Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A to C ) Interactions of CDK4 (A) or CDK6 (B) with SMYD2, and SMYD2 with CDK4 or CDK6 (C) in RCTE cells were detected by immunoprecipitation (IP) and immunoblotting (IB). Immunoglobulin G (IgG) was used as a negative control. ( D ) Schematic of GST-SMYD2 fusion protein constructs (top), which was detected using Coomassie blue staining (middle). GST pull-down assays indicated the interaction of GST-SMYD2 fusion proteins with CDK4 and CDK6 (bottom). ( E ) GFP-tagged SET domain–deleted SMYD2 cannot pull down CDK4 and CDK6 in HEK293T cells. ( F to I ) The phosphorylation of SMYD2 was decreased in RCTE cells transfected with siRNAs to CDK4 (F), CDK6 (G), or both (H), and in RCTE cells treated with Abe (10 μM) (I). Knockdown of CDK4 decreased the expression of CDK6, and vice versa for CDK6 in these cells. The quantification analysis ( n = 3) of band intensities was shown in the graphs (bottom), in that the density of each protein band was normalized to actin, and then the value was divided by the value of corresponding siRNA and vehicle band density to actin. The value of the control band to actin was set to 1.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Construct, Staining, Transfection, Expressing

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A to D ) Western blot analysis indicated that knockdown of CDK4 and CDK6 with siRNAs (A) or knockdown of SMYD2 with siRNA (B) as well as inhibition of CDK4/6 with Abe (C) or inhibition of SMYD2 with AZ505 (D) decreased the mono-, di-, and trimethylation of histone H3 at lysine 4 (H3K4) and lysine 36 (H3K36) in RCTE cells. The quantification and statistical analysis ( n = 3) were shown in the graph (bottom). * P < 0.01 as compared to each control. ( E and F ) Knockdown (E) or inhibition (F) of SMYD2 decreased the mRNA and protein levels of CDK4 and CDK6 in RCTE cells, examined with qRT-PCR and Western blotting. * P < 0.01 as compared to each control ( n = 3). ns, not significant. ( G ) SMYD2 bound to the promoter of CDK4 and CDK6. ChIP-qPCR analysis was performed with an SMYD2 antibody, or normal rabbit IgG in RCTE cells. ( H ) ChIP assay was performed with mono-, di-, and trimethylated H3K4 and H3K36 antibodies and normal rabbit IgG in RCTE cells.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Western Blot, Inhibition, Quantitative RT-PCR

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A ) Immunofluorescence staining of primary cilia with α-acetyl-tubulin antibody (α-ac-tubulin) in primary renal epithelial cells isolated from kidneys of wild-type (WT) and Smyd2 flox/flox : Ksp-Cre mice, in which Smyd2 was specifically knocked out (KO) by the kidney-specific promoter (Ksp)–driven Cre recombinase in renal epithelial cells and cultured in serum-free medium for 72 hours before subjected to staining. The percentage of ciliated cells and cilia length was measured and statistically analyzed. Scale bar, 5 μm. Error bars represent the SD. N values represent the numbers of cilia (left graph) and cells (right graph), respectively, for each group. ( B to E ) Knockdown of Smyd2 with siRNA (B) or inhibition of Smyd2 with AZ505 (C) as well as knockdown of CDK4/CDK6 with siRNAs (D) or inhibition of CDK4/CDK6 with Abe (E) in mIMCD3 cells resulted in longer cilia compared to control cells as examined by immunofluorescence with α-acetyl-tubulin and SMYD2 antibodies. Scale bar, 5 μm. Error bars represent the SD. N values represent cilia numbers for each group.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Immunofluorescence, Staining, Isolation, Cell Culture, Inhibition

