Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 1. CARM1 associates with p/CIP on DNA. (A) HeLa nuclear extracts were separated by gel filtration chromatography, and fractions were assayed by immunoblotting using the antibodies indicated on the left. (B) Fractions corresponding to peaks I and II shown in panel A were pooled and passed through an anti-p/CIP immunoaffinity column or immunoglobulin G (IgG) column as control. After extensive washing, bound proteins were eluted using low pH buffer and assayed by immunoblotting using antibodies specific for p/CIP, CBP, or CARM1. (C) p/CIP and CARM1 bind to the endogenous PS2 promoter in vivo. ChIP analysis of the PS2 promoter in MCF-7 cells using antibodies against p/CIP and CARM1 demonstrates that both p/CIP and CARM1 are recruited to the PS2 promoter in response to estradiol. MCF-7 cells were stimulated for various time periods. Cells were fixed, and sequential ChIPs were performed using an anti-p/CIP antibody, followed by reimmunoprecipitation (ReChIP) using a CARM1-specific antibody. Samples were analyzed by PCR using specific primers flanking the PS2 promoter, and the relative abundance was quantified by densitometry.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Chromatography, Western Blot, Control, In Vivo

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 2. In vitro methylation of p/CIP. (A) Epitope-tagged recombi- nant proteins were generated in Sf9 cells using baculovirus. Cells were then harvested, and proteins were immunopurified using anti-FLAG Sepharose. Approximately 500 ng of various purified proteins was then analyzed by SDS-PAGE and Coomassie blue staining. (B) Approximately 500 ng of purified recombinant CBP, p/CIP, or specific histones was incubated in the presence of 100 ng of either CARM1 or PRMT1 and [3H]SAM for 1 h. Proteins were then separated by SDS-PAGE and analyzed by fluorography. (C) In vitro methylation assays were performed using 500 ng of p/CIP, 100 ng of recombinant CARM1, and increasing concentrations of histones as indicated at the top of the panel. The reactions were terminated, proteins were separated by SDS-PAGE, and methylation of p/CIP was monitored by fluorography. BSA, bovine serum albumin.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: In Vitro, Methylation, Generated, SDS Page, Staining, Recombinant, Incubation

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 3. p/CIP is methylated in intact cells. (A) Extracts from CARM1/ MEFs do not support methylation of p/CIP in vitro. Ap- proximately 500 ng of purified recombinant p/CIP protein was incu- bated with extracts derived from either CARM1/ or CARM1/

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Methylation, In Vitro, Recombinant, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 4. Molecular mapping of CARM1-dependent methylation of p/CIP. p/CIP truncation mutants were generated using a baculovirus system and purified by immunoaffinity chromatography. Approximately 500 ng of the various truncation mutants was then tested as a substrate for CARM1 by in vitro methylation assay using 100 ng of purified CARM1. On the left is the Coomassie-stained gel of the purified proteins resolved on an 8% SDS-PAGE gel, and on the right is the corresponding fluorograph. (B) One microgram of biotinylated peptides corresponding to the regions of p/CIP indicated was used as a substrate, and the incorporation of [3H]SAM was measured by in vitro methylation in the presence of purified CARM1. The asterisk indicates the three dimethylated arginines identified by mass spectrometry. (C) Homology of MD1 within the SRC family of proteins. SRC3 corresponds to the human homologue of p/CIP. The shaded areas indicate conserved arginines, and asterisks indicate arginines identified by mass spectrometry in p/CIP.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Methylation, Generated, Chromatography, In Vitro, Staining, SDS Page, Mass Spectrometry

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 5. Phosphorylation at S847 antagonizes methylation of p/CIP at R839. (A) GST recombinant proteins containing various regions of p/CIP spanning the full-length protein were generated in bacteria, purified, and used as substrates (500 ng) for purified CARM1 (100 ng) by in vitro methylation. At left is a Coomassie-stained gel of the GST fusion proteins used in the methylation assay resolved on a 12% SDS-PAGE gel, and at right is the corresponding fluorograph of the in vitro methylation reactions. (B) Comparison of the MD2 domains between p/CIP and SRC3. The asterisk corresponds to the phosphorylation site at aa 847. The shaded areas contain the methylation sites defined by in vitro methylation reactions using recombinant GST proteins. (C) Biotinylated peptides corresponding to the regions P1, P2, and P3 (shaded areas in panel B) were used as substrates, and the incorporation of [3H]SAM was measured by in vitro methylation in the presence of purified CARM1. Peptides corresponding to P1R839A and P1R844A contain substitutions of arginine to alanine. The peptide P1S847(p) is identical to P1 but contains a phosphorylated serine at aa 847.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Phospho-proteomics, Methylation, Recombinant, Generated, Bacteria, In Vitro, Staining, SDS Page, Comparison

