Journal: Journal of molecular biology

Article Title: CARM1 (PRMT4) acts as a transcriptional coactivator during retinoic acid-induced embryonic stem cell differentiation

doi: 10.1016/j.jmb.2018.08.014

Figure Lengend Snippet: A) J1 WT mESCs were either treated with vehicle (Veh. Ctl.) or 1 μM RA for 24–72 hrs. CARM1 mRNA levels were measured by semi-quantitative PCR (semi-qPCR). Images are from one experiment of three biological repeats and HPRT is used as the loading control to normalize CARM1 mRNA values. B) J1 WT mESCs were untreated (No Tx) or treated as in A) and CARM1 protein levels were measured using western blot (WB) analysis (n=3). Actin is used as the loading control to normalize CARM1 protein values. C) Stable CARM1 knockdown (KD, #9117) and knockout (KO, #23) mESCs were generated as mentioned in the methods section. We used WB analysis to confirm the CARM1 KD and KO cell lines. The images are from one experiment of three biological repeats starting from the generation of lentiviral particles in HEK293T cells for the CARM1 KD cell lines. D) me-Pabp1 protein levels were measured by WB analysis in the CARM1 KD (#9117) and CARM1 KO (#23) cell lines to compare CARM1 depletion efficiency (n=3). E) Cell lines were plated in 12-well plates and counted 24hrs after initial plating for three consecutive days (n=3). ImageJ was used to measure mRNA band densities and Image Lab was used to measure protein band densities. Fold change is represented as the difference between each sample relative to WT Veh. Ctl, which is set to 1. Statistical significances were calculated using one-way ANOVA followed by the Tukey post hoc test (****p<0.0001).

Article Snippet: Cells were sonicated and the precleared lysates (25 μg DNA) were immunoprecipitated using 0.5–2.0 μg of antibodies specific for CARM1 (Epicypher, Research Triangle Park, NC, cat# 13–0006, lot# 13281001), H3K27ac (Abcam, cat# 4729, lot# GR28147), H3K27me3 (Abcam, cat# 6002, lot# 2736613), H3R17me2a (Abcam, cat# 8284, lot# {"type":"entrez-nucleotide","attrs":{"text":"GR295369","term_id":"239851048"}} GR295369 –1), Suz12 (Cell Signaling, cat# D39F6, lot# 3), or IgG (Santa Cruz, cat# sc2027, lot# L2414) (negative control).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Knock-Out, Generated

Journal: Journal of molecular biology

Article Title: CARM1 (PRMT4) acts as a transcriptional coactivator during retinoic acid-induced embryonic stem cell differentiation

doi: 10.1016/j.jmb.2018.08.014

Figure Lengend Snippet: A) J1 WT, shCtl, CARM1 KD (# 9117), and CARM1 KO (#23) cells were plated in 6-well plates and treated with 1 μM RA for 48 hrs following 24 hrs after initial plating for each biological repeat (n=3). mRNA levels were measured by qRT-PCR and normalized to 36B4 control mRNA levels using the delta CT method. To determine relative mRNA levels, we compared values to the highest signal, which was set to 1. B) J1 WT and CARM1 KO (#23) cells were plated in 6mm plates and harvested for protein isolation. Nanog and Oct4 protein levels were measured using western blot (WB) analysis. Images are from one experiment of three biological repeats. Actin is used as the loading control and Image Lab was used to measure protein band densities to generate the bar graphs. Statistical differences were calculated using one-way ANOVA followed by the Tukey post hoc test (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Cells were sonicated and the precleared lysates (25 μg DNA) were immunoprecipitated using 0.5–2.0 μg of antibodies specific for CARM1 (Epicypher, Research Triangle Park, NC, cat# 13–0006, lot# 13281001), H3K27ac (Abcam, cat# 4729, lot# GR28147), H3K27me3 (Abcam, cat# 6002, lot# 2736613), H3R17me2a (Abcam, cat# 8284, lot# {"type":"entrez-nucleotide","attrs":{"text":"GR295369","term_id":"239851048"}} GR295369 –1), Suz12 (Cell Signaling, cat# D39F6, lot# 3), or IgG (Santa Cruz, cat# sc2027, lot# L2414) (negative control).

Techniques: Quantitative RT-PCR, Isolation, Western Blot

Journal: Journal of molecular biology

Article Title: CARM1 (PRMT4) acts as a transcriptional coactivator during retinoic acid-induced embryonic stem cell differentiation

doi: 10.1016/j.jmb.2018.08.014

Figure Lengend Snippet: A) J1 WT, shCtl, CARM1 KD (# 9117), and CARM1 KO (#23) cells were plated in 6-well plates and treated with RA for 48 hrs following 24 hrs after initial plating for each biological repeat (n=3). mRNA levels were measured by semi-quantitative PCR (semi-qPCR) and 36B4 is used as the loading control. B) ImageJ was used to measure the semi-qPCR band densities shown in A) to generate the bar graphs. Band densities for each gene were normalized to 36B4. To determine relative mRNA expression, the most intense band was set to 1 (n=3, except Cyp26b1 is n=1). Statistical differences were calculated using one-way ANOVA followed by the Tukey post hoc test (**p<0.01, ***p<0.001, ****p<0.0001). These data were then replicated by using genome-wide RNA transcriptomic profiling (Supplemental Table 1).

