Fig. 7: RNF128 promotes Tollip-mediated selective autophagic degradation of S100A8. | Cell Death & Disease

Fig. 7: RNF128 promotes Tollip-mediated selective autophagic degradation of S100A8.

From: RNF128 deficiency in macrophages promotes colonic inflammation by suppressing the autophagic degradation of S100A8

Fig. 7

A Representative TEM images of Rnf128+/+ and Rnf128–/– BMDMs treated with or without EBSS for 2 h. The blue arrows indicate autophagic structures. Scale bars = 2 µm. B The number of autophagosomes per cell in (A) was quantified. The data are shown as mean ± SD. Five cells were counted. **P < 0.01. C Western blot analysis of LC3 and Sqstm1/p62 protein levels in BMDMs from Rnf128+/+ and Rnf128–/– mice stimulated with LPS (200 µg/ml) for 8 h. D Rnf128+/+ and Rnf128–/– BMDMs were transfected with GFP-LC3B for 48 h. GFP-LC3B distribution was observed by confocal microscopy. Scale bar, 5 μm. E The number of autophagosomes per cell in (D) was quantified. Data shown as mean ± SD. Ten cells were counted. Statistical data are presented as mean ± SD. **P < 0.01. F Rnf128+/+ and Rnf128–/– BMDMs were transfected with GFP-mCherry-LC3B for 48 h. GFP-mCherry-LC3B distribution was observed via confocal microscopy. Scale bar, 5 μm. G The numbers of autophagosomes and autolysosomes per cell in (F) were quantified. The data are shown as mean ± SD. Ten cells were counted. Statistical data are presented as mean ± SD. *P < 0.05; ***P < 0.001. H 293T cells were co-transfected with the indicated autophagy cargo receptors and S100A8-GFP for 48 h. Total cell lysates were immunoprecipitated with anti-Flag antibody. The immunoprecipitation complex was detected with anti-GFP and anti-Flag antibodies. I RNF128-Flag stably overexpressing or control THP-1 cells were treated with CQ (50 μM) for 4 h, and total cell lysates were immunoprecipitated with an anti-Tollip antibody. S100A8 and Tollip were detected by western blot. J THP-1 cells stably overexpressing RNF128 were transfected with Flag-Tollip and treated with CQ (50 μM) for 4 h. The co-localization of Flag-Tollip and S100A8 was observed by confocal microscopy. Representative confocal microscopy images were shown. Scale bars, 5 μm. K The colocalization Pearson correlation of Tollip-Flag and S100A8 in (J) was quantified. Statistical data are presented as mean ± SD. **P < 0.01. L Tollip stable knockout THP-1 or control cells were treated with EBSS for the indicated time, and the expression of S100A8 was detected by western blot.

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