Fig 1: L3MBTL3 regulates HIF-1α stability under hypoxia in vitro A. L3MBTL3 knockdown did not influence the transcription of HIF-1α no matter in normoxia or hypoxia conditions. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the two-way ANOVA test. **p < 0.01; ***p < 0.001; ns for non-significant. B. L3MBTL3 overexpression mediated instability of HIF-1α was abolished by MG132 inhibition. C. L3MBTL3 reduced the HIF-1α stability. 293T cells were transfected with control or Flag-L3MBTL3 plasmids, and then cultured under hypoxia for 6 h followed by treatment with cycloheximide inhibitor for 0 h, 0.5 h, 1 h or 2 h. The cell lysates were analyzed by Western blot. D. L3MBTL3 enhances HIF-1α ubiquitination. Hypoxic conditions cultured 293T cells transfected with control vector or HA-L3MBTL3 constructs together with Flag–HIF–1α and His-Ub plasmids, 20 h after transfection, the cells were treated with MG132 inhibitor for additional 4 h. Then cell lysates were immunoprecipitated with the anti-Flag (M2) beads. An anti-His antibody was used to explore the HIF-1α ubiquitination. E. L3MBTL3 enhances K48-linked polyubiquitination of HIF-1α. Hypoxia cultured 293T cells co-transfected with Flag–HIF–1α, HA-Ub (R48K) or HA-Ub (R63K) and Prk5-L3MBTL3 plasmids were also treated with MG132 as above. Denatured cell lysates were subjected to Western blot analysis. An anti-HA antibody was employed to do the ubiquitination analysis. The raw figures for Western blot were shown in the Supplementary Figure S4.
Fig 2: Hypoxic conditions induces L3MBTL3 upregulation A. The protein level of L3MBTL3 was upregulated under hypoxia. H1299, HepG2 and HeLa cells were treated with low oxygen conditions (1% O2) for the displayed time points. The cells lysates were analyzed by Western blot using the indicated antibody. The experiment was repeated at least once. B-G. L3MBTL3 upregulation caused by low oxygen conditions was suppressed by reduction of HIF-1α. In respect of normoxia or hypoxia, Western blot or real-time PCR analysis of HepG2 (B, C and D) and HeLa (E, F and G) cells transfected with siRNA of control or HIF-1α. H. HIF-1α enhanced L3MBTL3 promoter activity. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed by the one-way ANOVA test followed by Dunnett's multiple comparisons test (C, D, F and G) or the Student's t-test (H). **p < 0.01; ***p < 0.001; ****P < 0.0001. N = 2, the N numbers indicated biological replicates. The raw figures for Western blot were shown in the Supplementary Figure S4.
Fig 3: L3MBTL3 downregulates HIF-1α protein level in hypoxic condition A, B. L3MBTL3 reduced HIF-1α expression induced by low oxygen conditions. H1299 (A) and HepG2 (B) cells were transfected with control or Flag-L3MBTL3 plasmids, and then treated with hypoxia for the indicated time points. Western blot analysis was used to analyze the protein level in cell lysate. The experiment was repeated at least once in HepG2 cells. C. L3MBTL3 inhibited HIF-1α protein levels in a dose dependent manner. D-F. L3MBTL3 knockdown enhanced protein levels of HIF-1α upregulated by hypoxia. H1299 (D), HepG2 (E) and HeLa (F) cells transfected with siRNA of control or L3MBTL3 were culture in hypoxic conditions for 6 h. And then the cell lysates were gone through Western blot analysis. G. L3MBTL3 knock-out upregulated HIF-1α protein levels induced by hypoxia. The raw figures for Western blot were shown in the Supplementary Figure S4.
Fig 4: L3MBTL3 interacts directly with HIF-1α inside the cell nucleus A. Mutual interaction between exogenous HIF-1α and L3MBTL3 was explored. HepG2 cells transfected with Flag-L3MBTL3, HA–HIF–1α or control plasmids were cultured in normoxic or hypoxic states for 12 h. The cell lysates were then undergone immunoprecipitation analysis with anti-Flag beads. B. The interaction of endogenous HIF-1α and L3MBTL3 appeared inside the nucleus. Hypoxia cultured HepG2 cells were harvested followed by nucleus and cytoplasm separation. The different part lysates were then gone through immunoprecipitation assay with IgG, anti-L3MBTL3, anti–HIF–1α antibodies. C. Immunostaining showing that the colocalization of HIF-1α (green) and L3MBTL3 (red) in the nucleus (blue), scale bar: 10 μm. D. SAM domain of L3MBTL3 interacted with HIF-1α. 293T cells were transfected with HA–HIF–1α plasmids together with the empty vector control, plasmids encoding wild-type L3MBTL3 or mutants with deletion of different functional domains (right). The anti-Flag (M2) beads were then utilized to do the cells lysate immunoprecipitation assay. E. The interaction between L3MBTL3 and HIF-1α mainly relied on HLH domain. The purified bacteria expressed GST protein or HIF-1α GST-tagged truncations were made mixture with 293T cell lysates. The exogenous L3MBTL3 were immunoprecipitation with anti-GST beads then subjected to GST-pull down. F. The 3MBT domain of L3MBTL3 reduced the stability of HIF-1α. The raw figures for Western blot were shown in the Supplementary Figure S4.
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