Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: LEDGF Binds H3R17me2a Promoting De Novo Nucleotide Biosynthesis in SETD2 Mutant Clear Cell Renal Cell Carcinoma.

doi: 10.1002/advs.202416809

Figure Lengend Snippet: Figure 7. LEDGF reads H3R17me2a regulating key enzymes in the de novo synthesis pathway. A) Heat maps and averaged CUT&Tag signals of H3R17me2a and LEDGF across ±5 kb from the transcription start site (TSS) in A498 cells. B) Distribution of H3R17me2a and LEDGF enrichment peaks on the genome. C) Motif analysis of high frequency enrichment of H3R17me2a and LEDGF shows that there is a high degree of co-enrichment in the genome. D) There are specific enrichment peaks at the TSS of PPAT, PAICS, GART, ADSL, and ADSS2 in indicated groups, suggesting a potential

Article Snippet: The antibodies used in this study are as follows: LEDGF (Abcam, ab177159), CARM1 (CST, #3379), H3R17me2a (Acive motif, #39 710), PRMT6 (Abcam, ab271091), H3K36me3 (CST, #4909), PPAT (Proteintech, #15401- 1-AP), PAICS (Proteintech, #12967-1-AP), GART (Proteintech, #13659-1- AP), ADSL (Proteintech, #15264-1-AP), ADSS2 (Proteintech, #16373-1- AP), Histone H3 (Proteintech, #17168-1-AP), Alpha Actin (Proteintech, #23660-1-AP), Flag (Abmart, #M20008), Goat Anti-Rabbit IgG (H + L) (Proteintech,#SA00001-2), and Goat Anti-Mouse IgG (H + L) (Proteintech, #SA00001-1).

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: LEDGF Binds H3R17me2a Promoting De Novo Nucleotide Biosynthesis in SETD2 Mutant Clear Cell Renal Cell Carcinoma.

doi: 10.1002/advs.202416809

Figure Lengend Snippet: Figure 8. Deficiency of LEDGF protects NKG mice against xenograft proliferation. A) Schematic diagram of subcutaneous tumor model in NKG mice in indicated treatment groups. All surviving mice were euthanized 8 weeks after tumor cell inoculation. B) Knock out of LEDGF effectively reduced the proliferation of xenografts in NKG mice. (n = 5) C) There was no significant difference in body weight between the two groups throughout the experiment. D–F) Elimination of LEDGF effectively reduced the volume (D-E) and weight (F) of NKG mice xenografts. G) QRT-PCR was used to demonstrate that decrease of LEDGF can reduce mRNA expression of PPAT, PAICS, GART, ADSL, and ADSS2 in xenograft tumors. H) The proliferation ability of xenografts in LEDGF-KO group was significantly reduced. The expression levels of PPAT, PAICS, GART, and ADSL were significantly decreased, while ADSS2 expres- sion was almost unchanged. Scale bar = 100 μm. I) A schematic model illustrating that LEDGF interacts with CARM1-mediated H3R17me2a to promote ccRCC progression. Data are shown as mean ± SD. ***p < 0.001. ns means no significance.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Abcam, ab177159), CARM1 (CST, #3379), H3R17me2a (Acive motif, #39 710), PRMT6 (Abcam, ab271091), H3K36me3 (CST, #4909), PPAT (Proteintech, #15401- 1-AP), PAICS (Proteintech, #12967-1-AP), GART (Proteintech, #13659-1- AP), ADSL (Proteintech, #15264-1-AP), ADSS2 (Proteintech, #16373-1- AP), Histone H3 (Proteintech, #17168-1-AP), Alpha Actin (Proteintech, #23660-1-AP), Flag (Abmart, #M20008), Goat Anti-Rabbit IgG (H + L) (Proteintech,#SA00001-2), and Goat Anti-Mouse IgG (H + L) (Proteintech, #SA00001-1).

Techniques: Knock-Out, Quantitative RT-PCR, Expressing

Journal: PLOS One

Article Title: Identification of novel antiviral host factors by functional gene expression analysis using in vitro HBV infection assay systems

doi: 10.1371/journal.pone.0314581

Figure Lengend Snippet: Primers used in this study. Primers used in custom TaqMan® Array Fast plate.

Article Snippet: 7 , ADSL , Human , FAM , Hs01075807_m1 , Applied Biosystems.