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A ) Coimmunoprecipitation of endogenous α- and γ-tubulin with SMYD2 in RCTE cells. IgG was used as a negative control. ( B to D ) Coimmunoprecipitation of endogenous α-tubulin (B), γ-tubulin (C), and β-tubulin (D) with overexpressed SMYD2 in HEK293T cells. GFP vector–transfected cells were used as a negative control. ( E ) GST pull-down assays were performed by incubation of GST-SMYD2 fusion protein with 1 μg of recombinant α- or γ-tubulin protein and immunoblotting with an α-tubulin (top) and γ-tubulin antibody (middle). The expression of GST-SMYD2 was detected with Coomassie blue staining (bottom). ( F ) The expression of GST-SMYD2 constructs was detected using Coomassie blue staining (top). GST pull-down assays of GST-SMYD2 fusion proteins incubated with 1 mg of cell lysate from RCTE cells and immunoblotting using α- and γ-tubulin antibodies (bottom). ( G and H ) In vitro methylation assay of α-tubulin (G), γ-tubulin (H), and recombinant SMYD2. Histone H3 was used as a positive control in the in vitro methylation assays. ( I ) Relative intensity of methylated α- and γ-tubulin bands (black box) compared to the intensity of methylated histone H3, which was set to 1 (open box), in the in vitro methylation assays.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Negative Control, Plasmid Preparation, Transfection, Incubation, Recombinant, Western Blot, Expressing, Staining, Construct, In Vitro, Methylation, Positive Control

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A and B ) Knockdown of SMYD2 with siRNA (A) or inhibition of SMYD2 with AZ505 (B) decreased methylation of α-tubulin at K394 but not at K40 as examined by Western blotting with our generated TubK40me3 and TubK394me3 antibodies in RCTE cells. The quantification and statistical analysis ( n = 3) were shown in the graphs (bottom), in which the band density of control siRNA (A) and DMSO (B) was set to 1. ( C ) Western blot of the methylation of α-tubulin at K40 and at K394 with the TubK40me3 and TubK394me3 antibodies in RCTE cells transfected with Flag-tagged SMYD2 and GFP-tagged α-tubulin. The quantification and statistical analysis ( n = 3) were shown in the graph (bottom). ( D ) Representative images of RCTE cells stained with TubK394me3 (left) or SMYD2 (right) and costained with α-acetyl-tubulin/γ-tubulin (red) antibodies and DAPI (blue). Scale bar, 5 μm. ( E ) Representative images of RCTE cells stained with TubK394me3 (left, green) or SMYD2 (right, green) and costained with α-acetyl-tubulin/γ-tubulin (red) and DAPI (blue). Scale bar, 5 μm. Error bars represent the SD. N values represent cilia numbers in (D) and (E).

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Inhibition, Methylation, Western Blot, Generated, Transfection, Staining

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A and B ) Knockdown (A) and inhibition of SMYD2 (B) increased the levels of IFT20 protein (top) and mRNA (bottom) as examined by Western blotting and qRT-PCR in RCTE cells. The quantification and statistical analysis ( n = 3) were shown in the graph (bottom), as are also shown in (B), (C), (E), and (F). * P < 0.01 as compared to controls. ( C ) Overexpression of SMYD2 decreased the levels of IFT20 protein (left) and mRNA (right) as examined by Western blotting and qRT-PCR in HEK293T cells. ( D ) SMYD2 and H3K36me3 antibodies bound to the promoter of IFT20 in RCTE cells as examined with ChIP assay. ( E and F ) Knockdown (E) and inhibition of CDK4/6 (F) increased the levels of IFT20 protein (top) and mRNA (bottom) as examined by Western blotting and qRT-PCR in RCTE cells. n = 3. ( G ) Representative images of RCTE cells stained with IFT20 and α-acetyl-tubulin/γ-tubulin antibodies and DAPI in the presence of AZ505 (middle), Abe (right), and vehicle (left). Scale bar, 5 μm. The statistical analysis was shown in the graph (right). Error bars represent the SD. N values represent cilia numbers.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Inhibition, Western Blot, Quantitative RT-PCR, Over Expression, Staining

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A ) Western blot of SMYD2, CDK4, CDK6, and IFT20 in normal mammary cells MCF10A and breast cancer cells, including MCF7, T47D, MDA-MB468, and MDA-MB231 cells. ( B ) Western blot of SMYD2, IFT20, CDK4, and CDK6 in PH2 and PN24 cells. ( C ) The quantification and statistical analysis ( n = 3) were shown in the graphs corresponding to (top) and (bottom). ( D to F ) Representative images of T47D (top) and MDA-MB468 cells (bottom) (D) as well as PN24 cells (E and F) stained with SMYD2 (green) and α-acetyl-tubulin/γ-tubulin (red) and costained with DAPI (blue) in the presence of the SMYD2 inhibitor AZ505 (middle) and the CDK4/6 inhibitor Abe as well as vehicle. Scale bar, 5 μm. Statistical analysis of the percentage of ciliated breast cancer cells ( n = 200) ( P < 0.01) as well as the percentage of ciliated PN24 cells ( n = 900) and their cilia lengths are shown in the graph (right). Error bars represent the SD.

