atcc 15264 t  (ATCC)

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: 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 transcriptional regulatory axis in A498 cells. E–H) The specific enrichment of H3R17me2a at the TSS of PPAT , PAICS (E), GART (F), ADSL (G), and ADSS2 (H) was verified by ChIP‐qPCR assay. I–L) The specific enrichment of LEDGF at the TSS of PPAT , PAICS (I), GART (J), ADSL (K), and ADSS2 (L) was verified by ChIP‐qPCR assay. M,N) QRT‐PCR was used to demonstrate that decrease of LEDGF or H3R17me2a can reduce mRNA expression of key enzymes in the de novo synthesis pathway. (N) Western blot was performed to detect that decrease of LEDGF or H3R17me2a can significantly reduce the protein expression of key enzymes of de novo synthesis, except ADSS2. O–R) Metabolomics results showed that reduction of H3R17me2a level or LEDGF significantly decreases IMP (O) and GMP (P) levels in A498 cells. While the AMP in A498 cells remain relatively stable (Q), there is a significant reduction in dAMP (R) level. Data are shown as mean ± SD. ** p < 0.01, *** 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: ChIP-qPCR, Quantitative RT-PCR, Expressing, Western Blot

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