anti carm1  (Cell Signaling Technology Inc)

Journal: Acta Pharmaceutica Sinica. B

Article Title: A novel small molecule inhibitor targeting KHSRP methylation suppresses colon cancer progression

doi: 10.1016/j.apsb.2026.06.031

Figure Lengend Snippet: DIQ01 inhibits PRMT5-mediated methylation of KHSRP. (A) Schematic representation of KHSRP domain architecture across multiple species, highlighting conserved arginine residues (3R: R411, R413, R415; and R442) and the four truncation mutants used for interaction mapping. (B) Representative mass spectra confirming arginine methylation sites on KHSRP. (C) Detection of monomethylarginine (MMA) levels in endogenous KHSRP (HCT116) and exogenously expressed FLAG-tagged KHSRP (HEK293T) following DIQ01 treatment. KHSRP was immunoprecipitated using anti-KHSRP or anti-FLAG antibodies prior to Western blot analysis of MMA. (D) Volcano plot of proteins identified as KHSRP interactors by IP–MS in HCT116 cells. (E) HCT116 and HEK293T cells were co-transfected with FLAG-tagged KHSRP and either HA-tagged PRMT4, PRMT5, or both, with empty vector (EV) as control. KHSRP was immunoprecipitated using anti-FLAG antibody, and monomethylarginine (MMA) levels were detected by Western blot to evaluate PRMT4/5-mediated methylation of KHSRP. (F) Detection of MMA levels in endogenous KHSRP (HCT116) and exogenously expressed FLAG-tagged KHSRP (HEK293T) following treatment with PRMT inhibitors, including a PRMT4-specific inhibitor (EZM2302), a PRMT5-specific inhibitor (GSK591), or their combination. DMSO served as a vehicle control. (G) DIQ01 directly blocked PRMT5-mediated methylation of KHSRP in vitro . Top: DIQ01 concentration series (0–10 μmol/L; “–” no PRMT5, “0” vehicle with PRMT5). Bottom: time course with 2 μmol/L DIQ01 for 2–8 h (“+” DIQ01, “–” vehicle). MMA levels were detected by anti-MMA blot; total KHSRP and PRMT5 served as controls. Data represent ≥3 independent repeats. (H) HCT116 cells and HEK293T (co-expressing FLAG-tagged KHSRP and HA-tagged PRMT5) cells were treated with increasing concentrations of DIQ01. Co-immunoprecipitation was performed using anti-KHSRP antibody (HCT116) or anti-FLAG antibody (HEK293T), followed by immunoblotting for MMA, PRMT5, KHSRP, and FLAG. (I) FLAG-tagged KHSRP mutants carrying single arginine-to-lysine substitutions (R411K, R413K, R415K, or R442K), as well as the combined 3RK mutant, were expressed in HCT116 and HEK293T cells. Immunoprecipitation was performed using anti-FLAG antibody, followed by Western blot analysis of MMA levels. (J) HEK293T cells expressing the same KHSRP mutants (R411K, R413K, R415K, R442K, or 3RK) were treated with or without DIQ01 (500 nmol/L). MMA levels were evaluated by Western blot following FLAG-based immunoprecipitation. (K–M) Functional assays evaluating DNA damage and cell proliferation in HCT116 cells with stable KHSRP knockdown reconstituted with EV, KHSRP WT or KHSRP 3RK . (K) Colony formation assay: cells were cultured for five days, stained with SRB, and relative colony numbers were quantified (mean ± SD, n = 3; ∗∗∗ P < 0.001). (L) Western blot analysis of γ -H2AX expression and (M) alkaline comet assay were performed after 48 h to assess DNA damage, scale bar = 5 μm.

Article Snippet: KHSRP antibody for Western blotting and immunoprecipitation (ABclonal, Cat. No. A9075, Wuhan, China), pan-mono-methyl arginine antibody for Western blotting (ABclonal, Cat. No. A17984), and PRMT4 antibody for Western blotting (ABclonal, Cat. No. A21106) were obtained from ABclonal.

