Journal: Nature

Article Title: SLC25A39 is necessary for mitochondrial glutathione import in mammalian cells

doi: 10.1038/s41586-021-04025-w

Figure Lengend Snippet: a, Gene enrichment analysis (Gorilla) for proteomics data from the immunopurified mitochondria of HeLa cells (top). Gene ontology analysis of proteins altered by BSO treatment (bottom). Log (FDR), log false discovery rate (bottom). Statistical significance was determined by Fisher’s exact or binomial distribution test. b, Immunoblot of SLC25A39, SLC25A12 and CS in HeLa cells treated with the indicated doses of BSO for 24 h. CS was used as the loading control. c, Immunoblot of indicated proteins in HEK-293T (top) and K562 (bottom) cells treated with BSO (1 mM;10 μM, respectively) for the indicated days. CS and GAPDH were used as loading controls. d, Immunofluorescence analysis of indicated proteins in HeLa cells treated with vehicle or BSO (1 mM) for 24 h. Micrographs are representative images. Scale bar, 10 μm (top). Immunofluorescence analysis of SLC25A39 in parental and SLC25A39 knockout HeLa cells treated with BSO (1 mM) and erastin (5 uM) for 24 h (bottom). Micrographs are representative images of three independent experiments. Scale bar, 20 μm. e, Relative abundance of SLC25A39, GCLM and SLC25A40 transcripts in HEK-293T cells treated with BSO (1 mM) for the indicated days using quantitative reverse transcription PCR (RT-qPCR), normalized to transcripts of β-ACTIN. Error bars represent mean ± s.d.; n = 3 biologically independent samples. Statistical significance was determined by one-way ANOVA followed by Bonferroni post-hoc analysis. f, Immunoblot of indicated proteins in HEK-293T SLC25A39 knockout cells expressing a vector control or SLC25A39 cDNA treated with BSO (1 mM) for the indicated times. GAPDH was used as the loading control. g, The design of 3xFLAG- SLC25A39-P2A-RFP construct (top). Immunoblot of indicated proteins in HEK-293T cells expressing 3xFLAG- SLC25A39-P2A-RFP treated with BSO (1 mM) and erastin (5 μM) for 48 h (bottom). α-Tubulin was used as the loading control. h, Immunoblot of indicated proteins in HeLa cells treated with indicated doses of H2O2 for 24 h. CS and GAPDH were used as loading controls. i, Immunoblot of indicated proteins in HeLa cells treated with erastin (5 μM) or indicated doses of KI696 (NRF2 activator) for 24 h. GAPDH were used as loading controls. j, Immunoblot of indicated proteins in HeLa cells treated with erastin (5 μM) or indicated doses of RSL-3, an inhibitor of glutathione peroxidase 4 (GPX4), for 24 h. CS was used as a loading control. k, Immunoblot of SLC25A39 and ATF4 proteins in HeLa cells treated with indicated doses of mitoCDNB, diamide and BSO (1 mM)/erastin (5 μM) for 24 h. GAPDH was used as loading control. l, Immunoblot of indicated proteins in HeLa cells treated with erastin (5 μM) and co-treated with either GSH (10 mM), GSHee (10 mM), Trolox (50 μM) or Ferrostatin-1 (5 μM) for 48 h. CS was used as a loading control. GSHee, glutathione ethyl ester.

Article Snippet: RFP antibody (600–401-379, 1:1,000) was from Rockland. α-Tubulin antibody (AA4.3, 1:2,000) was from DSHB.

Techniques: Western Blot, Immunofluorescence, Knock-Out, Quantitative RT-PCR, Expressing, Plasmid Preparation, Construct

Journal: Scientific Data

Article Title: ALFI : Cell cycle phenotype annotations of label-free time-lapse imaging data from cultured human cells

doi: 10.1038/s41597-023-02540-1

Figure Lengend Snippet: Example of fluorescence images from the MI01 sequence. ( a ) H2B-GFP, ( b ) RFP-alpha-tubulin, ( c ) DIC, and ( d ) merged fluorescence signals. For better visualization, contrast enhancement has been applied to ( a – c ). Scale bar: 25 μ m.

Article Snippet: The ALFI image dataset is made available as an open-source dataset, which contains 29 original time-lapse microscopy videos and image sequences, named MI01-MI08, CD01-CD09, and TP01-TP12, of the following human cell lines: retinal pigment epithelial nontransformed immortalized hTERT RPE-1, uterine cervical adenocarcinoma HeLa, osteosarcoma U2OS (ATCC: HTB-96) cells and U2OS cells stably expressing histone H2B-GFP and RFP-alpha-tubulin (indicated as “U2OS fluo” in Tables – ).

Techniques: Fluorescence, Sequencing

Journal: Communications Biology

Article Title: Modular tissue-in-a-CUBE platform to model blood-brain barrier (BBB) and brain interaction

doi: 10.1038/s42003-024-05857-8

Figure Lengend Snippet: a The structure of the BBB was reconstructed in a CUBE using primary astrocytes and pericytes, and iPSC-derived brain microvascular endothelial cells (BMECs). (i) Differentiation of iPSC to BMEC was based on the protocol by Lippmann et al. (ii) BBB was assembled in the CUBE by first seeding astrocytes and pericytes embedded in Matrigel in the CUBE, then seeding BMECs with ROCK inhibitor Y27632 on the surface of the Matrigel after it has been cured. b The 3D structure of the BBB-in-a-CUBE was visualized on Day 6 by labelling BMECs with von Willebrand Factor (vWF), astrocytes with GFAP or CX3CR1, and pericytes with PDGFRβ or NG2, then counterstaining with DAPI. (i) was imaged at 63× magnification with 0.75 zoom factor and (ii) was imaged at 25× magnification with 1.0 zoom factor. c BBB-in-a-CUBE on Days 2, 4, and 6 from the side view was visualized by labelling astrocytes with CellLight Tubulin-RFP and pericytes with CellLight Actin-GFP, then counterstaining with DAPI. On Day 2 after seeding, the astrocytes and pericytes were evenly distributed within the Matrigel, but from Day 4 and Day 6, they can be seen to elongate towards and populate the region beneath the BMEC sheet, indicating self-organisation of the BBB structure. Scalebar = 200 µm for enlarged figure and 100 µm for all others.

Article Snippet: For experiments to visualize the movement of astrocytes and pericytes in the BBB model over the course of the culture period, astrocytes were labelled with CellLight Tubulin-RFP (Invitrogen, C10614) and pericytes with CellLight Actin-GFP (Invitrogen, C10582), overnight prior to making BBB-in-a-CUBE, according to the manufacturer’s protocol.

Techniques: Derivative Assay