Journal: Science Advances

Article Title: NRF2 coordinates cystine import and glutathione use to sustain ferroptosis resistance and formaldehyde tolerance in cancer

doi: 10.1126/sciadv.aea1431

Figure Lengend Snippet: ( A ) Volcano plot showing dysregulated genes in lung adenocarcinoma tumors (TCGA-LUAD cohort) carrying driver mutations in NFE2L2 or KEAP1 [cutoffs: Log2(FC) = ±0.6 and P -adj ≤ 0.05]. ( B ) Gene Set Enrichment Analysis (GSEA) using Kyoto Encyclopedia of Genes and Genomes (KEGG) gene sets in the TCGA-LUAD cohort with mutations in NFE2L2 or KEAP1 . ( C ) mRNA expression of selected genes involved in formaldehyde (FA) detoxification in KEAP1-NFE2L2 mutated and nonmutated (WT) LUAD tumors. ( D ) mRNA expression of selected genes in normal lung and whole blood obtained from GTEX. ( E ) Single-cell RNA-seq data from mouse hematopoietic and progenitor cells (HPC) plotted with kleintools ( https://kleintools.hms.harvard.edu/tools/spring.html ) . ( F ) Representative images of Western blots of selected factors from (A) performed in different cell lines. ( G ) Cystine uptake assay performed in selected cell lines (mean ± SEM, n = 3). ( H ) FA tolerance of A549 cells lacking SLC7A11 (sg SLC7A11 ) complemented with a wild-type copy of SLC7A11 (pSLC7A11) grown in the presence of 100 μM BSO (mean ± SEM, n = 3 and two-way ANOVA, **** P < 0.0001). ( I ) FA tolerance for H1299 cells harboring empty vector (pEV) or pSLC7A11 in the presence of 100 μM BSO [mean ± SEM, n = 3 and two-way ANOVA (Tukey test between untreated (UT) and BSO at each point); for H1299 pEV: **** P < 0.0001 and *** P = 0.0002]. ( J ) Viability of A549 cells carrying sg SLC7A11 and sgNTC (nontargeting control) under cystine deprivation [one-way ANOVA (Tukey test)]. ( K ) Glutathione (GSH) levels of the indicated cell lines maintained during 48 hours under cystine deprivation and exposing to FA during the last 24 hours [one-way ANOVA (Tukey test), ** P = 0.0058]. ( L ) FA tolerance of A549 cells lacking ADH5 in the presence of BSO (100 μM) or ERA (5 μM) [mean ± SEM, n = 6 and two-way ANOVA (Tukey test between UT and BSO or ERA at each point for multiple comparison); * P = 0.0300 and **** P < 0.0001].

Article Snippet: ADH5 -deficient cells were generated in A549 cells stably expressing Cas9 (Addgene, catalog no. 78546) infected with a lentiviral vector coding for an sgRNA against the exon 3 of ADH 5 previously published ( ).

Techniques: Analysis, Expressing, Single Cell, RNA Sequencing, Western Blot, Plasmid Preparation, Control, Comparison

Journal: Aging Cell

Article Title: A Cilia–Centered Nicotinamide Adenine Dinucleotide ( NAD + ) Signaling Network Regulates Hypothalamic Lysosomal Homeostasis and Metabolic Aging

