netn buffer containing protease inhibitor cocktail  (Bimake Inc)

Journal: The Journal of Biological Chemistry

Article Title: The dePARylase NUDT16 promotes radiation resistance of cancer cells by blocking SETD3 for degradation via reversing its ADP-ribosylation

doi: 10.1016/j.jbc.2024.105671

Figure Lengend Snippet: . The E3 ubiquitin ligase CHFR binds to PARylated SETD3 and promotes its ubiquitination and degradation. A and B , the E3 ubiquitin ligase CHFR interacts with PARylated SETD3. HeLa cells were transiently transfected with the indicated plasmids for 24 h, and then, the cell lysates were subjected to Co-IP with anti-S beads followed by Western blotting analysis with the indicated antibodies ( A ). HeLa cells were lysed with NETN buffer and subjected to Co-IP using anti-IgG or anti-CHFR antibodies followed by western blotting analysis with the indicated antibodies ( B ). C and D , colocalization of GFP-SETD3 with mCherry-LacI-CHFR. U2OS cells with stable integration of LacO arrays (U2OS-265) were cotransfected with GFP-SETD3 and mCherry-LacI-CHFR for 24 h followed by immunostaining with DAPI and visualization by confocal microscopy ( C ). The fluorescence intensity of GFP-SETD3 foci was quantified using ImageJ software and normalized to the nuclear background (n > 30). The data are means ± SD of biological triplicate experiments. ∗∗∗∗ p < 0.0001, Mann‒Whitney test. Scale bars, 5 μm ( D ). E and G , depletion of PARP1 impairs the interaction of SETD3 with CHFR. U2OS-265 cells were transfected with control siRNA or PARP1 siRNAs for 24 h followed by cotransfection of GFP-SETD3 and mCherry-LacI-CHFR for another 24 h, and the harvested cells were subjected to immunostaining and confocal microscopy imaging ( E ). The immunofluorescence intensity of GFP-SETD3 foci was quantified and normalized to the nuclear background (n > 30). The data are means ± SD of biological triplicate experiments. ∗∗∗∗ p < 0.0001, Mann‒Whitney test. Scale bars, 5 μm ( F ). PARP1-depleted HeLa cells were subjected to a Co-IP assay using anti-IgG or anti-CHFR antibodies followed by western blotting analysis with the indicated antibodies ( G ). H – L , CHFR depletion suppresses SETD3 ubiquitination and enhances its protein stability. CHFR-knockout HeLa cells were lysed with NETN buffer and then subjected to western blotting with the indicated antibodies ( H ). HeLa cells were transfected with SFB-CHFR for 24 h followed by incubation with 10 μg/ml cycloheximide (CHX) for the indicated periods of time. The cell lysates were harvested and analyzed by western blotting assay ( I ). Quantification of SETD3 protein levels in ( J ) using ImageJ software. K , HeLa cells were transfected with the indicated siRNAs for 24 h followed by cotransfection of SFB-SETD3 and HA-ubiquitin plasmids for another 24 h, and then, MG132 (10 μM) was added and incubated for another 4 h. The harvested cell lysates were subjected to a Co-IP assay using anti-S beads and analyzed by Western blotting with the indicated antibodies. L , the indicated plasmids were cotransfected into HeLa cells for 24 h followed by incubation with MG132 (10 μM) for an additional 4 h. The cell lysates were subjected to a Co-IP assay using anti-S beads and analyzed by Western blotting with the indicated antibodies.

Article Snippet: Briefly, cells were lysed with NETN buffer containing a protease inhibitor cocktail (Bimake) and benzonase nuclease (Merck Millipore) at 4 °C for 30 min. Then, the cells were lysed, and the lysates were separated by SDS‒PAGE and analyzed with the indicated antibodies.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Immunostaining, Confocal Microscopy, Fluorescence, Software, Cotransfection, Imaging, Immunofluorescence, Knock-Out, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The dePARylase NUDT16 promotes radiation resistance of cancer cells by blocking SETD3 for degradation via reversing its ADP-ribosylation

doi: 10.1016/j.jbc.2024.105671

Figure Lengend Snippet: NUDT16 exerts radioresistance effects via positive regulation of SETD3 expression. A and B , Control cells or indicated gene-deficient HeLa cells were either untreated or treated with IR (6 Gy) and allowed to recover for the indicated times followed by alkaline comet assay ( A ). Scale bar, 40 μm. The level of DNA breakage was determined by measuring the lengths of comet tail areas. The comet tail moment was quantified using CASP software. At least 60 comet tails were analyzed in each group ( B ). C and D , depletion of both NUDT16 and SETD3 did not further increase the accumulation of γH2AX foci. Representative micrographs showing the immunofluorescence assay results after the depletion of NUDT16, SETD3 or both in HeLa cells are shown in ( C ). Scale bar, 5 μm. Quantification of the γH2AX foci per cell using ImageJ software is presented in ( D ), and at least 100 cells were analyzed. E – H , depletion of both NUDT16 and SETD3 did not further enhance the sensitivity of either HeLa or MDA-MB-231 cells upon IR treatment. The indicated cells were first irradiated at different doses as indicated and subjected to a cell survival assay ( E and G ). The experiments were performed in triplicate, and the resulting colonies were quantified using ImageJ software. The results are shown as the averages of three independent experiments ( F and H ). I , parental or radiotherapy-resistant (RR) cells were either untreated or treated with IR (8 Gy) and then allowed to recover for 72 h, followed by Annexin V/PI staining and flow cytometry analysis. J , the results of apoptotic cell quantification in both groups are shown as the mean ± SD (n = 3). K and L , IR-resistant MDA-MB-231 cells exhibit stronger survival capability to IR treatment compared with the parental cells. The indicated cells were irradiated at different doses as indicated and then subjected to a cell survival assay ( K ). The resulting colonies were quantified using ImageJ software and the results are shown as the averages of three independent experiments ( L ). M , parental and RR cells were lysed with NETN buffer and analyzed by Western blotting with the indicated antibodies. N – P , the depletion of endogenous SETD3 in RR cells significantly increased the percentage of apoptotic cells after IR treatment. Control or SETD3-depleted RR cells were either untreated or treated with IR (8 Gy) and then allowed to recover for 72 h, followed by Annexin V/PI staining and flow cytometry analysis ( N and O ). P , results of apoptotic cell quantification in both groups are shown as the mean ± SD (n = 3).

Article Snippet: Briefly, cells were lysed with NETN buffer containing a protease inhibitor cocktail (Bimake) and benzonase nuclease (Merck Millipore) at 4 °C for 30 min. Then, the cells were lysed, and the lysates were separated by SDS‒PAGE and analyzed with the indicated antibodies.

Techniques: Expressing, Alkaline Single Cell Gel Electrophoresis, Software, Immunofluorescence, Irradiation, Clonogenic Cell Survival Assay, Staining, Flow Cytometry, Western Blot