Techniques: Methylation, Immunoprecipitation, Western Blot, Protein-Protein interactions, Transfection, Plasmid Preparation, Control, In Vitro, Concentration Assay, Expressing, Mutagenesis, Functional Assay, Knockdown, Colony Assay, Cell Culture, Staining, Alkaline Single Cell Gel Electrophoresis

Journal: Advanced Science

Article Title: KDM4A Erases the H3R17me2a Mark, Facilitating Chromosome Condensation

doi: 10.1002/advs.202514281

Figure Lengend Snippet: H3R17me2a reversibly decreases to facilitate chromosome condensation during mitosis. (A), Confocal images of H3R17me2a (green) and DAPI (blue) in 10T1/2 cells at interphase and metaphase. (B), Western blots of histones from cells arrested in late G2 (RO‐3306 or BI2536), prometaphase (nocodazole), or metaphase (nocodazole release with MG132). (C), In vitro methylation assay using biotinylated histone H3 peptides (1–21 aa; AR * T * KQTARKSTGGKAPRKQLA‐GGK(biotin)) and beads‐captured PRMT. (D–F), Confocal images of H3R17me2a (green), Flag (red), and DAPI (blue) in cells overexpressing H3‐Flag‐HA‐WT or ‐R2K (D). Quantification of the chromosome width (E) and H3R17me2a intensity (F) in the metaphase plate of Flag‐positive cells. Scale bars, 5 µm. (G), Western blots of lysates from cells overexpressing H3‐Flag‐HA‐WT, ‐R2K, or ‐R2A. Red arrowheads indicate Flag‐HA‐tagged H3, whereas black arrowheads indicate endogenous H3. (H), Western blots of lysates from cells treated with nocodazole after the knockdown of CARM1 and/or PRMT6. (I), Western blots of lysates from cells treated with nocodazole for 6, 12, or 24 h after CARM1 knockdown. (J), Levels of H3S10ph measured by FACS in cells treated with CARM1 siRNA or inhibitor for 72 h. Data are presented as the mean ± SD (n = 3). (K,L), After treatment with the CARM1 inhibitor for 72 h, 10T1/2 cells stably expressing GFP‐H2B were filmed for 5 h. Representative images are shown (K), and the duration of mitosis was determined (L).

Article Snippet: The following antibodies were used for immunoblotting, immunoprecipitation, or immunostaining: actin (Santa Cruz Biotechnology, Dallas, TX, USA, sc‐47778, 1:10000), Aurora B (Abcam, Cambridge, UK, ab2254, 1:5000), Borealin (Novus Biologicals, Centennial, CO, USA, NBP1‐89951, 1:2000), CARM1 (Bethyl Laboratories, Montgomery, TX, USA, A300‐421A, 1:5000), Cyclin B1 (Cell Signaling Technology, Danvers, MA, USA, #12231, 1:2000), Flag (Cell Signaling Technology, #2368 and #8146, 1:5000), HA (Cell Signaling Technology, #3724, 1:5000), histone H3 (Cell Signaling Technology, #9715, 1:5000), H3K9me2 (EpigenTek, Farmingdale, NY, USA, #A‐4035, 1:5000), H3K9me3 (Merck Millipore, Billerica, MA, USA, 07–442, 1:5000 and Diagenode, Denville, NJ, USA, C15200146, 1:500), H3R2me2a (Merck Millipore, 07–585, 1:2000), H3R17me2a (Abcam, ab8284, 1:2000), H3R26me2a (EpigenTek, #A‐3707, 1:2000), H3S10ph (Cell Signaling Technology, #9701, 1:5000), H3T3ph (Abcam, ab78351, 1:5000), INCENP (Thermo Fisher Scientific, 39–2800, 1:2000), KDM1A (Cell Signaling Technology, #2184, 1:5000), KDM3A (Proteintech, Rosemont, IL, USA, 12835‐1‐AP, 1:2000), KDM4A (Proteintech, 29977‐1‐AP, 1:5000), KDM4C (Novus Biologicals, NBP1‐49600, 1:5000), PRMT6 (Bethyl Laboratories, Montgomery, A300‐929A, 1:2000), p‐Ser (Santa Cruz Biotechnology, sc‐81514, 1:1000), p‐Ser PKC substrate (Cell Signaling Technology, #6967, 1:2000), Sp1 (Merck Millipore, CS200631 , 1:5000), Survivin (Abcam, ab76424, 1:2000), Suv39h1 (Cell Signaling Technology, #8729, 1:2000 and Santa Cruz Biotechnology, sc‐23961, 1:500), Suv39h2 (Proteintech, 11338‐1‐AP, 1:1000), and α‐tubulin (Santa Cruz Biotechnology, sc‐5286, 1:500).