Article Snippet: Cells were sonicated and the precleared lysates (25 μg DNA) were immunoprecipitated using 0.5–2.0 μg of antibodies specific for CARM1 (Epicypher, Research Triangle Park, NC, cat# 13–0006, lot# 13281001), H3K27ac (Abcam, cat# 4729, lot# GR28147), H3K27me3 (Abcam, cat# 6002, lot# 2736613), H3R17me2a (Abcam, cat# 8284, lot# {"type":"entrez-nucleotide","attrs":{"text":"GR295369","term_id":"239851048"}} GR295369 –1), Suz12 (Cell Signaling, cat# D39F6, lot# 3), or IgG (Santa Cruz, cat# sc2027, lot# L2414) (negative control).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Genome Wide

Journal: Journal of molecular biology

Article Title: CARM1 (PRMT4) acts as a transcriptional coactivator during retinoic acid-induced embryonic stem cell differentiation

doi: 10.1016/j.jmb.2018.08.014

Figure Lengend Snippet: A) J1 WT and CARM1 KO (#23) cells were plated in 6-well plates and treated with RA for 48 and 72 hrs following 24 hrs after initial plating for each biological repeat (n≥3). Images are from one experiment. mRNA levels were measured by semiquantitative PCR (semi-qPCR) and 36B4 is used as the loading control. B) ImageJ was used to measure the semi-qPCR band densities for each repeat to generate the bar graphs. Band densities for each gene were normalized to 36B4. To determine relative mRNA levels, we compared values to the most intense band, which was set to 1. For Sox17, a band was only detected in the J1 parental cells at 72 hrs after RA treatment, as is seen in the gel image; therefore, significant changes could not be calculated. Statistical differences were calculated using one-way ANOVA followed by the Tukey post hoc test (****p<0.0001).

Article Snippet: Cells were sonicated and the precleared lysates (25 μg DNA) were immunoprecipitated using 0.5–2.0 μg of antibodies specific for CARM1 (Epicypher, Research Triangle Park, NC, cat# 13–0006, lot# 13281001), H3K27ac (Abcam, cat# 4729, lot# GR28147), H3K27me3 (Abcam, cat# 6002, lot# 2736613), H3R17me2a (Abcam, cat# 8284, lot# {"type":"entrez-nucleotide","attrs":{"text":"GR295369","term_id":"239851048"}} GR295369 –1), Suz12 (Cell Signaling, cat# D39F6, lot# 3), or IgG (Santa Cruz, cat# sc2027, lot# L2414) (negative control).

Techniques:

Journal: Journal of molecular biology

Article Title: CARM1 (PRMT4) acts as a transcriptional coactivator during retinoic acid-induced embryonic stem cell differentiation

doi: 10.1016/j.jmb.2018.08.014

Figure Lengend Snippet: A) A representative scheme showing the regions used for chromatin immunoprecipitation (ChIP) relative to each transcriptional start site (TSS) for each gene. The bent arrows indicate the TSS. PRefSeq is a putative TSS previously identified and characterized in our lab. B) We plated J1 WT cells in 150mm plates and 24 hrs after initial plating for each biological repeat we added 1 μM RA for 24, 48, and 72 hrs. The vehicle control (Veh. Ctl.) plates were plated at the same time as the 48hr RA plates. 25μg of ChIP lysate was used with 5μL of CARM1 antibody for the immunoprecipitation (IP). qPCR was used to measure CARM1 occupancy at each gene region shown. IPs for IgG and CARM1 KO Veh. Ctl. were used as negative controls. Graphs represent the average of three biological repeats. To determine relative occupancy, the J1 WT Veh. Ctl. samples were set to 1 for each IP. The percent input values set to 1 for each J1 WT Veh. Ctl. are 0.03 (Hoxa1), 004 (NR2F1), and 0.03 (CRABP2). Statistical differences were calculated using one-way ANOVA followed by the Tukey post hoc test (*p<0.005, (**p<0.01, ***p<0.001).

Article Snippet: Cells were sonicated and the precleared lysates (25 μg DNA) were immunoprecipitated using 0.5–2.0 μg of antibodies specific for CARM1 (Epicypher, Research Triangle Park, NC, cat# 13–0006, lot# 13281001), H3K27ac (Abcam, cat# 4729, lot# GR28147), H3K27me3 (Abcam, cat# 6002, lot# 2736613), H3R17me2a (Abcam, cat# 8284, lot# {"type":"entrez-nucleotide","attrs":{"text":"GR295369","term_id":"239851048"}} GR295369 –1), Suz12 (Cell Signaling, cat# D39F6, lot# 3), or IgG (Santa Cruz, cat# sc2027, lot# L2414) (negative control).

Techniques: Chromatin Immunoprecipitation, Immunoprecipitation

Journal: Journal of molecular biology

Article Title: CARM1 (PRMT4) acts as a transcriptional coactivator during retinoic acid-induced embryonic stem cell differentiation

doi: 10.1016/j.jmb.2018.08.014

Figure Lengend Snippet: A) J1 WT and CARM1 KO (# 23) cells were plated and treated as in Fig. 5B. 25μg of ChIP lysate were used with 5μL of Suz12 antibody for the immuniprecipitation (IP). qPCR was used to measure Suz12 occupancy at each gene region shown. The IP for IgG is used as a negative control and the J1 WT Veh. Ctl. samples were set to 1 for each IP to determine relative occupancy. The percent input values set to 1 for each J1 WT Veh. Ctl. are 0.70 (Hoxa1), 5.97 (NR2F1), and 0.37 (CRABP2). B) Cells were plated as in A) and 2μL of the H3K27me3 antibody were used. The percent input values set to 1 for each J1 WT Veh. Ctl. are 1.44 (Hoxa1), 0.28 (NR2F1), and 0.14 (CRABP2). C) Cells were plated as in A) and 0.5μL of the H3K27ac antibody were used. The percent input values set to 1 for each J1 WT Veh. Ctl. are 0.48 (Hoxa1), 0.31 (NR2F1), and 0.43 (CRABP2). All graphs represent the average of at least three biological repeats. We used Student’s t-test to determine statistical differences at each time point after RA treatment between the two cell lines for NR2F1 PRefSeq and CRABP2 RARE1 (*p<0.05, **p<0.01, ****p<0.0001). For the Hoxa1 RARE, statistical differences were calculated using one-way ANOVA followed by the Tukey post hoc test to compare the J1 and KO Veh. Ctl. to J1 and KO 24–72 hrs RA samples (****p<0.0001).

