Fig 1: Blockade of miR-382 inhibits M2 polarization in the kidney and attenuates AA-induced CKD. (A) Images of co-staining for F4/80, CD206, and DAPI in renal sections from WT-14 days and KO-14 days. F4/80 was marked as green. CD206 was marked as red. Nucleic was blue. Scale bars, 200 µm. (B) Representative immunostaining for F4/80, CD206, Arg-1, and Ym-1 in WT and KO mice of 14 days of AAN. Scale bars, 20 µm. (C) Representative Sirius Red staining and immuno-histochemistry staining with antibodies against a-SMA, collagen I, fibronectin, and vimentin in renal sections from WT and KO of 14d AAN. Scale bars, 200 µm. (D) Serum creatinine of wild-type (WT) and miR-382 knockout (KO) mice with AA treatment for 7 and 14 days; saline was administered as control treatment. (E–H) Quantification of positive area for a-SMA, collagen I, fibronectin, and vimentin. Five microscopical fields were randomly selected per section, and the average positive area was calculated; n = 6 mice per group. (I) Quantification of mean positive area for Sirius Red staining. Five microscopical fields were randomly selected per section, and the average positive area was calculated; n = 6 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001; ANOVA.
Fig 2: Suppression or overexpression of miR-382 is implicated in M2-like macrophage activation in vitro. (A) Immunofluorescence staining for Arg-1, Ym-1, CD206, SIRP-a, and p-STAT3 in Raw264.7 cells with anti-scramble or anti-miR-382 with AA (10 µg/ml 48 h) induction. Scale bars, 20 µm. (B) Representative Western blot images and relative quantification for Ym-1, Arg-1, and CD206 in Raw264.7 cells between anti-scramble+ AA and anti-miR-382+ AA groups. (C) Abundance of miR-382 in anti-scramble or anti-miR-382 with AA (10 µg/ml 48 h) induction. U6 was used as an endogenous control. (D) MFI of CD206+ macrophages among control, AA, anti-scramble +AA and anti-miR-382+ AA groups in Raw264.7 cells. (E) MFI of CD206+ macrophages among control, IL-4, anti-scramble +IL-4, and anti-miR-382+ IL-4 groups in Raw264.7 cells. (F) mRNA level of Fizz1 in BMDMs from WT and KO mice following AA treatment. 18s served as standard. (G) MFI of CD206+ macrophages in BMDMs from WT and KO mice following AA treatment. (H) Images and quantification of Western blot for p-STAT3 Y705, p-STAT3 S727, and STAT3 in BMDMs between WT and KO mice following AA treatment. GAPDH was used an endogenous control. (I) Images and quantification of Western blot for SIRP-a in Raw264.7 cells in anti-scramble and anti-miR-382 with AA treatment. GAPDH served as standard. (J) Images of Western blot for p-STAT3 Y705, p-STAT3 S727, STAT3, and SIRP-a in NC and miR-382 mimic groups in Raw264.7 cells. GAPDH served as standard. (K) Abundance of miR-382 between NC and Mimics groups in Raw264.7 cells. U6 was used as an endogenous control. (L, M) Ratio of p-STAT3 Y705/STAT3 and p-STAT3 S727/STAT3 in NC and miR-382 mimic groups in Raw264.7 cells. (N) Quantification of Western blot for SIRP-a in Raw264.7 cells in NC and Mimics groups. (O) MFI of CD206+ macrophages between NC and Mimics groups in Raw264.7 cells. (P) Relative mRNA levels of Arg-1, Fizz1, and Ym-1 in Raw264.7 cells after overexpression of miR-382. *P < 0.05; **P < 0.01; ***P < 0.001; ANOVA.
Fig 3: Phi inhibited the inflammatory response of TBI-activated microglia. The TBI mouse model was constructed. The mice subjected to TBI were treated with Phi (10 mg/kg) and, or GW9662 (1 µmol/kg) 1 h before surgery and thereafter daily for seven days by intraperitoneal injection. The same volume of the solvent was given for the mice in the TBI or sham group. (A,B) RT-PCR was conducted to measure IL-1ß, IL-6, TNFa, IL-4, IL-10, and TGF-ß in the brain lesions seven days after TBI. (C) Western blot was conducted to detect the protein levels of iNOS, COX2, CD86, Arg1, Ym1, and CD206 in TBI lesions seven days after TBI. (D,E) Immunofluorescence was used to detect Iba1+iNOS+ and Iba1+Arg1+microglia in the brain lesions seven days after TBI. (F) Western blot was conducted to measure phospho-NK-?B p65 and PPAR-? in the brain lesions seven days after TBI. The values are expressed as mean ±SD. NS p > 0.05, ***p < 0.001 vs. the sham group; NS p > 0.05, ###p < 0.001 vs. the TBI group; NS p > 0.05, &&p < 0.01, &&&p < 0.001 vs. the TBI+Phi group. n = 5/group.
Fig 4: PGRN up-regulates PD-L1 expression on TAMs. a-b M2 was treated with PGRN recombinant protein; then PD-L1 expression was measured by flow cytometry and PCR. c-d. IL-4 was used to induce WT, PGRN KO peritoneal macrophages into M2; then WB and PCR were used to detect the difference in PD-L1 expression between them. e. After being treated with PGRN, the proportion of CD206+ PD-L1+ cells in M2 were measured by flow cytometry. F. Immunofluorescence was performed to analyze colocalization of F4/80 (red), iNOS (red), CD206 (red), Arg1 (red) and PD-L1 (green) in WT and PGRN KO mice breast cancer sections, and the nucleus was stained with DAPI (blue). **p < 0.01
Fig 5: Phi inhibited the inflammatory response of LPS-activated microglia via the PPAR? signaling pathway. The microglia were treated with LPS (10 ng/ml) or phillyrin (40 µg/ml) or GW9662(1 µM) for 4 h. (A,B) RT-PCR was conducted to measure the “M1” markers of microglia, including IL-1ß, IL-6, and TNFa, as well as “M2” markers, including IL-4, IL-10, and TGF-ß. (C,D) RT-PCR or Western blot was conducted to detect the mRNA or protein levels of iNOS, COX2, CD86, Arg1, Ym1, and CD206 in microglia. (E) Western blot was conducted to measure phospho-NK-?B p65 and PPAR-? in microglia. (F,G) Immunofluorescence was carried out to detect phospho-NK-?B p65, PPAR-? in microglia. The values are expressed as mean ± SD. NS p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the control group; NS p > 0.05, #p < 0.05, ##p < 0.01, ###p < 0.001 vs. the LPS group; NS p > 0.05, &p < 0.05, &&p < 0.01, &&&p < 0.001 vs. the LPS+Phi group. n = 5/group.
Supplier Page from Abcam for Anti-Liver Arginase antibody [EPR22033-369]