Glutaredoxin Deletion Shortens Chronological Life Span in <i>Saccharomyces cerevisiae</i> via ROS-Mediated Ras/PKA Activation

Glutaredoxins (GRXs), small redox proteins that use reduced glutathione as an electron donor, are key components of the cellular antioxidant system. In this study, we used <i>Saccharomyces cerevisiae</i> as a model system to investigate the effects of GRX deletion on yeast chronological life span (CLS). Deletion of either Grx1 or Grx2 shortened yeast CLS. Quantitative proteomics revealed that GRX deletion decreased the expression of stress-response proteins, leading to increased cellular reactive oxygen species accumulation and, subsequently, intracellular acidification. This activated the Ras/protein kinase A (PKA) signaling pathway. Genetic and biochemical analyses demonstrated that Ras/PKA activation decreased stress resistance and increased biosynthesis, requiring yeast cells to grow under unfavorable conditions and resulting in a shortened CLS. Our results provided new insights into mechanisms underlying exacerbation of the aging process by oxidative stress

Glutaredoxin Deletion Shortens Chronological Life Span in <i>Saccharomyces cerevisiae</i> via ROS-Mediated Ras/PKA Activation

Glutaredoxins (GRXs), small redox proteins that use reduced glutathione as an electron donor, are key components of the cellular antioxidant system. In this study, we used <i>Saccharomyces cerevisiae</i> as a model system to investigate the effects of GRX deletion on yeast chronological life span (CLS). Deletion of either Grx1 or Grx2 shortened yeast CLS. Quantitative proteomics revealed that GRX deletion decreased the expression of stress-response proteins, leading to increased cellular reactive oxygen species accumulation and, subsequently, intracellular acidification. This activated the Ras/protein kinase A (PKA) signaling pathway. Genetic and biochemical analyses demonstrated that Ras/PKA activation decreased stress resistance and increased biosynthesis, requiring yeast cells to grow under unfavorable conditions and resulting in a shortened CLS. Our results provided new insights into mechanisms underlying exacerbation of the aging process by oxidative stress

Additional file 1 of Comparison of swept-source OCTA and indocyanine green angiography in central serous chorioretinopathy

Additional file 1: Supplementary Table 1. The mean areas of type A abnormalities in acute and chronic CSC on SS-OCTA and ICGA for the two graders

