Phospho-PPAR gamma (Ser112) Antibody - #AF3284

Fig. 3. Effects of PPARγ activation on microglial phenotype switching. Levels of mRNAs 23 encoding (A) PPARα, (B) PPAR β/δ or (C) PPARγ in hippocampus of rat offspring. (D) Representative western blots and (E-F) quantitation of phospho- and non-phospho-PPARγ protein in hippocampus of rat offspring (n = 5 offspring per group). (G) Immunofluorescence staining of PPARγ located in microglia. Scale bars: 20 μm. (H) Expression of PPARγ in primary microglia after poly(I:C) stimulation. (I) Heat map of mRNA expression of M2 microglia markers Arg1, IL-4, and IL-10 as well as M1 microglia markers IL-6, TNFα, and IL- 1β (n = 5 replicates per group). Statistical analysis is shown in Table S4. *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: Ctrl, control; MSD, maternal sleep deprivation; Veh(Vehicle), treatment with normal saline; Pio, pioglitazone.

Figure 7 Effect of emodin on PPARγ-NF-κB pathway in macrophages and lung tissues. (A) Western blot analysis of p-PPARγ, PPARγ, p-NF-κB, and NF-κB in alveolar macrophages exposed to plasma exosomes from sham rats, SAP rats, and emodin-treated SAP rats and subsequently treated with the PPARγ agonist rosiglitazone or the antagonist GW9662. GAPDH was used as the loading control. (B) Quantitative analysis of p-PPARγ, PPARγ, and p-NF-κB expression in alveolar macrophages (n = 3). (C) Levels of the pro-inflammatory factors NO, TNF-α, and MIP-2 in culture media as detected by ELISA (n = 3). (D) Western blot analysis of p-PPARγ, PPARγ, p-NF-κB, and NF-κB in the lung tissues of healthy rats administered plasma exosomes from sham rats, SAP rats, and emodin-treated SAP rats and subsequently treated with the PPARγ agonist rosiglitazone or the antagonist GW9662. GAPDH was used as the loading control. (E) Quantitative analysis of p-PPARγ, PPARγ, and p-NF-κB expression in the lung tissues (n = 3). (F) Levels of the pro-inflammatory factors NO, TNF-α, and MIP-2 in the lung tissues as detected by ELISA (n = 3). All experiments were independently performed three times. All data were expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. the sham group, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. the Emo-exos-AMs or Emo-exos-lung; #P < 0.05, ##P < 0.01, ###P < 0.001 and ####P < 0.0001 vs. the SAP group, by one-way ANOVA followed by Tukey tests.

Fig. 7: Inhibition of EGFR phosphorylation promote M2 polarization by regulating glutamine metabolism through activation of PPARγ. A–F RAW264.7 macrophages were treated with LPS (1 μg/mL) for 24 h, with or without Erlotinib (20 μM) pretreatment for 30 min. A RT-qPCR analysis of mRNA expression of M2-related genes Mcr1 (n = 3). B RT-qPCR analysis of mRNA expression of M2-related genes Ym1 (n = 3). C Representative western blot of Arg1. D Flow cytometry analysis showing the level of M2 macrophage-associated markers CD206. E Percentage of CD206-positive RAW264.7 is shown (n = 3). I Mean fluorescence intensity (MFI) is shown (n = 3). G–I Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. G CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. H Percentage of CD206-positive alveolar macrophage is shown (n = 9). I Mean fluorescence intensity (MFI) is shown (n = 9). J Immunoblot analysis of p-PPARγ (Ser112), t-PPARγ in RAW264.7 cells treated with LPS (1 μg/mL) for 30 min with or without indicated concentration of PD168393 (10 μM) pretreatment for 30 min. K Fluorescence images depicting PPARγ translocation (left panel, scale bar, 50 μm; right panel, scale bar, 5 μm). L–N RAW264.7 cells were treated with LPS (1 μg/mL) for 24 h with or without Erlotinib (10 μM) or Rosiglitazone (Rosi (20 μM) pretreatment. L Cell surface CD206 were analyzed by flow cytometry. M Percentage of CD206-positive RAW264.7 is shown (n = 3). N Mean fluorescence intensity (MFI) is shown (n = 3). O–Q Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into Sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. O CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. P Percentage of CD206-positive alveolar macrophage is shown (n = 9). Q Mean fluorescence intensity (MFI) is shown (n = 9). R–V RAW264.7 cells were treated with LPS (1 μg/mL) for 30 min with or without PD168393 (10 μM) pretreatment for 30 min. R Flow cytometry analysis of JC-1 for the detecting the change of mitochondrial membrane potential (ΔΨm) (left panel, JC-1 aggregates; right panel, JC-1 monomers). S The ratio of JC-1 aggregates /JC-1 monomers was calculated as Δψm. T Total cellular ATP level was detected (n = 3). U Immunoblot analysis of IRG1, ATP5A, SDHA, Tubulin as a loading control. V Immunoblot analysis of SDHA and IRG1 in Control or IRG1 silenced RAW264.7 cells, Tubulin as a loading control. The graphs depict mean ± SD based on three independent experiments.

Fig. 6 Characterization of lipid and glucose metabolism in intermuscular adipose tissues (IMATs). Representative western blots (A, C, E, G, I) and quantification of the gene-expression levels of peroxisome proliferators-activated receptor-γ (PPAR-γ) (B), phosphorylated PPAR-γ (p-PPAR-γ; Ser112) (F), nuclear respiratory factor-1 (NRF-1) (J), glucose transporter-4 (GLUT-4) (D), glucose transporter-1 (GLUT-1) (H), and perilipin-5 (Plin-5) (K). The data represent the mean ± the standard error of the mean (n = 5–6/group). CON control, EMA combined therapies + compound-c, EMC combined therapies, EXE moderate exercise, GAPDH glyceraldehyde-3-phosphate dehydrogenase, MET metformin, PRE prediabetes. ap 

Figure 7. Effects of d-ECM on the ERK1/2-PPARγ pathway and the expression of adipocyte secreting factors ADIPOQ and aP2 in the fully differentiated ADSCs. After 3-days treatments and 14-days adipogenic induction, ADSCs at the 5th passage were collected and used for Western blotting analysis (a). Protein levels of ERK1/2 (b), p-ERK1/2 (c), PPARγ (d), p-PPARγ (e), ADIPOQ (f) and aP2 (g) were examined. GAPDH demonstrated the equal loading of protein samples. N = 3. *, p < 0.05, vs. 10% FBS group; **, p < 0.01, vs. 10% FBS group; #, p < 0.05, vs. 2% FBS group; ##, p < 0.01, vs. 2% FBS group; $, p < 0.05, vs. 2% FBS + d-ECM group; $$, p < 0.01, vs. 2% FBS + d-ECM group. ADIPOQ, adiponectin; aP2, adipocyte fatty-acid binding protein