Techniques: Western Blot, In Vitro, Methylation, Knockdown, Stable Transfection, Expressing

Journal: Advanced Science

Article Title: KDM4A Erases the H3R17me2a Mark, Facilitating Chromosome Condensation

doi: 10.1002/advs.202514281

Figure Lengend Snippet: Reduction in H3R17me2a increases Aurora B levels and chromatin binding, inducing H3S10ph. (A), Western blots of lysates from 10T1/2 cells with CARM1 knockdown or overexpression. (B), Western blots of lysates from cells incubated with CARM1‐WT or ‐E266Q for 48 h. (C,D), mRNA levels of Aurkb and Carm1 in cells transfected with CARM1 siRNA for 72 h (C) or plasmid for 48 h (D). (E,F), Immunoprecipitation using an anti‐HA antibody (E) and measurement of Aurkb mRNA levels (F) in cells overexpressing H3‐Flag‐HA‐WT, ‐R17K, ‐R17A, or ‐R17F. Red arrowheads indicate Flag‐HA‐tagged H3, whereas black arrowheads indicate endogenous H3. (G–J), Representative immunofluorescence images and quantitative intensity analyses of Aurora B (G,H) and H3S10ph (I,J) in cells overexpressing H3‐Flag‐HA‐WT, ‐R17K, or ‐R17F. (K), Levels of H3S10ph measured by FACS in cells overexpressing H3‐Flag‐HA‐WT, ‐R17K, or ‐R17F. Data are presented as the mean ± SD (n = 3). (L,M), Confocal images of HA (red), α‐tubulin (green), and DAPI (blue) in cells overexpressing H3‐Flag‐HA‐WT, ‐R17K, ‐R17A, ‐R17F, or ‐S10A. Scale bars, 5 µm (L). Quantification of metaphase plate chromosome width in HA‐positive cells (M). Cells were synchronized at metaphase by treatment with nocodazole and MG132. Chromosome width was determined by measuring spindle width, defined as the distance between the two outermost edges of the α‐tubulin signal perpendicular to the spindle axis. Measurements were based on the spatial extent of the signal rather than fluorescence intensity. (N), In vitro kinase assay using histone extracts from cells overexpressing H3‐Flag‐HA (WT, R17K, R17A, R17F, or S10A) and recombinant Aurora B protein.

Article Snippet: The following antibodies were used for immunoblotting, immunoprecipitation, or immunostaining: actin (Santa Cruz Biotechnology, Dallas, TX, USA, sc‐47778, 1:10000), Aurora B (Abcam, Cambridge, UK, ab2254, 1:5000), Borealin (Novus Biologicals, Centennial, CO, USA, NBP1‐89951, 1:2000), CARM1 (Bethyl Laboratories, Montgomery, TX, USA, A300‐421A, 1:5000), Cyclin B1 (Cell Signaling Technology, Danvers, MA, USA, #12231, 1:2000), Flag (Cell Signaling Technology, #2368 and #8146, 1:5000), HA (Cell Signaling Technology, #3724, 1:5000), histone H3 (Cell Signaling Technology, #9715, 1:5000), H3K9me2 (EpigenTek, Farmingdale, NY, USA, #A‐4035, 1:5000), H3K9me3 (Merck Millipore, Billerica, MA, USA, 07–442, 1:5000 and Diagenode, Denville, NJ, USA, C15200146, 1:500), H3R2me2a (Merck Millipore, 07–585, 1:2000), H3R17me2a (Abcam, ab8284, 1:2000), H3R26me2a (EpigenTek, #A‐3707, 1:2000), H3S10ph (Cell Signaling Technology, #9701, 1:5000), H3T3ph (Abcam, ab78351, 1:5000), INCENP (Thermo Fisher Scientific, 39–2800, 1:2000), KDM1A (Cell Signaling Technology, #2184, 1:5000), KDM3A (Proteintech, Rosemont, IL, USA, 12835‐1‐AP, 1:2000), KDM4A (Proteintech, 29977‐1‐AP, 1:5000), KDM4C (Novus Biologicals, NBP1‐49600, 1:5000), PRMT6 (Bethyl Laboratories, Montgomery, A300‐929A, 1:2000), p‐Ser (Santa Cruz Biotechnology, sc‐81514, 1:1000), p‐Ser PKC substrate (Cell Signaling Technology, #6967, 1:2000), Sp1 (Merck Millipore, CS200631 , 1:5000), Survivin (Abcam, ab76424, 1:2000), Suv39h1 (Cell Signaling Technology, #8729, 1:2000 and Santa Cruz Biotechnology, sc‐23961, 1:500), Suv39h2 (Proteintech, 11338‐1‐AP, 1:1000), and α‐tubulin (Santa Cruz Biotechnology, sc‐5286, 1:500).