Additional file 1 of A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

Additional file 1: Fig. S1. AML1-ETO promotes expression of FTO via PU.1. (A) Comparison of overall survival of patients with de novo t(8;21) AML (n = 26) using the Kaplan–Meier method grouped by the expression of FTO (high vs. low). p value was evaluated using the log-rank test. (B) Kaplan–Meier analysis of event-free survival (left) and overall survival (right) of patients with AML (n = 344, data from GSE6891) based on the expression of FTO. (C) Comparison of the expression of FTO in Kasumi-1 cells with or without AML1-ETO knockdown (shAE vs. shNS) detected by RNA-seq in the GSE115121 data set. (D) ChIP-seq of GSE65427 depicting FTO loci in Kasumi-1 cells targeting C- terminus of ETO (upper panel) and N-terminus of AML1(lower panel), representing AML1-ETO peaks on FTO. (E) Schematic diagrams showing the amplified regions on the promoter of SPI1 for the ChIP-qPCR showed in Fig. 1I and Fig. S1F. The location of targeted amplified region (named ‘Target’) and negative control site (NC) are indicated with blue horizontal lines. The red triangle indicates the location of peak summit of AML1-ETO on the promoter of SPI1 detected by the ChIP-seq of GSE65427. (F) ChIP-qPCR assays showing no direct binding of AML1 or ETO within 200 bp upstream of the SPI1 promoter in SKNO-1-siAE cells. (G and H) Pearson correlation of the expression of FTO and SPI1 in (G) normal blood tissues from the Genotype-Tissue Expression Project (GTEx, n = 444) or (H) BM samples of patients with AML from TCGA database (n = 173). (I) Sequences of the 4 FTO promoter fragments (the P1 to P4 showed in Fig. 1P) and putative PU.1 binging sites (sites 1, 2, and 3). Fig. S2. FTO upregulated AML1-ETO in a m6A-dependent manner. (A–C) The level of AML1-ETO mRNA detected by qPCR in SKNO-1 and Kasumi-1 cells (A) transduced with wild-type FTO (wt-FTO), mutant FTO (mut-FTO), or mock vectors; (B) transduced with FTO-knockdown (shFTO#1 and shFTO#2) or scramble shRNA (shNS) vectors; (C) treated with DMSO or FB23-2 treatment for 72 h. Fig. S3. Oncogenic role of FTO in t(8;21) AML cells and AML1-ETO9a driven AML mice. (A) Effects of forced expression or knockdown of FTO on cell cycle in SKNO-1 and Kasumi-1 cells. (B) Effects of forced expression or knockdown of FTO on colony-forming capacity of Kasumi-1 cells. (C) The effect of FTO knockdown on differentiation of Kasumi-1 cells. The percentage of CD11b+ cells was quantified (right panel). (D) Wright-Giemsa staining of Kasumi-1 cells with or without FTO knockdown. (E) Spleen size in AML1-ETO9a-driven AML mice with or without Fto knockdown or treatment with DMSO or FB23-2 (6 mg/kg) 7 weeks after transplantation (n = 6 for each group). (F) Spleen weight of AML1-ETO9a-driven AML mice from (E). (G) Wright-Giemsa staining of bone marrow of AML1-ETO9a-driven AML mice. (H–J) Percentage of GFP+ AML1-ETO9a AML cells in the (H) peripheral blood (PB), (I) bone marrow (BM), and (J) spleen (SP) of the mice treatment with DMSO or FB23-2 by flow cytometric analysis. (K–M) Flow cytometric analysis of the distribution of anti-CD11b-stained GFP + AML1-ETO9a AML cells in PB (K), BM (L), and SP (M) of mice treatment with DMSO or FB23-2. Fig. S4. Suppression of FTO resensitizes resistant cells to Ara-C in vitro and in vivo. (A and B) Apoptosis measured by flow cytometry for SKNO-1 (A) and Kasumi-1 (B) cells treated with DMSO, 30 µM Ara-C alone, 10 µM FB23-2 alone or combination of Ara-C and FB23-2 for 48 h with FB23-2 pretreatment for 6 h. (C and D) Percentage of GFP+ AML cells in peripheral blood (C) and spleen (D) of NOD/SCID/γcnull immunodeficient mice injected with SKNO-1 cells through tail vein treated with DMSO, Ara-C, FB23-2, or a combination of Ara-C and FB23-2 (n = 6 for each group). Fig. S5. Transcriptome-wide identification of FTO targets in t(8;21) AML. (A and B) Proportion of the distribution of m6A peaks in exon, intron, and intergenic regions across entire mRNA transcripts (A) or in the 5′-UTR, first exon, other exon, and 3′-UTR of mRNA transcripts (B) detected by m6A-seq assays in Kasumi-1 cells transduced with wild-type FTO or empty vector. (C and D) Gene set enrichment analysis (GSEA) of genes with a significant decrease in m6A levels as well as a significant increase (Hypo-up) or decrease (Hypo-down) in overall transcript levels in FTO-overexpressing Kasumi-1 cells. (E) Comparison of IGFBP2 expression between human primary AML cases with t(8;21) (n = 30, data from GSE30285) or normal controls (NC) (n = 9, data from GSE34814). (F) Kaplan–Meier analysis of event-free survival (left) and overall survival (right) of patients with AML (n = 344, data from GSE6891) based on the expression of IGFBP2. (G) Pearson correlation of the expression of FTO and IGFBP2 in BM samples of patients with AML from TCGA database (n = 173). Fig. S6. Luciferase reporter construction and identification of specific m6A readers targeting the 3′-UTR of IGFBP2 mRNA. (A) Construction of luciferase reporter vectors. Synthesized wildtype (wt) or mutant (mut) 3′ coding sequences of IGFBP2 were inserted into the XhoI and NotI site of the psiCHECK2 luciferase reporter. Putative m6A consensus motifs are shown in bold, whereas mutation sites (A to T mutation) are shown in red. (B and C) Identification of m6A specific binding proteins on 3′-UTR of IGFBP2 by RNA pull-down using 4 pairs of single-stranded RNA (ssRNA) baits containing the 4 m6A consensus motif on the 3′-UTR sequence of IGFBP2 respectively, with methylated (green) or unmethylated (red) adenosine (B). The iBAQ value of previously reported m6A readers (including YTHDFs, YTHDCs, IG2FBPs and hnRNPs) enriched by the 4 pairs of ssRNA probes detected by mass spectrometry analysis are shown (C). The YTHDC1, YTHDC3, and IGF2BP1 proteins that could not be enriched by all 4 pairs of ssRNA probes are not shown in the Figure (see Additional file 3: Table S4). (D and E) Western blot analysis of the expression of IGFBP2 with or without silencing of YTHDF2 (D) or YTHDF3 (E) by siRNA in SKNO-1 or Kasumi-1 cells. siNS, scramble siRNA. Fig. S7. Functional role of IGFBP2 in t(8;21) AML. (A and B) Effects of IGFBP2 knockdown on colony-forming capacity (A) and cell cycle (B) in SKNO-1 and Kasumi-1 cells. (C) Western blot analysis of silencing IGFBP2 by siRNA in SKNO-1 and Kasumi-1 cells. siNS, scramble siRNA. (D and E) External views (D) and weight (E) of the spleens from AML1-ETO9a-driven AML mice with or without Igfbp2 knockdown (n = 6 for each group) 7 weeks after transplantation. Fig. S8. FTO regulates leukemogenesis and sensitivity of t(8;21) AML cells to Ara-C through IGFBP2. (A) Effects of FTO knockdown with IGFBP2 overexpression after Ara-C treatment on colony-forming capacity of SKNO-1 and Kasumi-1 cells. (B–D) External views (B), weight of the spleens (C) and flow cytometric analysis of CD11b+ AML cells in PB, BM, and SP (D) of AML1-ETO9a-driven AML mice in Fig. 8E–G

Role of Nrf2 in dh404-induced suppression of cardiomyocyte hypertrophy and death, and cardiac fibroblast proliferation in vitro.