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Western Blot, Staining

Journal: Science Advances

Article Title: Cross-talk between CDK4/6 and SMYD2 regulates gene transcription, tubulin methylation, and ciliogenesis

doi: 10.1126/sciadv.abb3154

Figure Lengend Snippet: ( A and B ) Representative images of RCTE (A) and PN24 (B) cells stained with GLI2 (green) and GLI3 (red), and costained with α-acetyl-tubulin/γ-tubulin (purple) antibodies and DAPI (blue). Scale bar, 5 μm. The statistical analysis of the cilia tip localization of GLI2 (left) and GLI3 (right) is shown in the graphs. * P < 0.01. Error bars represent the SD. n = 100 cilia for each group. ( C and D ) Western blot analysis of Ptch1 and GLI1 in RCTE cells, which were treated with or without SAG and cotreated with or without the SMYD2 inhibitor AZ505 (C) and the CDK4/CDK6 inhibitor Abe (D) in serum-free medium for 48 hours. Statistical analysis of the expression ( n = 3) of Ptch1 and GLI1 proteins is shown in the graphs (right). ( E and F ) Western blot analysis of Ptch1 and Gli1 in PN24 cells, which were treated with or without SAG and cotreated with or without the SMYD2 inhibitor AZ505 (E) and the CDK4/CDK6 inhibitor Abe (F) in serum-free medium for 48 hours. Statistical analysis of the expression ( n = 3) of Ptch1 and Gli1 proteins is shown in the graphs (bottom).

Article Snippet: GST-Smyd2 fusion protein–expressing constructs were generated in a pGEX-6p-1 (GE Healthcare) vector by cloning fragments of human Smyd2 complementary DNA (cDNA) (GenScript) into the Bam HI and Xho I sites via polymerase chain reaction (PCR) using the In-Fusion HD Cloning Kit (Clontech).

Techniques: Staining, Western Blot, Expressing

Journal: Science Advances

Article Title: SMYD2 inhibition–mediated hypomethylation of Ku70 contributes to impaired nonhomologous end joining repair and antitumor immunity

doi: 10.1126/sciadv.ade6624

Figure Lengend Snippet: ( A ) Immunoblot analysis of pan-methylation (mono/dimethylation and trimethylation) of Ku70 in the anti-GFP immunoprecipitates HEK293FT cells cotransfected with GFP-Ku70 and FLAG-SMYD2-WT/Y240F. ( B ) Immunoblot analysis of pan-methylation of Ku70 in the anti-GFP immunoprecipitates SMYD2 knockdown HEK293FT cells, which transfected with GFP-Ku70. ( C ) Mass spectrometry analysis of methylation sites of Ku70 following transfection with SMYD2 and GFP-Ku70. The table in the lower graph shows the times of unique peptides and the detailed lysine methylation sites. ( D ) Immunoblot analysis of mono/dimethylation of Ku70-WT/mutants in anti-GFP immunoprecipitates. HEK293FT cells cotransfected with GFP-Ku70-WT or K74R/K516R/K539R and FLAG-SMYD2-WT. ( E ) Immunoblot analysis of mono/dimethylation of Ku70-WT/3KR mutants in anti-GFP immunoprecipitates HEK293FT cells cotransfected with GFP-Ku70-WT or 3KR and FLAG-SMYD2-WT. ( F ) Left graph: Accumulation of exogenous GFP-Ku70-WT/3KR mutant in BrdU-sensitized U2OS cells. Scale bars, 10 μm. Right graph: Relative intensity of GFP-Ku70-WT/3KR at micro-irradiated sites in the experiments described in the left graph. ( G ) Effects of overexpression of Ku70-WT or 3KR mutant on the efficiency of NHEJ in I9a cells. The I9a cells were transfected with FLAG-Ku70 WT/3KR lentivirus with puromycin selection. ( H ) Immunoblot analysis of mono/dimethylation and trimethylation of Ku70 and the interaction between Ku70 and Ku80 or DNA-PKcs in anti-GFP immunoprecipitates. Plasmids encoding GFP-Ku70 and FLAG-SMYD2 were cotransfected in HEK293FT for 72 hours, treated with irradiation at 10 Gy, and released for the indicated time. ( I ) Immunoblot analysis of the interaction between Ku70 and Ku80 or DNA-PKcs in anti-GFP immunoprecipitates. Plasmids encoding GFP-Ku70 and FLAG-SMYD2-WT or Y240F were cotransfected in HEK293FT for 72 hours. ( J ) Immunoblot analysis of the interaction between WT/3KR-Ku70 and Ku80 or DNA-PKcs in anti-GFP immunoprecipitates. Plasmids encoding GFP-Ku70-WT/3KR were transfected in HEK293FT for 72 hours.

