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: LEDGF Binds H3R17me2a Promoting De Novo Nucleotide Biosynthesis in SETD2 Mutant Clear Cell Renal Cell Carcinoma.

doi: 10.1002/advs.202416809

Figure Lengend Snippet: Figure 4. CARM1 is highly expressed in ccRCC and promotes proliferation. A,B) CARM1 is highly expressed in ccRCC patients and is correlated with poor prognosis on the UALCAN database. C) CARM1 and H3R17me2a are highly expressed in ccRCC cells such as 786-O, A498, and Caki-1. D,E) Immunofluorescence staining using the tissue microarray revealed that CARM1 is highly expressed in ccRCC tissue samples. Scale bar = 100 μm. (Tumor n = 79, Normal n = 79) F) CcRCC patients with advanced T stage have higher CARM1 expression, which may be associated with poor prognosis.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Abcam, ab177159), CARM1 (CST, #3379), H3R17me2a (Acive motif, #39 710), PRMT6 (Abcam, ab271091), H3K36me3 (CST, #4909), PPAT (Proteintech, #15401- 1-AP), PAICS (Proteintech, #12967-1-AP), GART (Proteintech, #13659-1- AP), ADSL (Proteintech, #15264-1-AP), ADSS2 (Proteintech, #16373-1- AP), Histone H3 (Proteintech, #17168-1-AP), Alpha Actin (Proteintech, #23660-1-AP), Flag (Abmart, #M20008), Goat Anti-Rabbit IgG (H + L) (Proteintech,#SA00001-2), and Goat Anti-Mouse IgG (H + L) (Proteintech, #SA00001-1).

Techniques: Staining, Microarray, Expressing

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

Article Title: LEDGF Binds H3R17me2a Promoting De Novo Nucleotide Biosynthesis in SETD2 Mutant Clear Cell Renal Cell Carcinoma.

doi: 10.1002/advs.202416809

Figure Lengend Snippet: Figure 5. Construction of the H3R17me2a-deficient cell lines. A,B) Validation of knockout efficiency of CARM1 and the reduction of H3R17me2a level in transduced pooled A498 cells (A) and 786-O cells (B). C–E) Validation of knockout efficiency of CARM1 and the reduction of H3R17me2a level in monoclonal A498 cells (C) and 786-O cells (D-E). F,G) Knockdown of PRMT6 using siRNA has little effect on H3R17me2a level in A498 cells (F) and 786-O cells (G). H) The combined knockdown of CARM1 and PRMT6 using siRNAs reduces H3R17me2a level in A498 cells; knockdown of PRMT6 in CARM1-KO cells significantly decreased H3R17me2a in A498 cells. I) Knockdown of PRMT6 in CARM1-KO cells significantly decreased H3R17me2a in A498 cells. J) The combination of CARM1 (MCE, EZM2302 and TP-064) and PRMT6 (MCE, EPZ020411) inhibitors can significantly reduce H3R17me2a level in A498 cells.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Abcam, ab177159), CARM1 (CST, #3379), H3R17me2a (Acive motif, #39 710), PRMT6 (Abcam, ab271091), H3K36me3 (CST, #4909), PPAT (Proteintech, #15401- 1-AP), PAICS (Proteintech, #12967-1-AP), GART (Proteintech, #13659-1- AP), ADSL (Proteintech, #15264-1-AP), ADSS2 (Proteintech, #16373-1- AP), Histone H3 (Proteintech, #17168-1-AP), Alpha Actin (Proteintech, #23660-1-AP), Flag (Abmart, #M20008), Goat Anti-Rabbit IgG (H + L) (Proteintech,#SA00001-2), and Goat Anti-Mouse IgG (H + L) (Proteintech, #SA00001-1).