Article Snippet: Cells were sonicated and the precleared lysates (25 μg DNA) were immunoprecipitated using 0.5–2.0 μg of antibodies specific for CARM1 (Epicypher, Research Triangle Park, NC, cat# 13–0006, lot# 13281001), H3K27ac (Abcam, cat# 4729, lot# GR28147), H3K27me3 (Abcam, cat# 6002, lot# 2736613), H3R17me2a (Abcam, cat# 8284, lot# {"type":"entrez-nucleotide","attrs":{"text":"GR295369","term_id":"239851048"}} GR295369 –1), Suz12 (Cell Signaling, cat# D39F6, lot# 3), or IgG (Santa Cruz, cat# sc2027, lot# L2414) (negative control).

Techniques: Negative Control

Journal: Journal of molecular biology

Article Title: CARM1 (PRMT4) acts as a transcriptional coactivator during retinoic acid-induced embryonic stem cell differentiation

doi: 10.1016/j.jmb.2018.08.014

Figure Lengend Snippet: In the absence of RA, CARM1 is present and both these gene sets are associated with co-repressors and repressive histone marks, such as H3K27me3. For those genes not affected upon CARM1 depletion (e.g. Hoxa1), Suz12 (representative of the PRC2 complex) and H3K27me3 are rapidly removed after RA addition and are no longer present at the RARE. Co-activators are then recruited to initiate transcription, along with an increase in activating histone marks (e.g. H3K27ac) independent of CARM1’s occupancy (i). Thus, CARM1 is bound at the Hoxa1 RARE +/− RA but does not influence RA-associated transcriptional activation, so CARM1 is not shown. For genes requiring CARM1 for their RA-induced transcriptional activation (i.e. NR2F1, CRABP2), Suz12 (representative of PRC2) and the H3K27me3 mark gradually decrease (NR2F1) or do not change (CRABP2) and are not completely removed with the addition of RA in WT cells. For NR2F1, lack of CARM1 prevents the decrease in Suz12 (representative of PRC2) and the H3K27me3 mark upon RA addition (ii). Lack of CARM1 also increases Suz12 level (representative of PRC2) at the RARE1 of CRABP2, but does not affect the H3K27me3 level. There is also an increase in the H3K27ac level at CRABP2 after RA addition in WT cells, and the absence of CARM1 blocks this increase in H3K27ac (iii). There are no changes in the H3K27ac level at NR2F1 upon RA addition. CARM1 facilitates this differential modulation of epigenetic regulators upon RA treatment to allow RA-induced transcriptional activation of CRABP2 and NR2F1.

Article Snippet: Cells were sonicated and the precleared lysates (25 μg DNA) were immunoprecipitated using 0.5–2.0 μg of antibodies specific for CARM1 (Epicypher, Research Triangle Park, NC, cat# 13–0006, lot# 13281001), H3K27ac (Abcam, cat# 4729, lot# GR28147), H3K27me3 (Abcam, cat# 6002, lot# 2736613), H3R17me2a (Abcam, cat# 8284, lot# {"type":"entrez-nucleotide","attrs":{"text":"GR295369","term_id":"239851048"}} GR295369 –1), Suz12 (Cell Signaling, cat# D39F6, lot# 3), or IgG (Santa Cruz, cat# sc2027, lot# L2414) (negative control).

Techniques: Activation Assay

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

Journal: EMBO Reports

Article Title: The L27 domain of MPP7 enhances TAZ-YY1 cooperation to renew muscle stem cells

doi: 10.1038/s44319-024-00305-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Myc-Carm1 (human cDNA) , Origene , Catalog #RC217483.

Techniques: Recombinant, Modification, Magnetic Beads, Sequencing, Blocking Assay, Plasmid Preparation, Electron Microscopy, Software, Reporter Assay, Imaging, In Situ

Journal: Thorax

Article Title: Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis.

doi: 10.1136/thoraxjnl-2021-217526

Figure Lengend Snippet: Figure 1 LPS and Escherichia coli increases PRMT4 protein expression in lymphocytes in vitro, and PRMT4 is increased in experimental septic models. (A–C) Jurkat cells (A), SKW6.4 cells (B) and THP-1 cells (C) were treated with LPS as indicated, and cell lysates were immunoblotted with PRMT4 and β-actin antibodies. Independent experiments, n=3. (D) Jurkat cells were treated with live E. coli as indicated, and cell lysates were immunoblotted with PRMT4 and β-actin antibodies. Densitometry was plotted in the lower panel. Independent experiments, n=3. (E) Lysates of peripheral blood leucocytes from deidentified human samples with or without sepsis were immunoblotting analysed with PRMT4 and β-actin. (F) PRMT4 protein levels were determined by ELISA from blood plasma from septic patients (n=53) and non-septic control patients (n=53). Lines indicate the median and IQR, Mann-Whitney U test, p=0.0004. (G) CLP procedures were subjected to C57BL/6 J mice for 48 hours; mice sera were collected from untreated controls (n=5) and polymicrobial infected mice (n=10) for PRMT4 ELISA analysis. (H,I) Leucocytes isolated from BALF in LPS-treated mouse were immunofluorescent stained with PRMT4 antibody. PRMT4 expression was visualised using confocal microscopy; the nuclei were stained by DAPI (H). Total cells were counted and positively stained granular and agranular cells were presented as percentage (I). A total of 300 granulocytes and 100 agranulocytes were counted. (J) Isolated CD4+ and CD8+ cells from LPS-treated mouse were lysed and immunoblotting analysed with PRMT4 antibody. Independent experiments, n=3. Scale bar=100 µm. *P=0.05–0.01, **P=0.01–0.001, ***P=0.001–0.0001, ****P<0.0001. BALF, bronchoalveolar lavage fluid; CLP, cecal ligation and puncture; DAPI, (4′,6-diamidino-2-phenylindole); LPS, lipopolysaccharide; PRMT4, protein arginine N-methyltransferase 4.