Techniques:

Journal: Biofactors (Oxford, England)

Article Title: Diverse Inhibitors of De Novo Purine Synthesis Promote AICAR ‐Induced AMPK Activation and Glucose Uptake in L6 Myotubes

doi: 10.1002/biof.70037

Figure Lengend Snippet: Assessment of sensitivity of L6 cells to inhibitors of purine metabolism. A: Purine metabolism and AMPK. The purine precursor ZMP is an AMPK activator. Methotrexate was shown to promote fatty acid oxidation (FAO) and glucose uptake via activation of AMPK in skeletal muscle tissue or cells [ , ]. Intermediates : 5,10‐CH 2 ‐THF, N 5 ,N 10 ‐methylene THF; 10‐CHO‐THF, N 10 ‐Formyl‐THF; AMP, adenosine monophosphate; DHF, dihydrofolate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; FGAR, formylglycinamide ribonucleotide; GAR, glycinamide ribonucleotide; GMP, guanosine monophosphate; Hx, hypoxanthine; IMP, inosine monophosphate; PRA, phosphoribosylamine; PRPP, 5‐phosphoribosyl‐1‐pyrophosphate; SAICAR, N‐succinyl‐5‐aminoimidazole‐4‐carboxamide ribonucleotide; THF, tetrahydrofolate; Xan, xanthine; ZMP, 5‐aminoimidazole‐4‐carboxamide ribonucleotide. Enzymes : ACC, acetyl‐coenzyme A carboxylase; ADSL, adenylosuccinate lyase; ADSS, adenylosuccinate synthetase; AMPK, AMP‐activated protein kinase; ATIC, 5‐aminoimidazole‐4‐carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase; DHFR, dihydrofolate reductase; GART, glycinamide ribonucleotide formyltransferase; GMPS, GMP synthetase; GPAT, glutamine phosphoribosylpyrophosphate amidotransferase; IMPDH, IMP dehydrogenase; TS, thymidylate synthetase; XDH, xanthine oxidase. Inhibitors : ALA, alanosine; ALO, allopurinol; FAO, fatty acid oxidation; MMF, mycophenolate mofetil; MP, mercaptopurine; MTX, methotrexate; TMP, trimethoprim; TMX, trimetrexate. “P” on AMPK and ACC indicates phosphorylation. (B, C) L6 Myotubes express enzymes of nucleotide and folate metabolism targeted by MTX, ALA, MMF, MP, TMX, and ALO. L6 cells were grown for 2 days in MEMα with nucleosides and 10% serum and then differentiated for 7 days in MEMα with nucleosides and 2% serum and for an additional day in MEMα without nucleosides and serum. Cells were then analyzed for expression of Gart , Atic , Adss1 , Adss2 , Adsl , Impdh1 , Impdh2 , Tyms , Dhfr , and Xdh genes (B). Expression of target genes was normalized to expression of Actin beta gene ( Actb ). Graphs show means with SD ( n = 2). Tyms : Thymidylate synthetase. In addition, cells were analyzed for protein expression of GART, ATIC, ADSS, IMPDH2, DHFR, and XDH on day 2 (myoblasts; MB), day 9 (myotubes after 7 days in MEMα with nucleosides and 2% serum; MT+) and day 10 (myotubes after 7 days in MEMα with nucleosides and 2% serum and 1 day in MEMα without nucleosides and serum; MT‐) of culture (C). Numbers next to blots indicate position and molecular weight (in kDa) of molecular weight markers. (D–J) Effect of MMF, ALA, MP, TMP, sulfamethoxazole (SMX), TMX and MTX on proliferation of L6 myoblasts in absence or presence of nucleosides. L6 myoblasts were grown in absence of nucleosides for 24 h and then treated with MMF (0.1–10 μM) (D), ALA (0.1–10 μM) (E), MP (1–100 μM) (F), TMP (1–100 μM) (G), SMX (10–1000 μM) (H), TMX (0.1–10 μM) (I), MTX (0.1–10 μM) (J) or vehicle (control, C) in absence or in presence of nucleosides for 48 h. Cell cultures before and after the treatment were analyzed for DNA content with Hoechst assay. Hoechst fluorescence (Hoechst FL) after the treatment was expressed relative to Hoechst fluorescence before the treatment (0 h). Graphs show means with SD ( n = 4–8). * p < 0.05 versus respective (without or with nucleosides) control, two‐way ANOVA with Dunnett's test.

