Fig 1: DOT1L activated AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway by direct regulation of Farnesoid X receptor (FXR). (A and B) qPCR was performed to detect FXR expression levels in SGC0946 treatment 786-O cells or DOT1L knockdown 786-O cells. (C and D) The protein levels of FXR were detected by Western blot in SGC0946 treatment 786-O cells or DOT1L knockdown 786-O cells. (E) H3K79me2 ChIP-seq in ENCODE database showing FXR promoter in humans. (F) Schematic diagram showed the location of 5 pairs of primers in FXR promoter regions. (G) ChIP assays were performed using H3K79me2 antibody in 786-O cells to detect the binding sites in FXR promoter regions. (H) ChIP-qPCR assays were performed using H3K79me2 antibody in 786-O cells. The normalized expression in 786-O cells was input. (I) 786-O cells were treated with SGC0946, then treated with Guggulsterone (20 µM) or INT767 (10 µM) for 24 h and harvested for Western blot.
Fig 2: DOT1L inhibition leads to abnormal mitochondrial dynamics in cell lines of renal cancer. (A) Representative picture of 786-O and 769P cells stained with Mito-Tracker Red CMXRos, captured using oil-Fluorescence microscope, which shows the morphology of mitochondria in each group (up, scale bar = 10 μm). (B) Data showing the mean branch length of mitochondrial in each group measured by Image J analysis. **P < .01 compared with 0 µM group. (C) Representative immunoblots showing SGC0946 increased the protein expression of Mitofusin 1 (MFN1) and Mitofusin 2 (MFN2) in SGC0946-treated 786-O cells.
Fig 3: DOT1L inhibition or silencing induces autophagy by activating the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. (A) Gene sets enriched 15 KEGG signaling pathways in phenotype DOT1l_high. (B) GSEA indicated that DOT1L expression was closely related to mTOR signaling in renal cancer samples (TCGA-KIRC). (C and D) Western blot analysis showing the total and phosphorylated levels of AMPK, mTOR in SGC0946 treated renal cancer cells. (E and F) Western blot analysis showing the total and phosphorylated levels of AMPK, mTOR in DOT1L knockdown renal cancer cells. *P < .05, **P < .01, and ***P < .001, compared with ctrl group.
Fig 4: DOT1L knockdown leads to autophagy increased in renal cancer. (A) Relative messenger RNA (mRNA) expression of DOT1L detected by using qRT-PCR in 786-O renal cancer cell lines after DOT1L knockdown. (B) Protein expression of DOT1L, H3K79me2, and H3K79me3 detected by Western blot in 786-O cells after DOT1L knockdown. (C) Monodansylcadaverine (MDC) staining was used to detect the autophagy of 786-O cells after DOT1L knockdown. Statistics of relative puncta are shown in the right histogram. Scar bar = 20 μm. (D) The LC3α/β and p62 expression were determined by Western blot in 786-O cells and 769P cells after DOT1L knockdown. (E) Representative pictures of 786-O cells contained with MitoTracker Red CMXRos, captured using oil-fluorescence microscope, which shows the morphology of mitochondria in control, sh1 and sh2 group (up, scale bar = 10 μM). ***P < .001, compared with ctrl group.
Supplier Page from Abcam for Anti-KMT4 / Dot1L antibody [EPR22936-138]