Journal: Nature Communications

Article Title: Xrn1 is a deNADding enzyme modulating mitochondrial NAD-capped RNA

doi: 10.1038/s41467-022-28555-7

Figure Lengend Snippet: a Schematic illustration of in vitro 5′-end RNA decay. b Time-course decay analysis of uniformly 32 P-labeled m 7 G- or NAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from n = 3 independent experiments (±SD denoted by error bars). Labeling as in the legend to Fig. . c Total NAD-capped RNA levels from WT and xrn1Δ were detected by NAD-capQ. Data represents average from n = 4 independent experiments. Error bars represent ±SEM; two sided unpaired t test; ** p = 0.0036). d qRT-PCR quantification of NAD-capped mRNA. NAD-capped RNA was enriched by NAD-capture, eluted from the beads, reverse transcribed and detected with gene-specific primers. Data are presented relative to the WT cells set to 1 and derived from n = 3 independent NAD-capture experiments; mean ± SEM; two-sided unpaired t test; ** p < 0.001, *** p < 0.0001. e Purified mitochondria derived from a strain harboring Xrn1 with a C-terminus Strep-tag II was analyzed by Western blot analysis with an anti-Strep-tag antibody. Western blotting analysis of whole cell extract (WCE) and extract prepared from purified mitochondria (mito) using Pgk1 (cytoplasmic protein) and Cox2 (mitochondrial protein) to determine the purity of mitochondria. Source data for panels b, c and d are provided in Source Data File . f Xrn1 knock-in strain containing the gene encoding green fluorescence protein (GFP) in frame with endogenous Xrn1 was generated into the strain carrying Edc3 fused to mCherry. Colocalization of Xrn1 with mitochondria (Mitotracker® Deep Red FM) and Edc3 (mCherry) by standard confocal (40X) microscopy is shown. g Reconstructed STORM images. Left panel shows images from a single optical section of RFP-labeled mitochondria and GFP-labeled Xrn1 in a single yeast cell obtained by spinning disk confocal (top) and STORM imaging (bottom). Merged image shows overlap of RFP and GFP signal. Right panel shows reconstructed STORM images obtained from sequentially acquired optical sections spaced 200 nm apart in the z axis. Merged images show colocalization of signals within the ~10 nm XY resolution. Scale bar is 100 nm.

Article Snippet: Mitochondrial preparation purity was assessed by Western blot analysis Anti-Cox2 (Anti-MTCO2 antibody [4B12A5] (ab110271)), and anti-Pgk1 ((PA5-28612) Invitrogen) antibodies were used to detect the respective proteins.

Techniques: In Vitro, Labeling, Quantitative RT-PCR, Derivative Assay, Purification, Strep-tag, Western Blot, Knock-In, Fluorescence, Generated, Microscopy, Imaging

Journal: Cell reports

Article Title: Circadian-clock-controlled endocrine and cytokine signals regulate multipotential innate lymphoid cell progenitors in the bone marrow

doi: 10.1016/j.celrep.2024.114200

Figure Lengend Snippet: (A) Expression of Rora at mRNA level in BM ILCPs from C57BL/6 mice based on publicly available scRNA-seq data (GSE193835). (B) Expression of Rora at the mRNA level in sorted BM ILCPs at different ZTs. RT-qPCR was performed. (C) Rora mRNA expression was determined at around ZT 5 using RT-qPCR in sorted BM Lin − CD127 + cells of Bmal1 f/f versus Plzf cre Bmal1 f/f mice. (D) RORα binding to the S1pr1 and Bmal1 genes in BM Lin − CD127 + ILC-lineage cells isolated from mice sacrificed at ZT 5 and 11. (E) The transcriptional activity of the S1pr1 promoter region was determined with a luciferase reporter assay. The reporter plasmid was transfected into pre-cultured Lin − BM cells for 10 h with or without regular or inverse RORα agonists and assayed for luciferase activity. (F) Expression of cell surface S1PR1 by in vitro cultured BM ILCPs with/without regular or inverse RORα agonists. (G) Chemotaxis of the cultured BM ILCPs with regular or inverse RORα agonists. BM ILCPs were cultured with or without regular or inverse RORα agonists (10 μM) and cytokines (SCF and IL-7 at 20 ng/mL) on OP9-DL1 cells as the feeder layer for 5 days. Cells from Rag1 −/− mice were used unless indicated otherwise. Pooled data obtained from at least three different experiments ( n = 3–6) are shown with SEM. p values from one- or two-way ANOVA (B and D–G) and two-tailed/paired Student’s t test (C) are shown.