Article Snippet: Quantitative real‐time polymerase chain reaction (qPCR) was performed with QuantStudio 3 Real‐Time PCR System (Thermo Fisher Scientific) using TaqMan Universal PCR Master Mix II and TaqMan gene expression assays for Gart (Rn01477298_m1), Atic (Rn00578818_m1), Adss1 (Rn01430183_m1), Adss2 (Rn02103847_s1), Adsl (Rn01768239_m1), Impdh1 (Rn01455843_g1), Impdh2 (Rn01640111_g1), Tyms (Rn01418709_m1), Dhfr (Rn04342282_g1), Xdh (Rn00567654_m1) and Actb (4352931).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Molecular Weight, Control, Fluorescence

Journal: Frontiers in Physiology

Article Title: Transcriptional Signature of an Altered Purine Metabolism in the Skeletal Muscle of a Huntington's Disease Mouse Model

doi: 10.3389/fphys.2017.00127

Figure Lengend Snippet: Transcriptional remodeling of genes involved in de novo purine synthesis and salvage pathway . Transcripts of genes involved in de novo purine biosynthesis. (A) Adsl , Adenylosuccinate lyase; (B) Adssl1 , Adenylosuccinate lyase 1; (C) Gart , Phosphoribosylglycinamide formyltransferase; (D) Ppat , Amidophosphoribosyltransferase; and the purine nucleotide salvage pathway (E) Aprt , Adenine phosphoribosyltransferase; were assessed in different types of skeletal muscle: EDL, Extensor Digitorum Longus; SOL, Soleus; TA, Tibialis Anterior; and G/P, Gastrocnemius and Plantaris complex. All Taqman qPCR-values were normalized to the geometric mean of three housekeeping genes as indicated in the Materials and Methods Section. Error bars are ± SEM ( n = 6). Student's t -test: * p < 0.05, ** p < 0.01; *** p < 0.001.

Article Snippet: Following Taq-man assays from Thermo Fisher Scientific were used in this study Adsl (Mm_00507759_m1); Adssl1 (Mm_00475814_m1); Gart (Mm_00599836_m1); Ppat (Mm_00549096_m1); Aprt (Mm_04207857_g1); Ak1 (Mm_00445475_m1); Gmpr (Mm_00499393_m1); Impdh2 (Mm_00496156_m1); Ada (Mm_00545720_m1); Adk (Mm_00612772_m1); Nt5e (Mm_00501910_m1); Ampd3 (Mm_00477495_m1); Entpd2 (Mm_00515450_m1); Pnp (Mm_00840006_m1); Xdh (Mm_00442110_m1); Prkaa1 (Mm_01296700_m1); Pdk4 (Mm_01166879_m1); Hk2 (Mm_00443385_m1). mRNA copy number was determined in triplicate for each RNA sample by comparison with the geometric mean of three endogenous housekeeping genes (Primer Design, UK), as described (Mielcarek et al., ).

Techniques:

Journal: Frontiers in Physiology

Article Title: Transcriptional Signature of an Altered Purine Metabolism in the Skeletal Muscle of a Huntington's Disease Mouse Model

doi: 10.3389/fphys.2017.00127

Figure Lengend Snippet: Summary of significantly deregulated transcripts in HD skeletal muscles that might alter purine homeostasis, leading to an energy imbalance . Adsl, Adenylosuccinate lyase ; Adssl1, Adenylosuccinate lyase 1 ; Gart, Phosphoribosylglycinamide formyltransferase ; Ak1, Adenylate kinase 1 ; Impdh2, inosine monophosphate dehydrogenase 2 ; Entpd2, Ectonucleoside triphosphate diphosphohydrolase 2 ; Ampd3, Adenosine deaminase 3 ; Pnp, Purine nucleoside phosphorylase ; Xdh, Xanthine dehydrogenase ; Hk2, Hexokinase 2 ; Prkaa1 ; 5 ′ -AMP-activated protein kinase catalytic subunit alpha-1 ; Pdk4, Pyruvate dehydrogenase, kinase isozyme 4 .

Article Snippet: Following Taq-man assays from Thermo Fisher Scientific were used in this study Adsl (Mm_00507759_m1); Adssl1 (Mm_00475814_m1); Gart (Mm_00599836_m1); Ppat (Mm_00549096_m1); Aprt (Mm_04207857_g1); Ak1 (Mm_00445475_m1); Gmpr (Mm_00499393_m1); Impdh2 (Mm_00496156_m1); Ada (Mm_00545720_m1); Adk (Mm_00612772_m1); Nt5e (Mm_00501910_m1); Ampd3 (Mm_00477495_m1); Entpd2 (Mm_00515450_m1); Pnp (Mm_00840006_m1); Xdh (Mm_00442110_m1); Prkaa1 (Mm_01296700_m1); Pdk4 (Mm_01166879_m1); Hk2 (Mm_00443385_m1). mRNA copy number was determined in triplicate for each RNA sample by comparison with the geometric mean of three endogenous housekeeping genes (Primer Design, UK), as described (Mielcarek et al., ).

Techniques: Muscles