐stimulated monocytes by immunoprecipitation (IP) followed by mass spectrometry (MS), showing the number of peptides detected for interacting proteins. (B) Reciprocal co‐IP in RAW264.7 cells co‐expressing HA‐tagged RAC2 and Myc‐tagged SIRT5 showing their direct interaction. (C) Endogenous interaction between SIRT5 and RAC2 confirmed by reciprocal co‐IP in mouse ovarian tissues. (D) SIRT5 deficiency increases RAC2 succinylation, as shown by elevated succinylation levels in SIRT5‐knockdown RAW264.7 cells (left) and Sirt5 −/− monocytes (right). (E) RAC2 was immunoprecipitated from mouse ovarian lysates, followed by a western blot to assess lysine succinylation. (F) Western blot analysis showing decreased RAC2 protein levels and increased global succinylation in RAW264.7 cells (left) with pharmacological SIRT5 inhibition (MC3482) or in Sirt5 −/− monocytes (right) compared to controls. (G) Co‐IF staining for RAC2 (red) and F4/80 (green) in ovarian sections reveals diminished RAC2 expression in F4/80⁺ macrophages from Sirt5 −/− mice compared to wild‐type controls. (H) RAC2 ubiquitination is elevated under SIRT5‐deficient conditions, as shown by increased ubiquitination in SIRT5‐knockdown or MC3482‐treated RAW264.7 cells. (I) Both ubiquitination and succinylation of RAC2 are elevated in monocytes from Sirt5 −/− mice compared with those from wild‐type controls. (J,K) Impaired monocyte‐to‐macrophage differentiation upon RAC2 inhibition. (J) qRT–PCR analysis shows reduced expression of macrophage markers ( Adgre1 , Fcgr1 , Cd68 ) in negatively selected monocytes following NSC23766 (RAC2 inhibitor) treatment ( n = 3 per group; one‐way ANOVA with Tukey's multiple comparisons test). Data are normalized to β‐actin and presented as Mean ± SD. ** p < 0.01; **** p < 0.0001. (K) IF staining for F4/80 (green) confirms impaired macrophage differentiation in the NSC23766‐treated group. Scale bar: 20 µm.

Article Snippet: For RAC2 inhibition assays, monocytes were treated from day 0 of differentiation with either DMSO (vehicle control) or 100 μ m NSC23766 (MedChemExpress, China) dissolved in DMSO.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Expressing, Knockdown, Western Blot, Inhibition, Staining, Quantitative RT-PCR

Journal: Cell Reports Medicine

Article Title: Macrophage efferocytosis mediated by the TP63-RAC2 pathway promotes immunosuppressive remodeling in esophageal cancer

doi: 10.1016/j.xcrm.2025.102529

Figure Lengend Snippet: Efferocytosis and polarization of macrophages were analyzed in vitro (A) Western blotting verifies the differences in TP63 and RAC2 protein expression levels in the shTP63 and shTP63+RAC2 groups compared with the control group. (B) Differences in the fluorescence signal intensity of CMFDA green among groups of human primary macrophages with EC9706. ∗ p < 0.05. (C) Differences in the fluorescence signal intensity of CMFDA green among groups in THP-1-induced macrophages with EC9706. ∗∗ p < 0.01. (D) The fluorescence signal intensity of CMFDA green in human primary macrophages treated with EC9706 was determined by flow cytometry. ∗∗∗ p < 0.001. (E) The fluorescence signal intensity of CMFDA green in THP-1-induced macrophages treated with EC9706 was determined by flow cytometry. ∗∗∗ p < 0.001. (F) The CD163 + cell level of each group in human primary macrophages with EC9706. ∗∗ p < 0.01, ∗∗∗ p < 0.001. (G) The CD163 + cell level of each group in THP-1-induced macrophages with EC9706. ∗∗∗ p < 0.001. (H) The CD86 + cells in each group of human primary macrophages treated with EC9706. ∗∗ p < 0.01, ∗∗∗ p < 0.001. (I) The CD86 + cells in each group of THP-1-induced macrophages treated with EC9706. ∗∗∗ p < 0.001. (J) Differences in IL-10, TGF-β, and TNF-α levels among the groups in human primary macrophages with EC9706. ∗∗∗ p < 0.001. (K) Differences in IL-10, TGF-β, and TNF-α levels among the groups in THP-1-induced macrophages with EC9706. Data are presented as the mean ± SD of five independent experiments. ∗∗∗ p < 0.001.

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. For mIHC analysis of human samples, a panel of primary antibodies were used, including CD68 (1:100, ab283654, Abcam), Annexin A1 (1:1000, ab214486, Abcam), TP63(1:200, ab124762, Abcam), RAC2(1:2000,60077-1-Ig, Proteintech).

Techniques: In Vitro, Western Blot, Expressing, Control, Fluorescence, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Macrophage efferocytosis mediated by the TP63-RAC2 pathway promotes immunosuppressive remodeling in esophageal cancer

doi: 10.1016/j.xcrm.2025.102529

Figure Lengend Snippet: Experimental study of shTP63 and shTP63+RAC2 in apoptotic AKR (apoptotic esophageal carcinoma) cells-C57BL/6 efferocytosis mouse model (A) Western blot verifies the difference in TP63 and RAC2 protein expression levels in shTP63 and shTP63+RAC2 groups compared with the control group. (B) The efferocytosis index level of each group was detected ( n = 5 per group). ∗∗ p < 0.01, ∗∗∗ p < 0.001. (C) The CD163 + cells in each group were detected ( n = 5 per group). ∗ p < 0.05, ∗∗ p < 0.01. (D) The CD86 + cells in each group ( n = 5 per group). ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Then, slides were treated by microwave to induce antigen retrieval using citric acid solution for 15 min. For mIHC analysis of human samples, a panel of primary antibodies were used, including CD68 (1:100, ab283654, Abcam), Annexin A1 (1:1000, ab214486, Abcam), TP63(1:200, ab124762, Abcam), RAC2(1:2000,60077-1-Ig, Proteintech).

Techniques: Western Blot, Expressing, Control