Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Histone demethylase LSD1 regulates hematopoietic stem cells homeostasis and protects from death by endotoxic shock

doi: 10.1073/pnas.1718759114

Figure Lengend Snippet: miRNA-mediated down-regulation of LSD1 in response to inflammatory cytokines. (A) FDCPmix cell (blue bar) or EML cell (orange bar) were treated with IL-1β. LSD1 mRNA levels are shown, measured by RT-qPCR. (B) FDCPmix cells were treated with IL-1β, and LSD1 expression was determined by Western blot; Ponceau S staining as loading control. (C) FDCPmix cells were transduced with lentiviral vectors carrying two independent shRNAs against argonout2 (Ago2) or DGCR8, and mRNA expression of LSD1 after IL-1β treatment was measured by RT-qPCR. (D) FDCPmix cells were stimulated by IL-1β or TNF-α for the indicated time, and the expression level of indicated miRNA was determined by qPCR. (E) Wild-type mice were reconstituted with BM cells transduced with pooled lentiviral vectors carrying antisense for indicated miRNAs (n = 12, group A+B, see text for grouping) or scramble control (n = 12). After reconstitution, mice were injected with 24 mg/kg LPS to induce sepsis, and the survival curve is shown. (F) Wild-type mice were reconstituted with BM cells transduced with pooled lentiviral vectors carrying antisense against miRNAs (scramble control, n = 12, group A, n = 12 or group B, n = 12). After reconstitution, mice were injected with 24 mg/kg LPS, and survival curve is shown. (G) Expression of LSD1 was determined in animals described in E. BM cells were isolated 36 h after LPS injection, and LSD1 expression is shown as an average of four animals. *P < 0.01; ns, not significant.

Article Snippet: Western blotting was performed as described before using anti-LSD1 (Cell Signaling for Western blotting; Abcam for ChIP); anti-Nurr1, anti-Nur77, and anti-Nor1 (Santa Cruz); and anti-actin (EMD Millipore). pGIPZ lentivirus vectors carrying shRNA and miR-Zip vectors for antisense miRNAs were purchased from Thermo Scientific and System Biosciences, respectively, and viral vector production was performed following the manufacturer’s protocol. microRNA Isolation and qPCR. miRNA isolation was performed using PureLink miRNA isolation kit and reverse transcription, and qPCR was performed using NCode Express SyberGreenER kit. qPCR data were normalized against U6.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining, Control, Transduction, Injection, Isolation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Histone demethylase LSD1 regulates hematopoietic stem cells homeostasis and protects from death by endotoxic shock

doi: 10.1073/pnas.1718759114

Figure Lengend Snippet: Model: Sepsis-induced miRNAs suppress expression of LSD1 and induce acute expansion of hyperproliferative and hyperinflammatory HSC, leading to septic shock.

Article Snippet: Western blotting was performed as described before using anti-LSD1 (Cell Signaling for Western blotting; Abcam for ChIP); anti-Nurr1, anti-Nur77, and anti-Nor1 (Santa Cruz); and anti-actin (EMD Millipore). pGIPZ lentivirus vectors carrying shRNA and miR-Zip vectors for antisense miRNAs were purchased from Thermo Scientific and System Biosciences, respectively, and viral vector production was performed following the manufacturer’s protocol. microRNA Isolation and qPCR. miRNA isolation was performed using PureLink miRNA isolation kit and reverse transcription, and qPCR was performed using NCode Express SyberGreenER kit. qPCR data were normalized against U6.

Techniques: Expressing