Image1_Comparative and evolutionary analysis of RIP kinases in immune responses.TIF

The group of receptor-interacting protein (RIP) kinases has seven members (RIPK1–7), with one homologous kinase domain but distinct non-kinase regions. Although RIPK1–3 have emerged as key modulators of inflammation and cell death, few studies have connected RIPK4–7 to immune responses. The divergence in domain structures and paralogue information in the Ensembl database have raised question about the phylogeny of RIPK1–7. In this study, phylogenetic trees of RIPK1–7 and paralogues constructed using full-length amino acid sequences or Kinase domain demonstrate that RIPK6 and RIPK7 are distinct from RIPK1–5 and paralogues shown in the Ensembl database are inaccurate. Comparative and evolutionary analyses were subsequently performed to gain new clues about the potential functions of RIPK3–7. RIPK3 gene loss in birds and animals that undergo torpor, a common physiological phenomenon in cold environments, implies that RIPK3 may be involved in ischemia-reperfusion injury and/or high metabolic rate. The negligible expression of RIPK4 and RIPK5 in immune cells is likely responsible for the lack of studies on the direct role of these members in immunity; RIPK6 and RIPK7 are conserved among plants, invertebrates and vertebrates, and dominantly expressed in innate immune cells, indicating their roles in innate immunity. Overall, our results provide insights into the multifaceted and conserved biochemical functions of RIP kinases.</p

Huaier extract induced autophagy in breast cancer cells.

<p>(A) Representative electron micrographs of breast cancer cells treated with or without 4 mg/ml Huaier extract for 48 h. Bars, 100nm. (B) Acidic vesicular organelles induced by Huaier extract were stained with MDC. Bars, 10 μm. (C) Aggregation of LC3B in Huaier-treated cells. Cells were treated with or without 4 mg/ml Huaier extract and stained with the LC3B antibodies using immunofluorescence staining. Puncta represent the autophagosome formation. Bars, 10 μm. (D) Cell lysates were harvested after incubation with different concentrations of Huaier extract for 48h. β-actin was used as a loading control. (E) Cells in suspension were labeled with acridine orange and quantified using flow cytometry. FL1-H indicates green color intensity (cytoplasm and nucleus), whereas FL3-H shows red color intensity (AVO). Cells in up quadrants were considered AVO-positive. Results shown are representative of three independent experiments.</p

Huaier extract reduced cell viability and triggered intracellular vesicular organelles in breast cancer cells.

<p>(A, C and E) The cytotoxic effect of Huaier extract was measured using the MTT assay. Cells were treated with different concentrations of Huaier extract over indicated time periods and subjected to the MTT assay. The viability of untreated cells was considered 100%. The experiments were performed in triplicate and data is presented as the mean ± SD of three separate experiments. ^*p < 0.05, ^**p < 0.01. (B, D and F) Micrographs show the appearance of vesicular organelles in breast cancer cells after 48 h of exposure to Huaier extract. Arrows indicate the intracellular vacuoles. Bars, 10 μm. (G) AO/EB staining of T47D cells was performed to detect apoptosis and necrosis induced by Huaier extract. Bars, 50 μm. (H) Representative TUNEL staining (red fluorescence) of MDA-MB-231 cells treated with or without Huaier extract. Bars, 50 μm.</p

LC3B siRNA blocks Huaier-mediated autophagic cell death.

<p>(A) LC3B expression was knocking-down by siRNA. MDA-MB-231 and MCF7 cells were transfected with siLC3B. siControl was used as the negative control. (B) Huaier-mediated autophagy was blocked by LC3B siRNA. MDA-MB-231 cells were treated with siControl, siControl+Huaier, siLC3B, or siLC3B+Huaier. Autophagy was detected by flow cytometry. (C and D) The cell viability was measured using the MTT assay. The experiments were performed in triplicate and data presented as the mean ± SD of three separate experiments. ^*p < 0.05, ^**p < 0.01.</p

Huaier extract inhibited the activity of mTOR/S6K pathway.

<p>(A and B) Cells were treated with various concentrations of Huaier extract for the indicated time. Cell lysates were prepared and detected via Western blotting. The effect of Huaier extract on the levels of mTOR, p70S6K, S6, 4E-BP1 and their phosphorylated forms are shown. (C) AMPK was activated after Huaier treatment in a dose-dependent manner. Results are representative of three independent experiments.</p

The effect of autophagy inhibition on the cytotoxicity of Huaier extract.

<p>MDA-MB-231 (A and B), MDA-MB-468 (C and D) and MCF7 (E and F) were pretreated with 3-MA or CQ before being exposed to Huaier extract for the indicated time. The cell viability was measured using the MTT assay. The experiments were performed in triplicate and data presented as the mean ± SD of three separate experiments. ^*p < 0.05, ^**p < 0.01.</p