Article Snippet: Immunoprecipitation was performed using rabbit control immunoglobulin G (IgG), GR (Cell Signaling Technology, D6H2L) or RORα (Santa Cruz Biotechnology sc-28612). qPCR analysis was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Isolation, Activity Assay, Luciferase, Reporter Assay, Plasmid Preparation, Transfection, Cell Culture, In Vitro, Chemotaxis Assay, Two Tailed Test

Journal: Cell reports

Article Title: Circadian-clock-controlled endocrine and cytokine signals regulate multipotential innate lymphoid cell progenitors in the bone marrow

doi: 10.1016/j.celrep.2024.114200

Figure Lengend Snippet: (A) Enhanced expression of Rora , Bmal1 , and Clock transcripts by IL-18. RT-qPCR on cultured ILCPs with IL-18 (20 ng/mL) for 5 days in the presence of IL-7 and SCF was performed. (B) The effect of IL-18 neutralization on the frequency of blood circulating ILCPs at different ZTs in Rag1 −/− mice. Mice were injected intraperitoneally with a neutralizing anti-IL-18 (YIGIF74–1G7) or isotype control antibody (Ab) on day 1 and 4 and sacrificed at the indicated ZTs on day 6. (C) S1PR1 expression on BM ILCPs cultured for 5 days with regular or inverse RORα agonists in the presence and absence of IL-18. (D) Expression of S1PR1 by BM ILCPs treated with or without 7α-OHC and/or IL-18. (E) Effects of regular or inverse RORα agonists on the frequency of BM and blood ILCPs. (F) Effects of regular or inverse RORα agonists on the emigration of BM ILCPs. (E and F) Rag1 −/− mice were treated with or without IL-18 (daily for 4 days) and Neo or SR3335 (2 times in 4 days) and examined at around ZT 5. (G) Impact of the circadian clock on the response of cultured BM ILCPs to RORα ligands and IL-18. BM ILCPs were cultured with SCF and IL-7 on OP9-DL1 cells for 5 days in the presence or absence of the indicated factors. Cells from Rag1 −/− mice were used unless indicated otherwise. Pooled data obtained from at least three different experiments ( n = 4–6) are shown with SEM. p values from two-tailed/paired Student’s t test (A) and one- or two-way ANOVA (B–G) are shown.

Article Snippet: Immunoprecipitation was performed using rabbit control immunoglobulin G (IgG), GR (Cell Signaling Technology, D6H2L) or RORα (Santa Cruz Biotechnology sc-28612). qPCR analysis was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Neutralization, Injection, Control, Two Tailed Test

Journal: Cell reports

Article Title: Circadian-clock-controlled endocrine and cytokine signals regulate multipotential innate lymphoid cell progenitors in the bone marrow

doi: 10.1016/j.celrep.2024.114200

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-mouse RORa (Clone H-65) , Santa Cruz Biotechnology , Cat# sc-28612; RRID: AB_2265518.

Techniques: Plasmid Preparation, Software

Journal: Journal of Fungi

Article Title: The Smk1 MAPK and Its Activator, Ssp2, Are Required for Late Prospore Membrane Development in Sporulating Saccharomyces cerevisiae

doi: 10.3390/jof7010053

Figure Lengend Snippet: Smk1 post-translational modification does not require the leading edge protein complex members ADY3 , IRC10 , and DON1 . Immunoblot of protein extracts examining Smk1-HA in an untagged strain lacking Smk1-HA (LH177), Smk1-HA (LH178), ady3∆ (LH1126), irc10∆ (LH1127), don1∆ (LH1128), and ssp2∆ (LH1129). Pgk1 is used as a loading control. The 50 kDa band from the Precision Plus Protein Markers (BioRad) is indicated on the left of each blot. The upper quantification corresponds to densitometry, in which the slower migrating (upper) Smk1-HA band was first normalized to Pgk1 levels, followed by the normalization of the slower migrating Smk1-HA band in each mutant to wild-type levels of expression. The lower quantification corresponds to densitometry, in which the slower migrating Smk1-HA band was normalized to the faster migrating Smk1-HA band for each genotype.

Article Snippet: Pgk1 was detected using a rabbit polyclonal anti-Pgk1 antibody (PA5-28612, ThermoFisher, Waltham, MA, USA) at a 1:5000 dilution.

Techniques: Modification, Western Blot, Control, Mutagenesis, Expressing