<p>(A) Effect of dh404 (200 nmol/L) on NE (20 µmol/L)-induced [³H]leucine uptake (left pannel) and H₂O₂ (100 µmol/L)-induced cell death of the cardiomyocytes infected with adenovirus of control (cont) shRNA and Nrf2 shRNA. *p<0.05 vs cont shRNA (-), n = 4. (B) Left pannel - Effect of dh404 (200 nmol/L) on Ang II (0.1 µmol/L)-, NE (20 µmol/L), and PE (20 µmol/L)-induced [³H]thymidine uptake in the cardiac fibroblasts. *p<0.05 vs control dh404 (-), n = 6. Right pannel – Effect of dh404 (200 nmol/L) on NE (20 µmol/L)-induced [³H]thymidine uptake in the cardiac fibroblasts infected with adenovirus of control (cont) shRNA and Nrf2 shRNA. *p<0.05 vs control dh404 (-), n = 4.</p

Effect of dh404 on myocardial fetal gene expression of mice after TAC.

<p>Hearts of mice were harvested, and left ventricles were dissected for RNA purification 4 weeks after the operation. Expression of ANF, BNP, αMHC, and βMHC was quantified by Q-PCR. *p<0.05 vs sham.</p

Echocardiography and pathology of mice treated with and without dh404 for 4 wks after TAC.

<p>Two dimension guided M-mode echocardiography and pathology were performed 4 weeks after the initiation of TAC or sham surgery in C57BL/6J mice treated with vehicle or dh404 as indicated.</p><p>Abbreviations: IVSd, interventricular septum diastolic; LVIDd, left ventricular internal dimension diastolic; LVIDs, left ventricular internal septum diastolic; LVPWd, left ventricular posterior wall diastolic; FS, fractional shortening; BW, body weight; HW/Tibia L, heart weight/tibia length ratio; LW/Tibia-L, lung weight/tibia length ratio.</p>a<p>p<0.05 vs sham (0);</p>b<p>p<0.05 vs TAC (0);</p>c<p>p<0.05 vs TAC + dh404 (5 mg/kg/d);</p>d<p>p<0.05 vs TAC + dh404 (10 mg/kg/d).</p

Effect of dh404 on myocardial fibrosis in mice after TAC.

<p>Myocardial fibrosis was assessed by staining of collagen with a Masson’s Trichrome Kit. In the left panels representative photomicrographs of a left ventricular (LV) and interventricular septum (IVS) sections from TAC hearts with and without dh404 treatment are presented. In the right panel the fibrotic areas of LV and IVS interstitial fibrosis as a percentage of total microscopic area per heart are presented. The numbers represent the number of sham and TAC hearts analyzed.</p

Effect of dh404 on myocardial apoptosis in mice after TAC.

<p>(A) In the left panel, the representative TUNEL staining of apoptotic cells in left ventricle are shown. TUNEL-positive, i.e., apoptotic nuclei in TAC hearts are stained red, nuclei blue and the myocardium is green (Alexa Fluor 488 Phalloidin to mark F-actin). Magnification, X 630. In the right panel, the apoptotic index results are given. TUNEL-positive cells were quantified as a percent of all nuclei in the section of LV. The number of hearts analyzed for each group is indicated. (B) In the left panel, the representative staining of cleaved caspase-3 positive cells in the left ventricle are shown. Cleaved caspase-3-positive, (i.e., cells with activated caspase-3) in TAC hearts are stained red and nuclei are blue. Cardiomyocytes were green utilizing an antibody of anti-cardiac myosin heavy chain. Magnification, X 630. In the right panel, the relative caspase-3 activity is summarized. Cleaved caspase-3-positive cells were quantified as a percent of all in a section of LV. The number of hearts analyzed for each group is indicated.</p

Effect of dh404 on myocardial Nrf2 expression of mice after TAC.

<p>C57BL/6J mice at age of 8 weeks were treated with vehicle or dh404 (10 mg/kg/d) and subjected to sham or TAC operations. Their hearts were harvested 4 weeks after the operation and analyzed for Nrf2 protein expression. Left panel - representative immunohistochemical staining of Nrf2 protein expression in left ventricles from TAC mice with or without dh404 treatment. Nrf2 nuclear translocation is indicated by arrows. Nrf2 staining was performed in 4 or more sections per heart. Nrf2 is stained red, nuclei blue and cardiomyocytes green utilizing an antibody of anti-cardiac myosin heavy chain. Right panel - a semi-quantitative analysis of Nrf2 protein levels in the heart. The number of hearts analyzed is indicated.</p