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 6. The relative abundance of p/CIP is regulated by arginine methylation. Epitope-tagged recombinant wild-type p/CIP (wt), or p/CIP mutants containing arginine substitutions at CARM1 methylation sites (p/CIPR3A or p/CIPR6A) were generated in Sf9 cells using baculovirus. Cells were then harvested, and proteins were immunopurified using anti-FLAG-Sepharose. Approximately 1 g of each purified protein was analyzed by SDS-PAGE and Coomassie blue staining (left) and used as substrates for in vitro CARM1-dependent methylation reactions (right). (B) COS-1 cells were transfected with expression vectors for wild-type p/CIP (wt), p/CIPR3A, or p/CIPR6A. After 24 h cells were lysed, and whole-cell extracts were prepared. Equal amounts of protein from each extract were then analyzed by Western blotting using an anti-FLAG antibody for p/CIP or anti-tubulin antibody (left). Transfected samples were immunopurified using anti-FLAG-Sepharose, followed by immuno- blotting with anti-FLAG antibody (right). (C) CARM1/ or CARM1/ MEFs were incubated with [35S]methionine for 40 min, followed by incubation with nonradioactive methionine-containing medium for the indicated time periods. Cells were then lysed, and p/CIP was immunopre- cipitated and then subjected to SDS-PAGE and fluorography. (D) U2OS cells were transfected with either p/CIP wild-type or p/CIP R6A mutant. After 24 h, cells were incubated with [35S]methionine for 40 min and then incubated with cold methionine-containing medium for the indicated time periods. Cells were then lysed, and p/CIP was immunoprecipitated using anti-FLAG-Sepharose and then subjected to SDS-PAGE and fluorography. Samples were quantified by densitometry and expressed as a percentage of p/CIP remaining normalized to time zero.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Methylation, Recombinant, Generated, SDS Page, Staining, In Vitro, Transfection, Expressing, Western Blot, Incubation, Mutagenesis, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 7. Methylation of p/CIP is associated with increased degradation. HeLa cells were grown in the presence of AdOx for 8 days, followed by immunoprecipitation of p/CIP. Equal concentrations of p/CIP were then incubated with extracts from CARM1/ or CARM1/ MEFs in the presence of SAM for the indicated time periods. Reactions were then terminated and analyzed by Western blotting using anti-p/CIP antibody. The graph on the right indicates the relative amounts of p/CIP remaining, based on densitometry.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: Methylation, Immunoprecipitation, Incubation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

doi: 10.1128/mcb.00815-06

Figure Lengend Snippet: FIG. 9. The association between CBP and p/CIP is decreased in CARM1/ MEFs. (A) Whole-cell extracts were prepared from CARM1/ MEFs or CARM1/ MEFs (at a similar passage num- ber). Approximately 20 g of protein from each extract was separated by SDS-PAGE, and Western blotting was performed using anti-p/CIP antibody or antitubulin as a control. (B) Whole-cell extracts were prepared from CARM1/ MEFs or CARM1/ MEFs, and the amount of starting material in the input was normalized so that each had approximately the same amount of p/CIP based on Western blot- ting. p/CIP was then immunopurified by passing the protein extracts through an anti-p/CIP immunoaffinity column. After extensive wash- ing, the bound proteins were eluted using 100 mM glycine (pH 3.0). Fractions were collected and assayed for p/CIP or CBP by SDS-PAGE followed by Western blotting.

Article Snippet: Antibodies against CARM1 and CBP were purchased from Santa Cruz Biotechnologies.

Techniques: SDS Page, Western Blot, Control

Journal: Journal of proteome research

Article Title: Combined Antibody/Lectin-Enrichment Identifies Extensive Changes in the O -GlcNAc Sub-proteome Upon Oxidative Stress

doi: 10.1021/acs.jproteome.6b00369

Figure Lengend Snippet: (A) OGT wild-type and null cells were treated with hydrogen peroxide (2.5mM; 1–3h). Proteins were enriched from nuclear and cytoplasmic lysates using the G5-lectibody column and eluted with 1M GlcNAc. 4% of the input or 20% of the eluate were separated by SDS-PAGE and the following proteins were detected by immunoblot: O-GlcNAc, OGT, hnRNP-U, Caprin, Carm1, PRMT1, VDAC, PRMT5, NFκB p65, NFκB p50, Fus, Actin, Nup62, 14–3-3 phospho-substrate, 14–3-3γ, and 14–3-3η. (B) The SILAC ratios for the proteins tested in Figure 5a are reported. (C) OGT, Carm1, hnRNP-U, 14–3-3 proteins, Caprin, and Fus, were immunoprecipitated from Control, Stressed cells (2.5 mM H2O2, 1–3 h) and OGT null cells. The levels of O-GlcNAc on the indicated proteins were assessed by immunoblot with CTD110.6.