doi: 10.1111/acel.70745

Figure Lengend Snippet: SIRT1 mediates the regulatory effect of NAD + on primary cilia. (a) SIRT1 activator resveratrol (20 μM) treatment elongates cilia length in N1 cells ( n = 8 wells per group). AC3: Adenylyl cyclase 3. (b) Effects of overexpression (OE) of wild–type SIRT1 and its catalytically inactive H363Y mutant on ciliary length in N1 cells ( n = 5 wells per group). (c) Effect of treatment with the SIRT1 inhibitor EX527 (100 μm for 16 h) on NR (100 μM)‐induced cilia growth ( n = 6 wells per group). (d) Sirt1 knockdown with small inhibitory RNA ( siSirt1 ) prevents NMN (100 μM)–induced cilia elongation ( n = 3 wells per group). (e) Effect of SIRT1 OE on mRNA expression levels of multiple cilia‐related genes ( n = 6 wells per group). (f) Sirt1 knockdown inhibits NMN‐induced upregulation of cilia‐related genes ( n = 6 wells per group). (g, h) SIRT1 and RFX1 cooperatively regulate the promoter activity of the Ift88 gene ( n = 3 wells per group). (i, j) Molecular interaction between SIRT1 and RFX1, and SIRT1‐mediated deacetylation of RFX1, demonstrated by sequential immunoprecipitation (IP) and immunoblotting (IB) in N1 cells with or without SIRT1 OE. Ac‐K, acetylated lysine. (k) Effect of autophagy inhibition by Atg5 knockdown or 3‐MA treatment on SIRT1‐induced changes in primary cilia ( n = 5). Data were obtained from three independent experiments and are presented as the mean ± SEM. Statistical analyses were performed using two‐sided unpaired Student's t‐ test (a, e), one‐way ANOVA (b, c, f, g, h, k), or two‐way (d) ANOVA with Tukey's post hoc test. n.s., not significant. Scale bars, 20 μm.

Article Snippet: Cotransfections were performed with or without expression plasmids for SIRT1 (Flag–SIRT1, 500 ng, Addgene #1791), Rfx1 (Psg5–Rfx1, 50 ng, provided by Dr. Hsu, National Health Research Institutes, Taiwan), or siSirt1 (100 pmol), as indicated.

Techniques: Over Expression, Mutagenesis, Knockdown, Expressing, Activity Assay, Immunoprecipitation, Western Blot, Inhibition

Journal: Aging Cell

Article Title: A Cilia–Centered Nicotinamide Adenine Dinucleotide ( NAD + ) Signaling Network Regulates Hypothalamic Lysosomal Homeostasis and Metabolic Aging

doi: 10.1111/acel.70745

Figure Lengend Snippet: Primary cilia are essential for NAD + ‐mediated regulation of lysosomal acidity. (a) Effects of NAD + elevation induced by NAD or its precursors (NMN, NR) treatment on lysosomal acidity, assessed by LysoSensor fluorescence intensity ( n = 6 wells per group). Scale bars, 20 μm. (b) Effect of overexpression of wild‐type SIRT1 and a catalytically inactive SIRT1 mutant ( H363Y ) on lysosomal acidity ( n = 5 wells per group). (c, d) Effects of NAD + depletion induced by FK866 treatment or Nampt knockdown on lysosomal acidity ( n = 6 wells per group). Scale bars, 20 μm. (e) Restorative effects of NMN co‐treatment on the FK866‐induced lysosomal deacidification in N1 cells transfected with control siRNA ( siControl ), Ift88 siRNA ( siIft88 ), or Ift20 siRNA ( siIft20 ) ( n = 4 wells per group). Scale bars, 20 μm. (f) Effects of NAD + depletion (FK866) and repletion (FK866 + NMN) on lysosomal cathepsin B activity, assessed by the Magic Red assay, in N1 cells with or without ciliary dysgenesis ( n = 6 wells per group). Data were obtained from three independent experiments and are presented as the mean ± SEM. Statistical analyses were performed using two‐sided unpaired Student's t ‐test (c, d), one‐way ANOVA (a, b), or two‐way ANOVA (e, f), followed by Tukey's post hoc test. n.s., not significant.

Article Snippet: Cotransfections were performed with or without expression plasmids for SIRT1 (Flag–SIRT1, 500 ng, Addgene #1791), Rfx1 (Psg5–Rfx1, 50 ng, provided by Dr. Hsu, National Health Research Institutes, Taiwan), or siSirt1 (100 pmol), as indicated.

Techniques: Fluorescence, Over Expression, Mutagenesis, Knockdown, Transfection, Control, Activity Assay