Journal: Advanced Science

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: CARM1 is highly expressed in ccRCC and promotes proliferation. A,B) CARM1 is highly expressed in ccRCC patients and is correlated with poor prognosis on the UALCAN database. C) CARM1 and H3R17me2a are highly expressed in ccRCC cells such as 786‐O, A498, and Caki‐1. D,E) Immunofluorescence staining using the tissue microarray revealed that CARM1 is highly expressed in ccRCC tissue samples. Scale bar = 100 µm. (Tumor n = 79, Normal n = 79) F) CcRCC patients with advanced T stage have higher CARM1 expression, which may be associated with poor prognosis. (T1 n = 53, T2 n = 23, T3 n = 3) G,H) Immunofluorescence staining using the tissue microarray revealed that H3R17me2a level is higher in ccRCC tissue samples. Scale bar = 100 µm. (Tumor n = 79, Normal n = 79) I) CcRCC patients with advanced T stage have higher H3R17me2a level, which may be associated with poor prognosis. (T1 n = 53, T2 n = 23, T3 n = 3) J) The siRNA targeting CARM1 mRNA can effectively interfere with CARM1 and decreases H3R17me2a in ccRCC cells. K–N) Reduction of CARM1 and H3R17me2a significantly inhibited the proliferation of ccRCC cells, as demonstrated by CCK‐8 (K), colony formation (L) and EdU proliferation assays (M‐N). Scale bar = 100 µm. Data are shown as mean ± SD. ** p < 0.01, *** p < 0.001.

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: Immunofluorescence, Staining, Microarray, Expressing, CCK-8 Assay

Journal: Advanced Science

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: Construction of the H3R17me2a‐deficient cell lines. A,B) Validation of knockout efficiency of CARM1 and the reduction of H3R17me2a level in transduced pooled A498 cells (A) and 786‐O cells (B). C–E) Validation of knockout efficiency of CARM1 and the reduction of H3R17me2a level in monoclonal A498 cells (C) and 786‐O cells (D‐E). F,G) Knockdown of PRMT6 using siRNA has little effect on H3R17me2a level in A498 cells (F) and 786‐O cells (G). H) The combined knockdown of CARM1 and PRMT6 using siRNAs reduces H3R17me2a level in A498 cells; knockdown of PRMT6 in CARM1‐KO cells significantly decreased H3R17me2a in A498 cells. I) Knockdown of PRMT6 in CARM1‐KO cells significantly decreased H3R17me2a in A498 cells. J) The combination of CARM1 (MCE, EZM2302 and TP‐064) and PRMT6 (MCE, EPZ020411) inhibitors can significantly reduce H3R17me2a level in A498 cells.

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: Biomarker Discovery, Knock-Out, Knockdown

Journal: Advanced Science

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: 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 expression 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: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: PRMT4/CARM1 Is a Novel Factor Promoting DNA Double-Strand Break Repair.

doi: 10.1111/gtc.70031

Figure Lengend Snippet: FIGURE 3 | Catalytic activity of PRMT4 was indispensable for releasing PRMT4 from the DSB sites. (A) U2OS cells expressing GFP-PRMT4 (wild-type or R168A mutant) were subjected to laser micro-irradiation and live-cell imaging. The track of laser micro-irradiation was indicated by white triangles. Scale bar: 10 μm. (B) Relative GFP signal intensity at DSB sites in (A) was quantified. The signal intensity 1 min after laser micro- irradiation when the accumulation of PRMT4 reached maximum was set to 1 and plotted (mean ± SEM, n = 10). *p < 0.05.

Article Snippet: Soluble fraction was subjected to immunoprecipitation with an anti- GFP antibody coupled with magnetic beads (GFP- Trap_MA, ChromoTek, PlaneggMartinsried, Germany) or anti- PRMT4 antibody (Cell Signaling Technology, 12495T, 1 μg/100 μg cell extract) by rotating overnight at 4°C.

Techniques: Activity Assay, Expressing, Mutagenesis, Irradiation, Live Cell Imaging

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: PRMT4/CARM1 Is a Novel Factor Promoting DNA Double-Strand Break Repair.

doi: 10.1111/gtc.70031

Figure Lengend Snippet: FIGURE 4 | PRMT4 was a novel factor promoting DSB repair. (A) Genotyping of a PRMT4 KO cell line. The target site of gRNA and primers used for genotyping are indicated with the dashed red line and black arrows, respectively (upper). The result of the agarose gel electrophoresis was shown (lower). Amplification of β-actin gene was the loading control. (B) Cell extracts prepared from U2OS (wild type) and PRMT4 KO cell lines were ex- amined by immunoblotting with the indicated antibodies. (C) Neutral comet assay was carried out with U2OS (wild type) and PRMT4 KO cell line. Relative tail moments that were obtained by normalizing with the tail moment of the undamaged sample were plotted for damaged and repaired samples. Mean and standard error are indicated with the horizontal bars. ***p < 0.0001. (D) U2OS (wild type) and PRMT4 KO cell line were treated with 150 μg/mL phleomycin for 2 h. After washing out phleomycin, cells were further cultured for the indicated periods. Immunoblotting analysis with the indicated antibodies was carried out. H2AX and α-tubulin were loading controls for γH2AX and PRMT4, respectively.

Article Snippet: Soluble fraction was subjected to immunoprecipitation with an anti- GFP antibody coupled with magnetic beads (GFP- Trap_MA, ChromoTek, PlaneggMartinsried, Germany) or anti- PRMT4 antibody (Cell Signaling Technology, 12495T, 1 μg/100 μg cell extract) by rotating overnight at 4°C.

Techniques: Agarose Gel Electrophoresis, Amplification, Control, Western Blot, Neutral Comet Assay, Cell Culture

Journal: Scientific Reports

Article Title: Discovery of an ApoE4-targeted small-molecule SirT1 enhancer for the treatment of Alzheimer’s disease

doi: 10.1038/s41598-025-96131-2

Figure Lengend Snippet: Antibodies and dilutions used for immunoblots.

Article Snippet: PRMT4 , Cell Signaling , 4438 S , 0.7361.

Techniques: Western Blot