hy 13867 atp competitive pkc inhibitor rbc  (MedChemExpress)

Journal: bioRxiv

Article Title: Resilience and restoration from fasting-refeeding mediated by a nutrient-regulated linker histone

doi: 10.1101/2025.04.14.648802

Figure Lengend Snippet: a , Schematic of candidate gene selection process for ARD-refeeding RNAi screen. b , Fold change (FC) of body size area relative to luci ctrl. for individual RNAi knockdown of 380 genes. Enhancement of regrowth over 15% are colored red, inhibition of regrowth by over 15% are colored blue. Dots indicate the mean body size of surviving worms from 30 refed worms distributed over three plates. c , Volcano plot of DEGs during ARD 10 d vs. Refed 1 d. d , Volcano plot of differentially expressed proteins during -AA 1 d Starvation vs. +AA 1 d (AA-replete) conditions. e , Volcano plot of differentially expressed proteins during Torin1 (250 nM) 1 d treatment vs. DMSO 1 d control. f , Images of HIL-1::GFP expression prior to and after ARD (1 d) induction. Scale bar 100 μm. g , Head images of HIL-1::GFP expression in WT and daf-16(mu86) ; hlh-30(tm1978) double mutant background. Scale bar 20 μm. Zoomed in images of some selected nuclei in middle panels. Zoomed in scale bar 2 μm. (Right) Quantification of number of head nuclei with HIL-1::GFP induction at ARD 1 d in WT and daf-16(mu86) ; hlh-30(tm1978) worms. N = 3. Error bars represent mean ± SEM. h , Summary of transcriptomic regulation of C. elegans linker histones during ARD and longevity contexts ( daf-2(e1370) , glp-1(e2141) , eat-2(ad465), raga-1(ok386) ). Significantly up- or downregulated genes (Adj. P < 0.05) are indicated with *. i , Summary of statistically significant hil-1 / H1 - 0 regulation in low nutrient conditions across organisms from both publicly available transcriptomics data and our RNA-sequencing analysis (marked with † ). For A. thaliana , HIS1-3 , a drought-induced linker histone with similarity to H1-0 is shown. Unpaired t-test **** P < 0.0001. In volcano plots, significantly upregulated genes/proteins (Adj. P < 0.05) are colored red; significantly downregulated genes/proteins are colored blue.

Article Snippet: For inhibition of mTOR, the ATP-competitive inhibitor Torin1 (cat. no. 4247, Tocris) was dissolved in DMSO and added to freshly prepared full media to attain a final concentration of 250 nM.

Techniques: Selection, Knockdown, Inhibition, Control, Expressing, Mutagenesis, RNA Sequencing

Journal: bioRxiv

Article Title: Resilience and restoration from fasting-refeeding mediated by a nutrient-regulated linker histone