Techniques: Binding Assay, Western Blot, Knockdown, Over Expression, Incubation, Transfection, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Fluorescence, In Vitro, Kinase Assay, Recombinant

Journal: Advanced Science

Article Title: KDM4A Erases the H3R17me2a Mark, Facilitating Chromosome Condensation

doi: 10.1002/advs.202514281

Figure Lengend Snippet: Reduction in H3R17me2a during mitosis enhances the binding of Suv39h1 to chromatin, leading to an increase in H3K9me3. (A,B), Western blots of lysates from 10T1/2 cells overexpressing the H3R17 methylation‐deficient mutant (A) and the methylation‐mimetic mutant (B). Red arrowheads indicate Flag‐HA‐tagged H3, whereas black arrowheads indicate endogenous H3. (C,D), Confocal images of H3K9me3 (green), Flag (red), and DAPI (blue) in cells overexpressing H3‐Flag‐HA‐WT or ‐R17F (C). H3K9me3 intensity in Flag‐positive cells was quantified (D). (E), The intensities of H3R17me2a and H3K9me3 throughout the cell cycle were analyzed via immunostaining. Quantification of representative images shown in Figure . (F,G), Immunoprecipitation using an anti‐H3K9me3 antibody (F) and chromatin fractionations (G) in CARM1‐WT or ‐KO cells treated with nocodazole. (H,I,J), Confocal images of chromosome spreads from nocodazole‐arrested CARM1‐WT or CARM1‐KO cells. Suv39h1 (green), Aurora B (red), and DAPI (blue) are shown (H). Quantification of the fluorescence intensities of chromosome‐bound Suv39h1 (I) and Aurora B (J). (K), Western blots of chromatin fractions from cells overexpressing H3‐Flag‐HA‐WT, ‐R17K, ‐R17A, or ‐R17F. Red arrowheads indicate Flag‐HA‐tagged H3, whereas black arrowheads indicate endogenous H3. (L–O), Representative images and quantification of chromosome‐bound Suv39h1 (green; L,M) and Aurora B (red; N,O) obtained by chromosome‐spreading assays in nocodazole‐arrested cells overexpressing H3‐Flag‐HA‐WT or ‐R17F. (P), In vitro methylation assay using histone extracts from cells overexpressing H3‐Flag‐HA (WT, R17K, R17A, or R17F) and beads‐captured Suv39h1. Red arrowheads indicate Flag‐HA‐tagged H3, whereas black arrowheads indicate endogenous H3. (Q,R), Levels of H3S10ph measured by FACS (Q) and western blots (R) in H3‐Flag‐HA‐WT or ‐R17F overexpressing cells under the indicated conditions. Red arrowheads indicate Flag‐HA‐tagged H3, whereas black arrowheads indicate endogenous H3. (S,T), Western blots (S) and cell cycle profiles analyzed using FACS (T) in CARM1‐WT or ‐KO cells under the indicated conditions.

Article Snippet: The following antibodies were used for immunoblotting, immunoprecipitation, or immunostaining: actin (Santa Cruz Biotechnology, Dallas, TX, USA, sc‐47778, 1:10000), Aurora B (Abcam, Cambridge, UK, ab2254, 1:5000), Borealin (Novus Biologicals, Centennial, CO, USA, NBP1‐89951, 1:2000), CARM1 (Bethyl Laboratories, Montgomery, TX, USA, A300‐421A, 1:5000), Cyclin B1 (Cell Signaling Technology, Danvers, MA, USA, #12231, 1:2000), Flag (Cell Signaling Technology, #2368 and #8146, 1:5000), HA (Cell Signaling Technology, #3724, 1:5000), histone H3 (Cell Signaling Technology, #9715, 1:5000), H3K9me2 (EpigenTek, Farmingdale, NY, USA, #A‐4035, 1:5000), H3K9me3 (Merck Millipore, Billerica, MA, USA, 07–442, 1:5000 and Diagenode, Denville, NJ, USA, C15200146, 1:500), H3R2me2a (Merck Millipore, 07–585, 1:2000), H3R17me2a (Abcam, ab8284, 1:2000), H3R26me2a (EpigenTek, #A‐3707, 1:2000), H3S10ph (Cell Signaling Technology, #9701, 1:5000), H3T3ph (Abcam, ab78351, 1:5000), INCENP (Thermo Fisher Scientific, 39–2800, 1:2000), KDM1A (Cell Signaling Technology, #2184, 1:5000), KDM3A (Proteintech, Rosemont, IL, USA, 12835‐1‐AP, 1:2000), KDM4A (Proteintech, 29977‐1‐AP, 1:5000), KDM4C (Novus Biologicals, NBP1‐49600, 1:5000), PRMT6 (Bethyl Laboratories, Montgomery, A300‐929A, 1:2000), p‐Ser (Santa Cruz Biotechnology, sc‐81514, 1:1000), p‐Ser PKC substrate (Cell Signaling Technology, #6967, 1:2000), Sp1 (Merck Millipore, CS200631 , 1:5000), Survivin (Abcam, ab76424, 1:2000), Suv39h1 (Cell Signaling Technology, #8729, 1:2000 and Santa Cruz Biotechnology, sc‐23961, 1:500), Suv39h2 (Proteintech, 11338‐1‐AP, 1:1000), and α‐tubulin (Santa Cruz Biotechnology, sc‐5286, 1:500).