Article Snippet: The lenti- PRMT4 shRNA was from Origene (Rockville, Maryland, USA). β-actin (cat# A3853) antibody, bacterial LPS) from E. coli O111:B4 (cat# L4391, lot 115M4090V) were from Sigma (Carlsbad, California, USA).

Techniques: Expressing, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Control, MANN-WHITNEY, Infection, Isolation, Staining, Confocal Microscopy, Ligation

Journal: Thorax

Article Title: Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis.

doi: 10.1136/thoraxjnl-2021-217526

Figure Lengend Snippet: Figure 2 PRMT4 gene expression increases on activation in CD4+ T cells. CD4+ cells were isolated from the spleen of a mouse (strain C57BL/6J). The mixture of naïve, unstimulated T cells and CD4 T cells activated with anti-CD3/CD28 comprising a total of 10 000 cells each were applied to single-cell RNA sequencing. UMAP lots as two dimensional were used to plot the expression of CD4-specific genes CD4 (A) and CD3e (B), naïve T cell- specific genes Sell (C) and IL7r (D), CD4+ cell activation increased PRMT4 (E), IL2 (F), IL2ra (G), as well as CD69 (H) gene expression. PRMT4, protein arginine N-methyltransferase 4; UMAP, uniform manifold approximation and projection.

Article Snippet: The lenti- PRMT4 shRNA was from Origene (Rockville, Maryland, USA). β-actin (cat# A3853) antibody, bacterial LPS) from E. coli O111:B4 (cat# L4391, lot 115M4090V) were from Sigma (Carlsbad, California, USA).

Techniques: Gene Expression, Activation Assay, Isolation, RNA Sequencing, Expressing

Journal: Thorax

Article Title: Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis.

doi: 10.1136/thoraxjnl-2021-217526

Figure Lengend Snippet: Figure 3 LPS increases PRMT4 expression and activates caspase 3 in lymphocytes. (A–C) Jurkat cells (A), SKW6.4 cells (B) and THP-1 cells (C) were treated with LPS as indicated. Cell lysates were subjected to immunoblotting for PRMT4, cleaved caspase 3, cleaved caspase 9 and β-actin. The densitometric results were plotted in the lower panels. Independent experiments, n=3. (D,E) Primary mouse splenic lymphocytes (D) and human peripheral blood T cells (E) were treated with LPS as indicated. Cell lysates were analysed by PRMT4, cleaved caspase 3 and β-actin immunoblotting. The plotted data are shown in the lower panels. Independent experiments, n=3. (F) The faecal material from mouse cecum was cultured in an LB plate overnight. Jurkat cells were treated with aforementioned gut-derived live bacteria for 2 hours. Cell lysates were immunoblotting analysed with PRMT4, cleaved caspase 3, cleaved caspase 9 and β-actin. The plotted data are shown in the lower panel. Independent experiments, n=3. *p=0.05– 0.01, **p=0.01–0.001, ***p=0.001–0.0002, **** p=0.0001. LPS, lipopolysaccharide; PRMT4, protein arginine N-methyltransferase 4.

Article Snippet: The lenti- PRMT4 shRNA was from Origene (Rockville, Maryland, USA). β-actin (cat# A3853) antibody, bacterial LPS) from E. coli O111:B4 (cat# L4391, lot 115M4090V) were from Sigma (Carlsbad, California, USA).

Techniques: Expressing, Western Blot, Cell Culture, Derivative Assay, Bacteria

Journal: Thorax

Article Title: Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis.

doi: 10.1136/thoraxjnl-2021-217526

Figure Lengend Snippet: Figure 4 Caspase 3 activation is PRMT4 dependent in lymphocytes. (A,B) Overexpression of PRMT4 increased cleaved caspase 3 baseline levels in Jurkat cells (A) and SKW6.4 cells (B). Relative expression of cleaved caspase 3 was plotted in the lower panel. (C) PRMT4 overexpression does not activate caspase 3 in THP-1 cells. (D) Ectopic expression of PRMT4 enhances LPS-induced caspase 3 activation in Jurkat cells. (E) KO of PRMT4 in Jurkat cells with the CRISPR/Cas9 technique. (F) KO of PRMT4 limits LPS-induced caspase 3 activation. (G) Lentiviral expression of PRMT4 enhances LPS-mediated caspase 3 activation and depletion of PRMT4 by lenti-shPRMT4 reduces cleaved caspase 3 in mouse splenic lymphocytes. Independent experiments, n=3. *P=0.05–0.01, **P=0.01–0.001, ***P=0.001–0.0001. KO, knockout; LPS, lipopolysaccharide; neg, negative; PRMT4, protein arginine N-methyltransferase 4.

Article Snippet: The lenti- PRMT4 shRNA was from Origene (Rockville, Maryland, USA). β-actin (cat# A3853) antibody, bacterial LPS) from E. coli O111:B4 (cat# L4391, lot 115M4090V) were from Sigma (Carlsbad, California, USA).