doi: 10.1101/2025.04.14.648802

Figure Lengend Snippet: a , Immunoblots of lysates from HEK293FT cells under -AA 1 d Starvation vs. +AA 1 d conditions and Torin1 1 d (250 nM) vs. DMSO 1 d control, probed with the indicated antibodies. The histone deacetylase inhibitor Vorinostat (10 µM) was used as a control for H1.0 induction . N = 3. b , Immunofluoresence staining of H1.0 in HEK293FT cells under -AA 1 d Starvation vs. +AA 1 d conditions. Scale bar, 50 µm; N = 3. c , Evaluation of cell death in HEK293FT H1-0 KO cells during -AA 10 d Starvation, as measured by the Incucyte Cytotoxicity Assay. Cell death was quantified by Red object area. N = 3. Representative images after -AA 10 d Starvation are shown to the right; scale bar, 150 µm. d , Evaluation of cell death in HEK293FT H1-0 KO cells upon AA addback after -AA 5 d Starvation, as measured by the Incucyte Cytotoxicity Assay. Cell death was quantified by Red object area. N = 3. Representative images of AA addback 2 d are shown to the right; scale bar, 150 µm. e , Transcriptomic comparison of log 2 (FC) of WT response to -AA 1 d Starvation vs. H1-0 KO response to -AA 1 d Starvation. Significant DEGs from the genotype-starvation interaction term are highlighted in colors according to category. Nonlinear fit analysis compared the observed correlation against a hypothetical correlation of x = y, Slope = 1. P < 0.0001. f , Volcano plot of significantly (Adj. P < 0.05) differentially expressed TEs after -AA 1 d Starvation in WT vs. H1-0 KO cells. g , Volcano plot of significantly (Adj. P < 0.05) differentially expressed intron reads after -AA 1 d Starvation in WT vs. H1-0 KO cells. h , Top 6 enriched GO:BP terms of significant genes from the genotype-starvation interaction analysis are displayed for each category. i , H1-0 mRNA levels were assessed by RT-qPCR in PBMCs isolated from five patients with autosomal dominant polycystic kidney disease (ADPKD) after a 3 d water fast. H1-0 expression FC was calculated relative to the mean of two AL baseline values. Ribosomal Protein Lateral Stalk Subunit P0 ( RPLP0 ) was used as a housekeeping gene. j , Graphical summary of HIL-1/H1.0 regulation and function under low-nutrient states and refeeding. Error bars represent mean ± SEM. Statistical significance between two groups was tested by unpaired one-sample t-test (comparing against a hypothetical value of one) while the Incucyte timecourse was assessed by multiple unpaired t tests with Holm-Šídák correction. ns = not significant. P * < 0.05; P ** < 0.01; P**** < 0.0001. ns = not significant.

Article Snippet: For inhibition of mTOR, the ATP-competitive inhibitor Torin1 (cat. no. 4247, Tocris) was dissolved in DMSO and added to freshly prepared full media to attain a final concentration of 250 nM.

Techniques: Western Blot, Control, Histone Deacetylase Assay, Staining, Cytotoxicity Assay, Comparison, Quantitative RT-PCR, Isolation, Expressing

Journal: bioRxiv

Article Title: Resilience and restoration from fasting-refeeding mediated by a nutrient-regulated linker histone

doi: 10.1101/2025.04.14.648802

Figure Lengend Snippet: a , Immunoblots of lysates from HEK293FT cells under -AA 1 d Starvation vs. +AA 1 d (AA-replete) conditions, probed with the indicated antibodies. N = 4. b , Immunoblots of lysates from HEK293FT cells under Torin1 1 d (250 nM) vs. DMSO 1 d control conditions, probed with the indicated antibodies. N=4. c , RNA-seq expression of H1-2 in HEK293FT cells under -AA 1 d Starvation vs. +AA 1 d. N = 4. d , RNA-seq expression of H1-2 in HEK293FT cells under Torin1 1 d (250 nM) vs. DMSO 1 d control conditions. N = 4. e , Immunoblots of lysates from WI-26 cells under -AA 1 d Starvation vs. +AA 1 d, and Torin1 1 d (250 nM) vs. DMSO 1 d. Vorinostat (10 µM) was used as a control for H1.0 induction . N = 3. f , Immunoblots of lysates from HEK293FT cells under AA addback after -AA 1 d Starvation (addback for 1 d and 2 d) vs. -AA 1 d Starvation, probed with the indicated antibodies. Displayed data was normalized to AA-replete +AA 1 d condition. N = 3. g , H1-0 mRNA levels were assessed by RT-qPCR in HEK293FT cells under -AA 1 d Starvation vs. +AA 1 d. Displayed data was normalized to +AA 1 d condition. CCR4-NOT transcription complex, subunit 4 ( CNOT4 ) was used as a housekeeping gene. N = 3. h , H1-0 mRNA levels were assessed by RT-qPCR in HEK293FT cells under AA addback after -AA 1 d Starvation (addback for 8 h and 1 d) vs. -AA 1 d Starvation. Displayed data was normalized to +AA 1 d condition. CCR4-NOT transcription complex, subunit 4 ( CNOT4 ) was used as a housekeeping gene. i , Immunoblots of lysates from HEK293FT cells under the indicated siRNA-mediated knockdown conditions, probed with the indicated antibodies. AA Starvation 1 d was initiated 2 d after transfection. CNOT4 was used as a housekeeping gene. Displayed data was normalized to luci ctrl. +AA 1 d condition. N = 3. j , H1-0 mRNA levels were assessed by RT-qPCR in HEK293FT cells under the indicated knockdown conditions. AA Starvation 1 d was initiated 2 d after transfection. CNOT4 was used as a housekeeping gene. Displayed data was normalized to luci ctrl. +AA 1 d condition. N = 3. k , ReMap ChIP-seq track in the UCSC Genome Browser, showing experimentally validated binding of FOXO1 (green peak, GSE80773, CD34 cells) and FOXO3 (yellow peak, GSE97661, Hep-G2 cells) in the H1-0 promoter region. Error bars represent mean ± SEM. Statistical significance between two groups was tested by unpaired t-test or one-sample t-test (comparing against a hypothetical value of one) while the one-way ANOVA was used for multiple comparisons. P * < 0.05; P ** < 0.01; P*** < 0.001; P**** < 0.0001. ns = not significant.