Techniques: Binding Assay, Western Blot, Methylation, Mutagenesis, Immunostaining, Immunoprecipitation, Fluorescence, In Vitro

Journal: Advanced Science

Article Title: KDM4A Erases the H3R17me2a Mark, Facilitating Chromosome Condensation

doi: 10.1002/advs.202514281

Figure Lengend Snippet: KDM4A is phosphorylated by PKCα during mitosis, acquiring H3R17me2a demethylation activity. (A), Western blots of the chromatin fractions from cells arrested at the G2 (RO‐3306 or BI2536), prometaphase (nocodazole), or metaphase (nocodazole release with MG132). (B–D), Immunoprecipitation using an anti‐KDM4A antibody (B) and intensities of H3R17me2a (C) and H3K9me3 as determined via immunostaining (D) in cells treated with 0.5 µ m calphostin C or 100 nm PMA for 12 h. (E), mRNA levels of Aurkb , Kdm4a , and Carm1 in cells treated with nocodazole, calphostin C, or PMA for 12 h. (F), Levels of H3S10ph measured by FACS in cells added with nocodazole after calphostin C or PMA pretreatment. Data are presented as the mean ± SD (n = 3). (G), Schematic representation of the function of KDM4A depending on PKC‐mediated phosphorylation. (H), Western blots of lysates from cells overexpressing PKCα‐WT, ‐CA, or ‐DN. (I), Western blots of histones from CARM1‐WT or ‐KO cells treated with nocodazole, calphostin C, or PMA for 12 h. (J,K), Western blots (J) and cell cycle profiles analyzed using FACS (K) in CARM1‐WT or ‐KO cells added with nocodazole after calphostin C or PMA pretreatment.

Article Snippet: The following antibodies were used for immunoblotting, immunoprecipitation, or immunostaining: actin (Santa Cruz Biotechnology, Dallas, TX, USA, sc‐47778, 1:10000), Aurora B (Abcam, Cambridge, UK, ab2254, 1:5000), Borealin (Novus Biologicals, Centennial, CO, USA, NBP1‐89951, 1:2000), CARM1 (Bethyl Laboratories, Montgomery, TX, USA, A300‐421A, 1:5000), Cyclin B1 (Cell Signaling Technology, Danvers, MA, USA, #12231, 1:2000), Flag (Cell Signaling Technology, #2368 and #8146, 1:5000), HA (Cell Signaling Technology, #3724, 1:5000), histone H3 (Cell Signaling Technology, #9715, 1:5000), H3K9me2 (EpigenTek, Farmingdale, NY, USA, #A‐4035, 1:5000), H3K9me3 (Merck Millipore, Billerica, MA, USA, 07–442, 1:5000 and Diagenode, Denville, NJ, USA, C15200146, 1:500), H3R2me2a (Merck Millipore, 07–585, 1:2000), H3R17me2a (Abcam, ab8284, 1:2000), H3R26me2a (EpigenTek, #A‐3707, 1:2000), H3S10ph (Cell Signaling Technology, #9701, 1:5000), H3T3ph (Abcam, ab78351, 1:5000), INCENP (Thermo Fisher Scientific, 39–2800, 1:2000), KDM1A (Cell Signaling Technology, #2184, 1:5000), KDM3A (Proteintech, Rosemont, IL, USA, 12835‐1‐AP, 1:2000), KDM4A (Proteintech, 29977‐1‐AP, 1:5000), KDM4C (Novus Biologicals, NBP1‐49600, 1:5000), PRMT6 (Bethyl Laboratories, Montgomery, A300‐929A, 1:2000), p‐Ser (Santa Cruz Biotechnology, sc‐81514, 1:1000), p‐Ser PKC substrate (Cell Signaling Technology, #6967, 1:2000), Sp1 (Merck Millipore, CS200631 , 1:5000), Survivin (Abcam, ab76424, 1:2000), Suv39h1 (Cell Signaling Technology, #8729, 1:2000 and Santa Cruz Biotechnology, sc‐23961, 1:500), Suv39h2 (Proteintech, 11338‐1‐AP, 1:1000), and α‐tubulin (Santa Cruz Biotechnology, sc‐5286, 1:500).

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Immunostaining, Phospho-proteomics