Fig. 3: TRPML1-mediated PHD activity modulates NF-κB signaling pathway, which in turn controls IL1B transcription in macrophages. | Nature Communications

Fig. 3: TRPML1-mediated PHD activity modulates NF-κB signaling pathway, which in turn controls IL1B transcription in macrophages.

From: Lysosomes finely control macrophage inflammatory function via regulating the release of lysosomal Fe2+ through TRPML1 channel

Fig. 3: TRPML1-mediated PHD activity modulates NF-κB signaling pathway, which in turn controls IL1B transcription in macrophages.The alternative text for this image may have been generated using AI.

a Relative NF-κB activity in LPS-stimulated BMDMs treated with either of ML-SA5 (0.1 μM) or Control for different treatment periods as indicated was assessed by a NF-κB reporter assay. n = 3. b Relative NF-κB activity in BMDMs treated with ML-SA5 for different treatment periods as indicated was assessed by a NF-κB reporter assay. n = 4. c Representative immunoblots and quantitative analysis of phosphorylated p65, IKBα and IKKα/β protein levels in BMDMs under control, LPS (100 ng/mL), LPS + ML-SA5 (0.1 μM), ML-SA5, LPS + ML-SA5 + ML-SI3 (10 μM), or ML-SA5 + ML-SI3. All treatments were for 30 min. n = 3–9. d Relative NF-κB activity in BMDMs under control, ML-SA5 (0.1 μM), ML-SA5 + ML-SI3 (10 μM), ML-SA5 + DXZ (200 μM), or ML-SA5 + Roxadustat (100 μM). All treatment were for 30 min. n = 5. e Relative NF-κB activity under control or ML-SA5 (0.1 μM) conditions in NC shRNA or PHD1 shRNA + PHD2 shRNA expressed RAW264.7 cells. All treatment were for 30 min. n = 3. f Relative IL1B luciferase activity in BMDMs under control, ML-SA5 (0.1 μM), LPS (100 ng/mL), LPS + ML-SA5 (0.1 μM), LPS + ML-SA5 + ML-SI3 (10 μM), LPS + ML-SA5 + DXZ (200 μM), or LPS + ML-SA5 + Roxadustat (100 μM) was measured by luciferase assays. All treatment were for 24 h. n = 3. g Relative IL1B luciferase activity under control, ML-SA5 (0.1 μM), LPS (100 ng/mL), or LPS + ML-SA5 (0.1 μM) in NC shRNA or p65 shRNA expressed BMDMs was measured by luciferase assays. All treatments were for 12 h. n = 3–4. h Chromatin immunoprecipitation (ChIP)-qPCR was used to amplify chromatin derived from immunoprecipitations with anti-p65 antibody. Co-precipitating chromatin fragments were analyzed by real time PCR to quantify enrichment of IL1B at the −688 of IL1B position in BMDMs treated with LPS (100 ng/mL), LPS + ML-SA5 (0.1 μM), LPS + ML-SA5 + ML-SI3 (10 μM), LPS + ML-SA5 + Roxadustat (100 μM), or LPS + ML-SA5 + 2,2’ Bipyridyl (150 μM). n = 3. All treatments were for 6 h. Means ± SEMs were shown in a–h. Significant differences were evaluated using one-way ANOVA followed by Tukey’s test.

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