Article Snippet: For inhibition of mTOR, the ATP-competitive inhibitor Torin1 (cat. no. 4247, Tocris) was dissolved in DMSO and added to freshly prepared full media to attain a final concentration of 250 nM.

Techniques: Western Blot, Control, RNA Sequencing, Expressing, Quantitative RT-PCR, Knockdown, Transfection, ChIP-sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Neutrophil extracellular traps have active DNAzymes that promote bactericidal activity

doi: 10.1093/nar/gkae1262

Figure Lengend Snippet: Peroxidase activity of G4/H DNAzyme in purified NETs DNA using AR. Peroxidase activity using equal masses of Telo G4 and purified NETs DNA were assayed with various combinations of individual components, G4 specific binding inhibitors (BRACO19 and NMM) and antioxidant (vitamin C). Telo G4 + hemin + H 2 O 2 and HRP + H 2 O 2 used have comparable activity. NETs + hemin + H 2 O 2 had greater activity than individual components and 7.6x higher than hemin alone. Peroxidase activity was abrogated by G4 inhibitors and antioxidant. All experiments were performed in triplicate with mean and standard deviation plotted. One-way ANOVA test with Tukey’s honestly significant difference test performed to compare the different combinations. **** indicates P -value < 0.0001.

Article Snippet: Four units DNase I, 20 units EcoR I, 150 μM G4-specific competitive inhibitor BRACO19 (#SML0560, MilliporeSigma), the G4-binding hemin analog 150 μM NMM (#396879, Santa Cruz Biotechnology) and 2 mM FR scavenger vitamin C (#11140, MilliporeSigma) were tested to abrogate the DNAzyme activity.

Techniques: Activity Assay, Purification, Binding Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Neutrophil extracellular traps have active DNAzymes that promote bactericidal activity

doi: 10.1093/nar/gkae1262

Figure Lengend Snippet: Ex vivo bactericidal activity of the G4/H DNAzyme against EC. ( A ) Schematic of the ex vivo bactericidal assay. Neutrophils from whole blood were stimulated with IL-8 and treated with various combinations of antiphagocytic cytochalasin D, G4 specific inhibitor BRACO19, G4 binding hemin analog NMM and DNase I with no additional hemin or H 2 O 2 . ( B ) Bactericidal activity of isolated neutrophils through NETs against EC. Approximately 80% of inoculated EC was killed by IL-8-stimulated NETs. Phagocytosis accounts for an additional ∼10% of killing. Abrogation of NETs killing by G4-specific inhibitors like BRACO19, NMM or antioxidant vitamin C (<20%). DNase I treatment only reduced killing by ∼20%. ( n = 3 biologically independent experiments; bars represent mean signal, and error bars denote s.e.m.; one-way ANOVA performed; **** indicates P -value < 0.0001).

Article Snippet: Four units DNase I, 20 units EcoR I, 150 μM G4-specific competitive inhibitor BRACO19 (#SML0560, MilliporeSigma), the G4-binding hemin analog 150 μM NMM (#396879, Santa Cruz Biotechnology) and 2 mM FR scavenger vitamin C (#11140, MilliporeSigma) were tested to abrogate the DNAzyme activity.

Techniques: Ex Vivo, Activity Assay, Serum Bactericidal Assay, Binding Assay, Isolation