Journal: Nature Communications

Article Title: Chronic activation of endothelial MAPK disrupts hematopoiesis via NFKB dependent inflammatory stress reversible by SCGF

doi: 10.1038/s41467-020-14478-8

Figure Lengend Snippet: a , b Immunoblot analysis and quantification demonstrating that expression of IkB-SS in BMECs isolated from CDH5-MAPK mice does not affect ERK1/2 or p65 phosphorylation. Black arrowhead represents endogenous IκBα whereas red arrowhead represents IkB-SS transgene. c , d Representative immunofluorescence images and quantification demonstrating increased levels of nuclear p65 in BMECs derived from CDH5-MAPK mice as compared to controls. Note that expression of IkB-SS in BMECs isolated from CDH5-MAPK mice ( CDH5-MAPK::IkB ) decreases nuclear p65 levels. Each dot within the bar graph represents nuclear p65 staining intensity per individual cell. e , f Heatmap and bar graph demonstrating increased expression of NF-κB-dependent inflammatory genes within BMECs of CDH5-MAPK mice. Crossing CDH5-MAPK with Tie2.IkB-SS mice ( CDH5-MAPK::IkB ) resulted in decreased expression of NF-κB-dependent target genes. Expression of Actb was used for normalization. Dendrograms represent unsupervised hierarchical clustering of the entire dataset ( n = 3 mice/cohort). Color scales represent relative mRNA abundance based on normalized gene expression. Raw data for Heatmaps included in Supplementary Table and Source Data. g Representative immunofluorescence images of femurs intravitally labeled with a vascular-specific CD144/VE-cadherin antibody (red) demonstrating that suppression of NF-κB signaling within endothelial cells of CDH5-MAPK mice resolves their vascular dilatation. Error bars represent sample mean ± SEM. One-way ANOVA for multiple comparisons and Tukey’s correction was performed to determine significance. ** P < 0.01; *** P < 0.001; n.s. P > 0.05.

Article Snippet: IkB-SS expressing lentiviral vector was generated by sub-cloning the sequence of human IκBα super-suppressor (Addgene #15264) into pLVX Puro Vector (Clontech #632164).

Techniques: Western Blot, Expressing, Isolation, Phospho-proteomics, Immunofluorescence, Derivative Assay, Staining, Gene Expression, Labeling

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