Techniques: Activation Assay, Over Expression, Expressing, CRISPR, Knock-Out

Journal: Thorax

Article Title: Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis.

doi: 10.1136/thoraxjnl-2021-217526

Figure Lengend Snippet: Figure 5 High protein level of PRMT4 causes lymphocyte death. (A,B) FACS analysis of apoptosis in PRMT4 KO or overexpressed Jurkat cells with or without LPS treatment. Data of (A) were quantitated in (B). (C) Lenti-PRMT4 or shRNA particles were delivered intratracheally into the mouse. Mouse splenic T cells were isolated and treated with LPS for 18 hours, and viable cells were counted. (D) Jurkat cells were treated with LPS and a range of PRMT4 inhibitors as indicated for 3 hours. Cell lysates were analysed for cleaved caspase 3. Relative expression of cleaved caspase 3 in each group is plotted in the lower panel. Independent experiments, n=3. (E) Isolated mouse splenic T cells were treated with LPS and TP064; cleaved caspase 3 was immunoblotting analysed and plotted in the lower panel. Independent experiments, n=3. *P=0.05–0.01, *P=0.01–0.001, ***P=0.001–0.0001. FACS, fluorescence-activated cell sorting; KO, knockout; LPS, lipopolysaccharide; neg, negative; OE, PRMT4 overexpression; PRMT4, protein arginine N-methyltransferase 4; sh, PRMT4 shRNA; Vec, vector.

Article Snippet: The lenti- PRMT4 shRNA was from Origene (Rockville, Maryland, USA). β-actin (cat# A3853) antibody, bacterial LPS) from E. coli O111:B4 (cat# L4391, lot 115M4090V) were from Sigma (Carlsbad, California, USA).

Techniques: shRNA, Isolation, Expressing, Western Blot, Fluorescence, FACS, Knock-Out, Over Expression, Plasmid Preparation

Journal: Thorax

Article Title: Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis.

doi: 10.1136/thoraxjnl-2021-217526

Figure Lengend Snippet: Figure 6 Inhibition of PRMT4 suppresses splenic lymphocyte death in an LPS challenged mouse model. (A) PRMT4 was knocked down or overexpressed by IT administrated lentiviral constructs for 14 D. LPS or PRMT4 inhibitor were given intratracheally) as indicated for 24 hours (n=8). Spleen tissues were stained with TUNEL. (B) TUNEL-positive cells in spleen tissues were quantitated. (C,D) CD4+ lymphocytes were isolated from splenic tissues in aforementioned PRMT4 knockdown or overexpression experiments (A) and analysed with flow cytometry. CD4 was used as a T-cell marker. Percentage of apoptosis was quantitated in (D) (n=3). (E) Survival studies were conducted in the LPS lung injury model; mice were observed for 48 hours (n=10). (F,G) Two-stage meta-analysis was conducted using two independent sets of murine data using LPS-only group as reference: PRMT4+LPS (F) and TP064+LPS (G). The data of shPRMT4 group are not shown because the HR was not computable. Two independent experiments were conducted (n=26, (10, 16)). Scale bar=100 µm. LPS, lipopolysaccharide; PRMT4, protein arginine N-methyltransferase 4.

Article Snippet: The lenti- PRMT4 shRNA was from Origene (Rockville, Maryland, USA). β-actin (cat# A3853) antibody, bacterial LPS) from E. coli O111:B4 (cat# L4391, lot 115M4090V) were from Sigma (Carlsbad, California, USA).

Techniques: Inhibition, Construct, Staining, TUNEL Assay, Isolation, Knockdown, Over Expression, Flow Cytometry, Marker

Journal: Thorax

Article Title: Protein arginine N-methyltransferase 4 (PRMT4) contributes to lymphopenia in experimental sepsis.

doi: 10.1136/thoraxjnl-2021-217526

Figure Lengend Snippet: Figure 7 Inhibition of PRMT4 suppresses splenic lymphocyte death in a polymicrobial sepsis model. (A) CP was performed in PRMT4 knocked down or overexpressed mice (n=8). TP064 (0.2 µg/mouse) was administrated intravenously in one group for 48 hours. Spleen tissues were stained with TUNEL. (B) TUNEL-positive cells in spleen tissues. (C,D) Isolated splenic CD4+ T cells were analysed by flow cytometry (C). CD4 was used as a T-cell marker. The data from (C) are plotted in (D). (E) Survival studies were conducted in the CLP model, and mice were observed for 5 days (n=16). (F–H) Meta-analysis was conducted among two independent sets of murine data using CLP only as reference group: PRMT4+CLP (F), shPRMT4+CLP (G) and TP064+CLP (H). Two independent experiments were conducted (n=26, (10, 16)). Scale bar=100 µm. CLP, cecal ligation and puncture; PRMT4, protein arginine N-methyltransferase 4.

Article Snippet: The lenti- PRMT4 shRNA was from Origene (Rockville, Maryland, USA). β-actin (cat# A3853) antibody, bacterial LPS) from E. coli O111:B4 (cat# L4391, lot 115M4090V) were from Sigma (Carlsbad, California, USA).

Techniques: Inhibition, Staining, TUNEL Assay, Isolation, Flow Cytometry, Marker, Ligation

Journal: American Journal of Physiology - Cell Physiology

Article Title: Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration

doi: 10.1152/ajpcell.00073.2017

Figure Lengend Snippet: Oxidative stress downregulates protein arginine methyltransferase 4 (PRMT4) protein stability. A: time course study of H2O2 in murine lung epithelial (MLE12) cells. MLE12 cells were treated with 200 μM H2O2 for different time durations. The cell lysates were analyzed with PRMT4 and glycogen synthase kinase 3β (GSK-3β) immunoblotting, and β-actin was used as a loading control. B: the densitometry results of A were plotted. C: concentration course study of H2O2 in MLE12 cells. MLE12 cells were treated with diverse concentration of H2O2 for 4 h. PRMT4, GSK-3β, and β-actin immunoblotting was conducted. D: the densitometry results of C were plotted. E: MLE12 cells were treated with 200 μM H2O2 for 4 h, and total RNA was isolated and analyzed with quantitative RT-PCR. Results are representative of 3 independent experiments. *P < 0.05 or **P ≤ 0.01 vs. corresponding control.

