Journal: Nature cell biology

Article Title: Adaptive endoplasmic reticulum stress signalling via IRE1α-XBP1 preserves self-renewal of haematopoietic and pre-leukaemic stem cells

doi: 10.1038/s41556-019-0285-6

Figure Lengend Snippet: a, Annexin V staining of HSC purified from NrasG12D and control mice at two weeks after pIpC injection, following 18 h of treatment with either 0.6 μg ml−1 Tm or 0.2 μg. Representative FACS plots (left) and a summary from n = 3 biological replicates from 3 independent experiments (right) are shown. Percentage of cells in each quadrant is indicated in FACS plots. b, HSCs (200 cells per test) were purified from NrasG12D and control mice two weeks after pIpC injection, and caspase 3/7 activity was measured after 18h of treatment with either 0.6 μgml−1 Tm or 0.2 μM Tg (n = 4 biological replicates from 4 independent experiments). c, Number of colonies obtained from HSCs (200 cells per test) purified from NrasG12D and control mice two weeks after pIpC injection; the cells were cultured in methycult M3434 (n = 4 biological replicates from 4 independent experiments). The colony numbers were counted after ten days of treatment with 25nM or 50 nM Tg. d, Levels of protein aggregates measured by ProteoStat (see Methods) in HSCs 24h after intraperitoneal injections with LPS (2 mgkg−1 body mass; n = 4 and 5 biological replicates for the PBS- and LPS-treated groups, respectively, from 4 independent experiments). e, Percentage of annexin V+ HSCs 24h after intraperitoneal LPS injection (2 mgkg−1 body mass; n = 3 independent experiments). f, LPS injection increased donor reconstitution from NrasG12D HSCs in transplants engrafted with mixed control and NrasG12D bone marrow cells. Bone marrow cells (0.25×106 cells) from NrasG12D and control CD45.2 mice (two weeks after pIpC injection) were transplanted with 0.5×106 competitor (CD45.1) bone marrow cells into recipient mice. Six weeks after transplantation, transplants were injected with 2 mgkg−1 LPS (three doses) every other day and bone marrow HSCs chimaerism was analysed 2–4 weeks after the LPS injections. n = 10 PBS-treated control, n = 14 PBS-treated NrasG12D, n = 4 LPS-treated control and n = 12 LPS-treated NrasG12D recipients pooled from 3 independent experiments. Data represent the mean ± s.d. for all panels. Two-sided Student’s t-tests were used for statistical analyses, except in f where one-sided f-tests were performed. The legend in a also applies to b-f.

Article Snippet: After washing with 1×Perm/Wash Buffer (BD, 5227892), the cells were resuspended with 1×Perm/Wash Buffer containing ProteoStat detection reagent (1/5,000; Enzo, ENZ-51023-KP050) and stained for 30 min. After washing with FACS Buffer, the cells were resuspended in FACS Buffer containing DAPI (2.5 μg ml −1 ).

Techniques: Staining, Purification, Injection, Activity Assay, Cell Culture, Transplantation Assay

Journal: G3: Genes|Genomes|Genetics

Article Title: Loss of N-Glycanase 1 Alters Transcriptional and Translational Regulation in K562 Cell Lines

doi: 10.1534/g3.119.401031

Figure Lengend Snippet: Correction of NGLY1-deficient phenotypes in K562 cell lines by exogenous expression of a DYK-tagged NGLY1 protein. (A) Western blot analysis of NGLY1-deficient K562 cell line's expression of plasmid-based N- or C-terminally tagged NGLY1. Antibodies probed are labeled along the left side of the figure along with corresponding kDa mol. weight markers. The same protein lysates were loaded on two gels. (B) FACS analysis of NGLY1-deficient K562 cell line's expression of plasmid-based N- or C-terminally tagged NGLY1. Results were quantified, averaged, and graphed. (C) Immunofluorescent staining of NGLY1-deficient K562 cell lines with DAPI (blue) and Proteostat (red). (D) Quantification of Proteostat staining of NGLY1-deficient K562 cells by flow cytometry analysis. Rescued lines include both C- and N-terminally tagged NGLY1.

Article Snippet: The Proteostat Dye (Enzo ENZ-51023-KP050 ) was used according to manufacturer’s protocol.

Techniques: Expressing, Western Blot, Plasmid Preparation, Labeling, Staining, Flow Cytometry