Article Snippet: Antibodies used in this study include: OGT (DM-17), heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) and actin (Sigma-Aldrich); Carm1 (coactivator-associated arginine methyltransferase 1), Caprin and FUS RNA binding protein (Fus; Bethyl Laboratories); heat shock cognate 71 kDa (Hsc70), Protein Arginine Methyltransferase 1 (PRMT1), Voltage-dependent anion channels (VDAC), Protein Arginine Methyltransferase 5 (PRMT5), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and Casein kinase 2 (CK2; Santa Cruz Biotechnology); AKT phospho-substrate, 14–3-3 substrate, 14–3-3γ, 14–3-3η (Cell Signaling); and mAb414 (Abcam).

Techniques: SDS Page, Western Blot, Immunoprecipitation

Journal: Journal of proteome research

Article Title: Combined Antibody/Lectin-Enrichment Identifies Extensive Changes in the O -GlcNAc Sub-proteome Upon Oxidative Stress

doi: 10.1021/acs.jproteome.6b00369

Figure Lengend Snippet: (A) Mouse embryonic fibroblasts (MEFs) were labeled with medium and heavy isotopes of arginine and lysine, and treated with 2.5 mM H2O2 for 1 and 2 h respectively. Equal amounts of protein were combined and O-GlcNAc-modified proteins were purified using a combination of immobilized O-GlcNAc-specific antibodies (CTD110.6, RL2, HGAC39 and HGAC89) and WGA referred to as the G5-lectibody resin/column. Eluted proteins were trypsin digested, separated into 12 fractions by basic reversed phase (bRP) chromatography and subjected to LC-MS/MS. (B) To reduce antibody contamination of the eluent, the G5-lectibody resin was packed on top of a trap column containing equal parts Protein A, Protein G and anti-IgM sepharose. (C) O-GlcNAc modified proteins were enriched from Nuc/Cyt lysates using sepharose-4B covalently coupled to the indicated antibodies and lectins at 2mg/ml. Immunoprecipitates were separated by SDS-PAGE and OGT, OGA, Carm1, Nup62, Caprin, hnRNP-U and Casein Kinase 2 α (CK2α) were detected by immunoblot. (D) O-GlcNAc modified proteins were enriched from wildtype (WT) or OGT null (KO) Nuc/Cyt lysates (400 μg) or buffer alone (B) using WGA sepharose (2.5 or 10 nmol of GlcNAc binding) and the G5-lectibody column (3 nmoles GlcNAc-binding). Input (5μg, 1.4%), unbound (5μg, 1.4%), and immunoprecipitated (5.25%) proteins were separated by SDS-PAGE. OGT, Carm1, and Caprin were detected by immunoblot (n=3).

Article Snippet: Antibodies used in this study include: OGT (DM-17), heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) and actin (Sigma-Aldrich); Carm1 (coactivator-associated arginine methyltransferase 1), Caprin and FUS RNA binding protein (Fus; Bethyl Laboratories); heat shock cognate 71 kDa (Hsc70), Protein Arginine Methyltransferase 1 (PRMT1), Voltage-dependent anion channels (VDAC), Protein Arginine Methyltransferase 5 (PRMT5), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and Casein kinase 2 (CK2; Santa Cruz Biotechnology); AKT phospho-substrate, 14–3-3 substrate, 14–3-3γ, 14–3-3η (Cell Signaling); and mAb414 (Abcam).

Techniques: Labeling, Modification, Purification, Chromatography, Liquid Chromatography with Mass Spectroscopy, SDS Page, Western Blot, Binding Assay, Immunoprecipitation