Article Snippet: PRMT4 and GSK-3β short hairpin RNA lenti-viral constructs were from Origene (Rockville, MD).

Techniques: Western Blot, Concentration Assay, Isolation, Quantitative RT-PCR

Journal: American Journal of Physiology - Cell Physiology

Article Title: Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration

doi: 10.1152/ajpcell.00073.2017

Figure Lengend Snippet: H2O2-induced PRMT4 instability is via proteasomal degradation. A–F: time and concentration studies of PRMT4 degradation under H2O2. MLE12 cells were treated with H2O2 with a range of concentrations in the absence (A and B) or presence of cycloheximide (CHX) for 4 h (C and D), or cells were treated with CHX for various time durations in the presence of 200 μM H2O2 (E and F). The cell lysates were subjected to PRMT4, GSK-3β, and γ-tubulin immunoblotting analysis. The densitometry results of A, C, and E were plotted, and the half-life of the proteins were calculated (B, D, and F). G: MLE12 cells were treated with diverse concentrations of H2O2 (0, 100, 200, and 300 μM) in the presence of MG132 (20 μM) for 4 h. Cell lysates were subjected to PRMT4, GSK-3β, and γ-tubulin immunoblotting analysis. H: the densitometry results of G were plotted. Results are representative of 3 independent experiments *P < 0.05 or **P ≤ 0.01 vs. corresponding control.

Article Snippet: PRMT4 and GSK-3β short hairpin RNA lenti-viral constructs were from Origene (Rockville, MD).

Techniques: Concentration Assay, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration

doi: 10.1152/ajpcell.00073.2017

Figure Lengend Snippet: GSK-3β interacts with PRMT4 and catalyzes PRMT4 phosphorylation at T132. A: cell lysates were applied with PRMT4 immunoprecipitation (IP) study. PRMT4 precipitates were analyzed with GSK-3β and PRMT4 antibodies subsequently. B and C: GSK-3β in vitro phosphorylation of PRMT4 at T132. Wild-type (WT), T132A, and S136A PRMT4 mutant recombinants were synthesized with TnT expression system and in vitro phosphorylation assay was conducted with enzymatic active GSK-3β. Samples were analyzed with phospho-threonine antibody (B) or phospho-serine antibody (C). The GSK-3β signal in IgG lanes in B and C may be from TnT in vitro protein synthetic system. D: PRMT4 was expressed in GSK-3β-silenced cells. V5 immunoprecipitates were analyzed with phosphor-threonine and V5 immunoblotting. The inputs were analyzed with GSK-3β and β-actin immunoblotting. E: WT and T132A mutant PRMT4 were expressed in MLE12 cells, respectively. V5 immunoprecipitates were analyzed with phospho-threonine. Inputs were analyzed with V5 immunoblotting. F: T132A mutant PRMT4 was coexpressed with GSK-3β in MLE12 cells for 48 h. V5 immunoprecipitates were analyzed with phospho-threonine and V5 immunoblotting, respectively. The inputs were analyzed with GSK-3β and β-actin immunoblotting. Results are representative of 3 independent experiments.

Article Snippet: PRMT4 and GSK-3β short hairpin RNA lenti-viral constructs were from Origene (Rockville, MD).

Techniques: Immunoprecipitation, In Vitro, Mutagenesis, Synthesized, Expressing, Phosphorylation Assay, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration

doi: 10.1152/ajpcell.00073.2017

Figure Lengend Snippet: T132 phosphorylation status determines PRMT4 ubiquitination. A and B: WT and T132A mutant PRMT4 were coexpressed with GSK-3β for 48 h. V5 immunoprecipitates were analyzed with ubiquitin immunoblotting. C and D: WT, T132C, and T132D mutant PRMT4 were introduced into MLE12 cells for 48 h, respectively. V5 immunoprecipitates were analyzed with ubiquitin and V5 immunoblotting. Results are representative of 3 independent experiments. *P < 0.05 or **P ≤ 0.01 vs. corresponding control.

Article Snippet: PRMT4 and GSK-3β short hairpin RNA lenti-viral constructs were from Origene (Rockville, MD).

Techniques: Mutagenesis, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration

doi: 10.1152/ajpcell.00073.2017

Figure Lengend Snippet: GSK-3β regulates PRMT4 protein stability. Empty vectors (A), GSK-3β expression (C), scramble (E), and shRNA (G) constructs were introduced into MLE12 cells, respectively. Cells were then treated with protein synthesis inhibitor CHX (20 μM) for different time intervals as indicated. PRMT4, GSK-3β, and β-actin immunoblotting was conducted. B, D, F, and H: the densitometry results of A, C, E, and G were plotted, respectively. Results are representative of 3 independent experiments. **P ≤ 0.01 vs. corresponding control.

Article Snippet: PRMT4 and GSK-3β short hairpin RNA lenti-viral constructs were from Origene (Rockville, MD).

Techniques: Expressing, shRNA, Construct, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration

doi: 10.1152/ajpcell.00073.2017

Figure Lengend Snippet: GSK-3β rescues oxidative stress-decreased PRMT4 at protein level. Empty vector (A) or GSK-3β constructs (C) were introduced into MLE12 cells for 48 h. H2O2 was added into the cells at variant amounts as indicated. Cell lysates were immunoblotting analyzed with PRMT4, GSK-3β, and γ-tubulin antibodies, respectively. B and D: the densitometry results of A and C were plotted. C: GSK-3β protein were ectopically expressed in MLE12 cells for 48 h. Results are representative of 3 independent experiments. *P < 0.05 or **P ≤ 0.01 vs. corresponding control.

