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MPs induce inflammation by <t>recognizing</t> <t>TLR2/4</t> in BMDMs. (A) Representative Western blot images of BMDMs treated with MPs or LPS (positive control) for 24 h. TLR2, TLR4, <t>TIRAP,</t> MyD88, TRAF6, P-JNK, and P-c-Jun protein expression levels, analyzed by Western blotting. β-actin expression was used as a loading CTL. (B) Relative mRNA expression levels of Il-1β , Il-6 , and Tnf-α , measured in the presence of MP. Gapdh was used for normalization. (C) Representative flow cytometry plots presenting BMDM M1 and M2 polarization upon MP treatment. The M1 and M2 macrophage populations were quantified as CD86 + CD206 – and CD86 – CD206 + , respectively. (D) Representative immunofluorescence images of BMDMs treated with or without MPs for 24 h, stained with MPO (green), CitH3 (red), and DAPI (blue). Scale bars, 10 μm. Fluorescence intensity-based MPO and CitH3 quantification in BMDMs. a.u., arbitrary unit. The data represent at least three independent experiments and are expressed as the mean ± SEM, using one-way ANOVA. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.
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MPs induce inflammation by <t>recognizing</t> <t>TLR2/4</t> in BMDMs. (A) Representative Western blot images of BMDMs treated with MPs or LPS (positive control) for 24 h. TLR2, TLR4, <t>TIRAP,</t> MyD88, TRAF6, P-JNK, and P-c-Jun protein expression levels, analyzed by Western blotting. β-actin expression was used as a loading CTL. (B) Relative mRNA expression levels of Il-1β , Il-6 , and Tnf-α , measured in the presence of MP. Gapdh was used for normalization. (C) Representative flow cytometry plots presenting BMDM M1 and M2 polarization upon MP treatment. The M1 and M2 macrophage populations were quantified as CD86 + CD206 – and CD86 – CD206 + , respectively. (D) Representative immunofluorescence images of BMDMs treated with or without MPs for 24 h, stained with MPO (green), CitH3 (red), and DAPI (blue). Scale bars, 10 μm. Fluorescence intensity-based MPO and CitH3 quantification in BMDMs. a.u., arbitrary unit. The data represent at least three independent experiments and are expressed as the mean ± SEM, using one-way ANOVA. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.
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MPs induce inflammation by recognizing TLR2/4 in BMDMs. (A) Representative Western blot images of BMDMs treated with MPs or LPS (positive control) for 24 h. TLR2, TLR4, TIRAP, MyD88, TRAF6, P-JNK, and P-c-Jun protein expression levels, analyzed by Western blotting. β-actin expression was used as a loading CTL. (B) Relative mRNA expression levels of Il-1β , Il-6 , and Tnf-α , measured in the presence of MP. Gapdh was used for normalization. (C) Representative flow cytometry plots presenting BMDM M1 and M2 polarization upon MP treatment. The M1 and M2 macrophage populations were quantified as CD86 + CD206 – and CD86 – CD206 + , respectively. (D) Representative immunofluorescence images of BMDMs treated with or without MPs for 24 h, stained with MPO (green), CitH3 (red), and DAPI (blue). Scale bars, 10 μm. Fluorescence intensity-based MPO and CitH3 quantification in BMDMs. a.u., arbitrary unit. The data represent at least three independent experiments and are expressed as the mean ± SEM, using one-way ANOVA. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Journal: ACS Nano

Article Title: Microplastic-Induced Macrophage Dysfunction Drives Lung Tumor Progression through Glutathione Imbalance

doi: 10.1021/acsnano.5c15425

Figure Lengend Snippet: MPs induce inflammation by recognizing TLR2/4 in BMDMs. (A) Representative Western blot images of BMDMs treated with MPs or LPS (positive control) for 24 h. TLR2, TLR4, TIRAP, MyD88, TRAF6, P-JNK, and P-c-Jun protein expression levels, analyzed by Western blotting. β-actin expression was used as a loading CTL. (B) Relative mRNA expression levels of Il-1β , Il-6 , and Tnf-α , measured in the presence of MP. Gapdh was used for normalization. (C) Representative flow cytometry plots presenting BMDM M1 and M2 polarization upon MP treatment. The M1 and M2 macrophage populations were quantified as CD86 + CD206 – and CD86 – CD206 + , respectively. (D) Representative immunofluorescence images of BMDMs treated with or without MPs for 24 h, stained with MPO (green), CitH3 (red), and DAPI (blue). Scale bars, 10 μm. Fluorescence intensity-based MPO and CitH3 quantification in BMDMs. a.u., arbitrary unit. The data represent at least three independent experiments and are expressed as the mean ± SEM, using one-way ANOVA. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: Each membrane was incubated overnight at 4 °C with primary antibodies against TLR2 (Cell Signaling Technology, 13744S), TLR4 (Proteintech, 66350–1-Ig), TIRAP (Cell Signaling Technology, 13077), MyD88 (Cell Signaling Technology, 4283), TRAF6 (Cell Signaling Technology, 67591), P-JNK (Cell Signaling Technology, 9251), P-c-Jun (Cell Signaling Technology, 3270), NRF2 (Cell Signaling Technology, 12721), GCLM (Abcam, ab126704), SLC7A11 (Novus Biologicals, NB300–318), NQO1 (Santa Cruz Biotechnology, sc-32793), GCLC (Cell Signaling Technology, 52183), GPX1 (R&D systems, AF3798), GPX3 (R&D systems, AF4199), GPX4 (R&D systems, MAB5457), FTL (Santa Cruz Biotechnology, sc-390558;), FTH1 (Santa Cruz Biotechnology, sc-376594), and β-actin (Cell Signaling Technology, 8457).

Techniques: Western Blot, Positive Control, Expressing, Flow Cytometry, Immunofluorescence, Staining, Fluorescence