Journal: Theranostics

Article Title: TFE3, a potential therapeutic target for Spinal Cord Injury via augmenting autophagy flux and alleviating ER stress

doi: 10.7150/thno.46566

Figure Lengend Snippet: The activity of TFE3 after SCI is regulated by AMPK-mTOR and AMPK-SKP2-CARM1 signaling pathways. ( A ) Western blot analysis of AMPK-mTOR signal pathway in the cytoplasm in spinal cords from the control mice, SCI mice, and SCI mice treated with DMSO or Compound C, at Day3. ( B ) Densitometric analysis of the AMPK, p-AMPK, p-mTOR and p-4EBP1 data from (A), normalized to the loading control GAPDH. ( C ) Western blotting of TFE3 nuclear translocation at the lesion for each group. ( D ) Corresponding densitometric analysis of the TFE3 bands in (C) normalized to the loading control H3. ( E ) Western blots of the AMPK-SKP2-CARM1 signaling pathway in the nucleus of the indicated groups at Day3 after SCI. ( F ) Densitometric analysis of AMPK, p-AMPK, p-FOXO3a, SKP2 and CARM1 bands from (E) normalized to control H3. ( G ) Nuclear CARM1-TFE3 complex was detected by IP in indicated groups at Day3 after SCI. ( H ) Densitometric analysis of TFE3 and CARM1 data from (G) normalized to loading control H3. ( I ) Western blot analysis of LC3, SQSTM1/p62 and UB in the spinal cord lesion of each group at Day3 after SCI. ( J ) Densitometric analysis of band data from (I) normalized to the loading control GAPDH. ( K ) Western blotting of LC3II in the indicated mice spinal cord slides cultured in the presence or absence of CQ at Day3. ( L ) Densitometric analysis of LC3II from (K) normalized to the loading control GAPDH. n=6, ns stands for not significant, *P<0.05, **P<0.01.

Article Snippet: Primary antibodies against Beclin1 (Cat. No. 3738), ATG5 (Cat. No. 12994), ATP6V1B2 (Cat. No. 14617), Ubiquitin (Cat. No. 3936), ATF4 (Cat. No. 11815), CHOP (Cat. No. 2895), AMPKα (Cat. No. 5832), p-AMPKα (Cat. No. 2535) p-FOXO3a (Cat. No. 9466), p-EIF2α (Cat. No. 3398), p-4EBP1 (Cat. No. 9456), mTOR (Cat. No.2983), p-mTOR (Cat. No.5536) and CARM1 (Cat. No. 4438) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Protein-Protein interactions, Western Blot, Control, Translocation Assay, Cell Culture

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 1. CARM1 was negatively associated with ferroptosis. a) HE staining of clinical specimens of colon cancer. Scale bar, 20 μm. b) Twenty-five pairs of colorectal cancer (CRC) tissues and adjacent tissues were digested into single-cell suspensions, and lipid ROS production was assayed via flow cytometry by using C11-BODIPY after RSL3 treatment for 4 h (n = 25). c) Malondialdehyde (MDA) levels were detected by using a lipid peroxidation MDA assay kit in single-cell suspensions treated with RSL3 for 4 h from 25 pairs of CRC tissues and adjacent tissues (n = 25). d) Heatmap of RNA-seq using six patient tumors with different lipid ROS levels showing changes in gene expression, including CARM1. e) Quantitative real-time PCR (qPCR) analysis of CARM1 mRNA levels in tumors from 10 patients. f) Western blot analysis of CARM1 in the same tissues as (e). g) Cell viability was assayed in vector- and CARM1-overexpressing LoVo and HCT116 cells treated with the indicated doses of RSL3 and erastin for 24 h. h) Scatter plot of the immunohistochemistry (IHC) staining score for CARM1, lipid ROS, and MDA levels in CRC tissues (n = 25). All p values and R values were calculated with Spearman’s r test. i) Representative results of immunohistochemical staining for CARM1 from 25 clinical CRC patients. Scale bars, 20 μm. j,k) Lipid ROS (left) and MDA (right) levels were compared in CARM1 high (CARM1 IHC score ≥6) and CARM1 low (CARM1 IHC score<6) groups. The data shown represent the mean ± SD. In (b) and (c), comparisons were made by using paired Student’s t-test, and in (j) and (k), comparisons were made by using the two-tailed, unpaired Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Staining, Cytometry, Multiple Displacement Amplification, RNA Sequencing, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Immunohistochemistry, Immunohistochemical staining, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 2. CARM1-KD enhances ferroptotic cell death. a) Cell viability was measured in siNC and siCARM1 LoVo cells treated with 2.5 × 10−6 m RSL3 or 5 × 10−6 m erastin for 12 h (n = 5 independent experiments). b) Cell viability was measured in siNC and siCARM1 LoVo cells treated with cell death inhibitors and 2. 5 × 10−6 m RSL3 or 5 × 10−6 m erastin for 12 h. Fer-1, 1 × 10−6 m ferrostatin-1; NAC, 5 × 10−3 m; Nec, 2 × 10−6 m necrostatin-1; Z-V, 20 × 10−6 m Z-VAD-FMK (n = 5 independent experiments). c,d) Malondialdehyde (MDA) levels and relative lipid ROS were assayed in the indicated LoVo cells treated with 2. 5 × 10−6 m RSL3 or 5 × 10−6 m erastin for 12 h (n = 3 independent experiments). e) Transmission electron microscopy (TEM) images of the indicated LoVo cells subjected to RSL3 (2. 5 × 10−6 m) for 12 h. White arrows indicate mitochondria. Scale bars, left, 2 μm; right, 500 nm. f) The indicated stable LoVo cells were used to evaluate mitochondrial membrane potential via fluorescence staining of mitochondria with JC-1 dye (n = 3 independent experiments). g) shNC and shCARM1 LoVo cells were subcutaneously injected into the mice. RSL3 was administered to all tumors with or without Fer-1. Tumor volumes (n = 5) were calculated every 4 days, and the growth curve was drawn. h) Images of tumors from LoVo xenograft mice with altered treatments are shown, and the tumor weights (n = 5) of the subcutaneous xenografts were measured. i) Representative immunohistochemical images of CARM1 and Ki67 in tumor sections are shown. Scale bars, 20 μm. j,k) MDA levels and relative lipid ROS in tumor cells isolated from (h) were assayed (n = 5 independent experiments). The data shown represent the mean ± SD. Comparisons were made by using one-way ANOVA with Tukey’s test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Transmission Assay, Electron Microscopy, Membrane, Staining, Injection, Immunohistochemical staining, Isolation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 4. CARM1 directly interacts with and decreases ACSL4 protein levels in colon cancer cells. a) Mass spectrometry analysis identified ACSL4 in the binding protein pool of CARM1. b) Immunoprecipitation (IP) analyses were performed to examine the endogenous interaction between CARM1 and ACSL4 by using antibodies against CARM1 and ACSL4 in LoVo cells. c) IP analyses were performed to examine the exogenous interaction between CARM1 and ACSL4 by using antibodies against Flag and HA, respectively, in HEK293T cells. d) In vitro GST pull-down assay to verify the binding of CARM1 and ACSL4. e) Immunofluorescence staining was performed to observe the colocalization of CARM1 (green) and ACSL4 (red) in LoVo and