Article Snippet: PRMT4 and GSK-3β short hairpin RNA lenti-viral constructs were from Origene (Rockville, MD).

Techniques: Plasmid Preparation, Construct, Variant Assay, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration

doi: 10.1152/ajpcell.00073.2017

Figure Lengend Snippet: Oxidative stress inhibits PRMT4-mediated lung epithelial cell migration. A: PRMT4 was ectopically expressed or knocked down with PRMT4-specific shRNA constructs in MLE12 cells for 24 h. Wound-healing assay was conducted as described in materials and methods. Images were acquired with light microscopy. Scar bar = 400 μm. B: the results of A were plotted in a bar graph. C: the cells used in A were analyzed with PRMT4 immunoblotting. D: PRMT4 overexpressed and knockdown cells were treated with 50 or 100 μM H2O2 in Transwells for 18 h. Migrated cells were assayed and the percentage of migration was plotted in a bar graph. E and F: above cells were analyzed with PRMT4 immunoblotting. G and H: MLE12 cells were transfected with, GSK-3β, shRNA against GSK-3β, or PRMT4 constructs as indicated. G and H: immunoblotting analysis (G) and Transwell assay (H) were conducted in the presence of 100 μM H2O2. Results are representative of 3 independent experiments. *P < 0.05 or **P ≤ 0.01 vs. corresponding control.

Article Snippet: PRMT4 and GSK-3β short hairpin RNA lenti-viral constructs were from Origene (Rockville, MD).

Techniques: Migration, shRNA, Construct, Wound Healing Assay, Light Microscopy, Western Blot, Transfection, Transwell Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Oxidative stress destabilizes protein arginine methyltransferase 4 via glycogen synthase kinase 3β to impede lung epithelial cell migration

doi: 10.1152/ajpcell.00073.2017

Figure Lengend Snippet: Schematic presentation of oxidative stress modulation of cell migration via GSK-3β/PRMT4 signaling. GSK-3β phosphorylates T132 within PRMT4 to stabilize PRMT4 protein from ubiquitin proteasomal degradation. Oxidative stress downregulates GSK-3β, which leads to PRMT4 degradation. Decreased PRMT4 protein impedes cell migration, which may affect repair and regeneration process after lung injury.

Article Snippet: PRMT4 and GSK-3β short hairpin RNA lenti-viral constructs were from Origene (Rockville, MD).

Techniques: Migration

Journal: Journal of Medicinal Chemistry

Article Title: Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction–Reconstruction and Fragment-Growing Approach

doi: 10.1021/acs.jmedchem.2c00252

Figure Lengend Snippet: Inhibitory activities of compounds 12a – 12h : the heatmaps depict the IC 50 values (nM) for compounds 12a – 12h (top panel) and the selectivity index (fold) for PRMT4 over the specified PRMT (bottom).

Article Snippet: Full-length recombinant PRMT4 (Active motif, # 81107) was diluted at the concentration of 50 μg/mL in 10 mM sodium acetate, pH 4.5, and then immobilized on a Series S Sensor Chip CM5 at a flow rate of 10 μL/min by using standard amine-coupling protocols to obtain densities of 4.6 kRU.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction–Reconstruction and Fragment-Growing Approach

doi: 10.1021/acs.jmedchem.2c00252

Figure Lengend Snippet: Sensorgrams obtained from the SPR interaction analysis of compounds 12a and 12f – 12h (panels a–d, respectively) binding to immobilized PRMT4. Each compound was injected at different concentrations (from 3 to 0.05 μM for 12a and from 640 to 10 nM for 12f – 12h ) with an association and a dissociation time of 90 s, with a flow rate of 30 μL/min. The equilibrium dissociation constants ( K D ) were derived from the ratio between kinetic dissociation ( k off ) and association ( k on ) constants.

Article Snippet: Full-length recombinant PRMT4 (Active motif, # 81107) was diluted at the concentration of 50 μg/mL in 10 mM sodium acetate, pH 4.5, and then immobilized on a Series S Sensor Chip CM5 at a flow rate of 10 μL/min by using standard amine-coupling protocols to obtain densities of 4.6 kRU.

Techniques: Binding Assay, Injection, Derivative Assay

Journal: Journal of Medicinal Chemistry

Article Title: Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction–Reconstruction and Fragment-Growing Approach

doi: 10.1021/acs.jmedchem.2c00252

Figure Lengend Snippet: Electron density (2 F obs – F calc ) weighted maps. Compound 12b (a) and compound 12h (b) bound to subunit B of mm PRMT4 (PDB IDs: 7PV6 and 7PUC ). PRMT4 is represented as a gray cartoon, and compounds are represented as cornflower blue sticks. Maps are represented as a mesh, with the contouring level set to 1σ. For clarity, N-terminal helices (residues 135–155) of PRMT4 are not shown. E258 and E267 belonging to the double-E loop and H415 of the THW loop are also displayed as sticks.