Article Snippet: The antibodies used were SMYD2 (A6474, ABclonal), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 10494-1-AP, Proteintech), tubulin (11224-1-AP, Proteintech), H3 (17168-1-AP, Proteintech), γ-H2AX (#2577S, Cell Signaling Technology), H2AX (#7631, Cell Signaling Technology), DNA-PKcs (28534-1-AP, Proteintech), Ku70 (A0883, ABclonal), Ku80 (A5862, ABclonal), LIG4 (A1743, ABclonal), XLF (A4985, ABclonal), XRCC4 (A1677, ABclonal), mono/dimethylation (ab23366, Abcam), trimethylation (PTM-601, PTM BIO), FLAG (AE063, AE005, ABclonal), GFP (66002-1-Ig, Proteintech), p-TBK1-Ser 172 (#5483S, Cell Signaling Technology), TBK1 (28397-1-AP, Proteintech), p-IRF3-Ser 396 (#29047, Cell Signaling Technology), IRF3 (11312-1-AP, Proteintech), p-STING-Ser 365 (#72971S, Cell Signaling Technology), p-STING-Ser 366 (50907, Cell Signaling Technology), and STING (19851-1-AP, Proteintech).

Techniques: Western Blot, Methylation, Knockdown, Transfection, Mass Spectrometry, Mutagenesis, Irradiation, Over Expression, Selection

Journal: Science Advances

Article Title: SMYD2 inhibition–mediated hypomethylation of Ku70 contributes to impaired nonhomologous end joining repair and antitumor immunity

doi: 10.1126/sciadv.ade6624

Figure Lengend Snippet: In response to DNA damage, SMYD2 is recruited to DNA damage sites and sequentially mediates Ku70 methylation at lysine-74, lysine-516, and lysine-539, which promotes the efficient recruitment of Ku70/Ku80/DNA-PKcs complex and efficient NHEJ repair. Genetic or pharmacological inhibition of SMYD2 lead to the lack of NHEJ repair and accumulation of DNA damage, which leads to the increased cytoplasmic dsDNA, and activates the cGAS-STING pathway, boosting the production of chemokines and then triggers the antitumor immunity via infiltration and activation of cytotoxic CD8 + T cells and NK cells. SMYD2 inhibitor AZ505-enhanced radiation therapy efficiency indicates its translational significance.

Article Snippet: The antibodies used were SMYD2 (A6474, ABclonal), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 10494-1-AP, Proteintech), tubulin (11224-1-AP, Proteintech), H3 (17168-1-AP, Proteintech), γ-H2AX (#2577S, Cell Signaling Technology), H2AX (#7631, Cell Signaling Technology), DNA-PKcs (28534-1-AP, Proteintech), Ku70 (A0883, ABclonal), Ku80 (A5862, ABclonal), LIG4 (A1743, ABclonal), XLF (A4985, ABclonal), XRCC4 (A1677, ABclonal), mono/dimethylation (ab23366, Abcam), trimethylation (PTM-601, PTM BIO), FLAG (AE063, AE005, ABclonal), GFP (66002-1-Ig, Proteintech), p-TBK1-Ser 172 (#5483S, Cell Signaling Technology), TBK1 (28397-1-AP, Proteintech), p-IRF3-Ser 396 (#29047, Cell Signaling Technology), IRF3 (11312-1-AP, Proteintech), p-STING-Ser 365 (#72971S, Cell Signaling Technology), p-STING-Ser 366 (50907, Cell Signaling Technology), and STING (19851-1-AP, Proteintech).

Techniques: Methylation, Inhibition, Activation Assay