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Mass Spectrometry, Binding Assay, Immunoprecipitation, In Vitro, Pull Down Assay, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 5. CARM1 methylates ACSL4 at R339. a,b) Co-immunoprecipitation (Co-IP) was performed to detect the methylation levels of ACSL4 with CARM1 attenuation (left) or upregulation (right). c) IP assay was performed for the enrichment of ACSL4 protein, staining was performed with Coomassie bright

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Methylation, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Inhibition of CARM1-Mediated Methylation of ACSL4 Promotes Ferroptosis in Colorectal Cancer.

doi: 10.1002/advs.202303484

Figure Lengend Snippet: Figure 7. RNF25 knockdown inhibits CARM1-induced ferroptosis resistance. a) Western blot analysis of LoVo and HCT116 cells transfected with the indicated plasmid and siRNAs. Protein levels of CARM1, RNF25, ACSL4 and ACSL4 R339me2a were assayed. b) Western blot analysis of vector- and RNF25-overexpressing LoVo and HCT116 cells treated with DMSO or 10 × 10−9 m EZM2302 for 24 h. Protein levels of RNF25, ACSL4, and H3R17me2a were assayed. c) HEK293T cells transfected with the indicated plasmids and treated with or without 10 × 10−9 m EZM2302 for 24 h. Immunoprecipitation (IP) with an anti-Flag antibody and Western blotting with an anti-Myc antibody were performed to detect the ubiquitination level of ACSL4. d) Cell viability was assayed in the indicated LoVo and HCT116 cells as (a) treated with 2. 5 × 10−6 m RSL3 for 12 h (n = 5 independent experiments). e,f) Malondialdehyde (MDA) levels and relative lipid ROS were assayed in the indicated LoVo and HCT116 cells treated with 2. 5 × 10−6 m RSL3 for 12 h (n = 3 independent experiments). g) Mitochondrial membrane potential was detected for the same cells as (e) by using fluorescence staining of mitochondria with JC-1 dye (n = 3 independent experiments). h) Schematic diagram of our hypothesis about this project. The data shown represent the mean ± SD. Comparisons were made by using one-way ANOVA with Tukey’s test; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: GST Pull-Down Assay: The proteins for CARM1 (OriGene, TP317483) and ACSL4 (H00002182-P01, Abnova) were commercially available.

Techniques: Knockdown, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Ubiquitin Proteomics, Membrane, Staining