Article Snippet: Full-length recombinant PRMT4 (Active motif, # 81107) was diluted at the concentration of 50 μg/mL in 10 mM sodium acetate, pH 4.5, and then immobilized on a Series S Sensor Chip CM5 at a flow rate of 10 μL/min by using standard amine-coupling protocols to obtain densities of 4.6 kRU.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction–Reconstruction and Fragment-Growing Approach

doi: 10.1021/acs.jmedchem.2c00252

Figure Lengend Snippet: Structures of mm PRMT4 in complex with compounds 12a – 12c and 12f – 12h (PDB IDs: 7PV6, 7PPY , 7PPQ , 7PU8 , 7PUQ , and 7PUC, respectively). (a) Superimposition (done on protein backbones) of compounds ( 12a , 12b , 12c , 12f , 12g , and 12h ) bound to subunit B of mm PRMT4. Each PRMT4 subunit is represented as a cartoon (shades of gray, lime, cyan, marine, yellow, gray, and pink ribbons), and compounds are represented as sticks (in lime, yellow, cyan, cornflower blue, sea blue, and pink, respectively). (b) Close-up view of bound compound conformations. (c) Binding interactions of compound 12a (lime sticks) with mm PRMT4 monomer B (ribbon). (d) Binding interactions of compound 12h (pink sticks) with mm PRMT4 monomer B (ribbon). Hydrogen bonds are shown as dashed lines. For clarity, N -terminal helices (residues 135–165) of PRMT4 are not shown.

Article Snippet: Full-length recombinant PRMT4 (Active motif, # 81107) was diluted at the concentration of 50 μg/mL in 10 mM sodium acetate, pH 4.5, and then immobilized on a Series S Sensor Chip CM5 at a flow rate of 10 μL/min by using standard amine-coupling protocols to obtain densities of 4.6 kRU.

Techniques: Binding Assay

Journal: Journal of Medicinal Chemistry

Article Title: Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction–Reconstruction and Fragment-Growing Approach

doi: 10.1021/acs.jmedchem.2c00252

Figure Lengend Snippet: (a) Electron density (2 F obs – F calc ) weighted maps of compound 12a (represented as cornflower blue sticks) bound to subunit B of mm PRMT6 (represented as a gray cartoon; PDB ID: 7NUD ). Maps are represented as a mesh, with the contouring level set to 1σ. For clarity, N-terminal helices (residues 42–56) of mm PRMT6 are not shown. (b) Superimposition (done on protein backbones) of the conformations of compound 12a (yellow or cornflower blue sticks, respectively) when bound to subunit B of mm PRMT4 (represented as a dark gray cartoon) and when bound to mm PRMT6 (represented as a light gray cartoon). For clarity, N-terminal helices of PRMT4 and PRMT6 are not shown.

Article Snippet: Full-length recombinant PRMT4 (Active motif, # 81107) was diluted at the concentration of 50 μg/mL in 10 mM sodium acetate, pH 4.5, and then immobilized on a Series S Sensor Chip CM5 at a flow rate of 10 μL/min by using standard amine-coupling protocols to obtain densities of 4.6 kRU.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction–Reconstruction and Fragment-Growing Approach

doi: 10.1021/acs.jmedchem.2c00252

Figure Lengend Snippet: Cellular effects of compounds 12f – 12h . (a, b) The viability of HEK293T cells was assessed by measuring the mitochondrial-dependent reduction of MTT to formazan, with respect to DMSO, after treatment with compounds 12f – 12h at three different concentrations (10, 50, and 100 μM) for (a) 24 h and (b) 72 h. Data are reported as the mean ± SD of four independent experiments. (c, d) Western blot analyses were performed (a) on lysates from HEK293T cells after treatment with compounds 12f – 12h at 10 and 50 μM for 24 h and (d) on lysates from MCF7 cells after treatment with compound 12h at 10 and 50 μM for 4 and 8 days. Methylation was detected by immunoblotting with a pan-PRMT4 substrate antibody (PRMT4 sub ; see the main text). Total histone H3 (c) or actin (d) was used to check for equal loading. The cell-permeable PRMT4 inhibitor TP064 (10 μM) was used as a reference compound. (e, f) Relative proliferation of (e) HEK293T and (f) MCF7 cells with different concentration of 12h for different time points. The medium was changed at day 4. All the data points represent the relative viability normalized to day 0. The error bars represent the standard deviation of three biological replicates performed at each time point.

Article Snippet: Full-length recombinant PRMT4 (Active motif, # 81107) was diluted at the concentration of 50 μg/mL in 10 mM sodium acetate, pH 4.5, and then immobilized on a Series S Sensor Chip CM5 at a flow rate of 10 μL/min by using standard amine-coupling protocols to obtain densities of 4.6 kRU.

Techniques: Western Blot, Methylation, Concentration Assay, Standard Deviation

Journal: Journal of Medicinal Chemistry

Article Title: Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction–Reconstruction and Fragment-Growing Approach

doi: 10.1021/acs.jmedchem.2c00252

Figure Lengend Snippet: Inhibitory Activities of Compounds 12a – 12h against Various PRMTs

Article Snippet: Full-length recombinant PRMT4 (Active motif, # 81107) was diluted at the concentration of 50 μg/mL in 10 mM sodium acetate, pH 4.5, and then immobilized on a Series S Sensor Chip CM5 at a flow rate of 10 μL/min by using standard amine-coupling protocols to obtain densities of 4.6 kRU.

Techniques:

Journal: Journal of Medicinal Chemistry

Article Title: Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction–Reconstruction and Fragment-Growing Approach

doi: 10.1021/acs.jmedchem.2c00252

Figure Lengend Snippet: X-ray Data Collection and Refinement Statistics for PRMT4 Complexes with Compounds 12a – 12c and 12f – 12h

Article Snippet: Full-length recombinant PRMT4 (Active motif, # 81107) was diluted at the concentration of 50 μg/mL in 10 mM sodium acetate, pH 4.5, and then immobilized on a Series S Sensor Chip CM5 at a flow rate of 10 μL/min by using standard amine-coupling protocols to obtain densities of 4.6 kRU.

Techniques: Solvent, Biomarker Discovery