Techniques: Biomarker Discovery, Knock-Out, Knockdown

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

Article Title: LEDGF Binds H3R17me2a Promoting De Novo Nucleotide Biosynthesis in SETD2 Mutant Clear Cell Renal Cell Carcinoma.

doi: 10.1002/advs.202416809

Figure Lengend Snippet: Figure 8. Deficiency of LEDGF protects NKG mice against xenograft proliferation. A) Schematic diagram of subcutaneous tumor model in NKG mice in indicated treatment groups. All surviving mice were euthanized 8 weeks after tumor cell inoculation. B) Knock out of LEDGF effectively reduced the proliferation of xenografts in NKG mice. (n = 5) C) There was no significant difference in body weight between the two groups throughout the experiment. D–F) Elimination of LEDGF effectively reduced the volume (D-E) and weight (F) of NKG mice xenografts. G) QRT-PCR was used to demonstrate that decrease of LEDGF can reduce mRNA expression of PPAT, PAICS, GART, ADSL, and ADSS2 in xenograft tumors. H) The proliferation ability of xenografts in LEDGF-KO group was significantly reduced. The expression levels of PPAT, PAICS, GART, and ADSL were significantly decreased, while ADSS2 expres- sion was almost unchanged. Scale bar = 100 μm. I) A schematic model illustrating that LEDGF interacts with CARM1-mediated H3R17me2a to promote ccRCC progression. Data are shown as mean ± SD. ***p < 0.001. ns means no significance.

Article Snippet: The antibodies used in this study are as follows: LEDGF (Abcam, ab177159), CARM1 (CST, #3379), H3R17me2a (Acive motif, #39 710), PRMT6 (Abcam, ab271091), H3K36me3 (CST, #4909), PPAT (Proteintech, #15401- 1-AP), PAICS (Proteintech, #12967-1-AP), GART (Proteintech, #13659-1- AP), ADSL (Proteintech, #15264-1-AP), ADSS2 (Proteintech, #16373-1- AP), Histone H3 (Proteintech, #17168-1-AP), Alpha Actin (Proteintech, #23660-1-AP), Flag (Abmart, #M20008), Goat Anti-Rabbit IgG (H + L) (Proteintech,#SA00001-2), and Goat Anti-Mouse IgG (H + L) (Proteintech, #SA00001-1).

Techniques: Knock-Out, Quantitative RT-PCR, Expressing

Journal: The Journal of Neuroscience

Article Title: Arginine Methyltransferase 1 in the Nucleus Accumbens Regulates Behavioral Effects of Cocaine

doi: 10.1523/JNEUROSCI.0246-15.2015

Figure Lengend Snippet: Comprehensive list of all primer sequences used

Article Snippet: Chemiluminescent assay to establish IC 50 values Chemiluminescent Assay Kits [PRMT3 (52005L), PRMT4 (52041L), PRMT5 (52002L), PRMT6 (52046), and PRMT8 (52058), BPS Bioscience) were used to determine the IC 50 of compounds.

Techniques: Sequencing

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: 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: ( A ) Flowchart for bulk RNA-sequencing (RNA-seq). ( B ) PCA analysis of transcriptome data of Con ( Pax7 CreERT2 /+ ), Mpp7 cKO, Amot cKO MuSCs. ( C ) Venn diagram summarizes (35) overlapping DEGs between the Mpp7 cKO (58 DEGs) and the Amot cKO (66 DEGs). ( D ) Hierarchical clustering and heatmap of RNA-seq expression z-scores for the 35 overlapping DEGs; red asterisk, Carm1 . ( E ) Expression changes (log2 FC, log2 fold change) of genes in GO-term enriched pathways (y-axis) are displayed for the Mpp7 cKO (grey bars) and the Amot cKO (black bars). ( F , G ) Representative IF of CARM1 in Con (as in Fig. ), Mpp7 cKO and Amot cKO MuSCs at 48 h in culture ( F ) and relative CARM1 fluorescent signals (in AU, G ). ( H ) Expressing a Myc-tagged Carm1 rescued the Mpp7 cKO in SM culture; Con (−) and Mpp7 cKO (−) from Fig. ; IF of Myc, PAX7, and MYOD was performed to determine transfected cells’ fates. Quantification of cell fate fractions is shown; keys at top. ( I ) V5-Mpp7 activated a Carm1-reporter (a luciferase reporter driven by a promoter region (−630 to +15) of Carm1 , depicted at the top) in 293T cells; (−), empty expression construct. RNA-seq data deposit (NCBI) and analyses are in Methods. Data information: Scale bar = 25 µm in ( F ). ( G ) 200 MuSCs from 2 to 3 mice in each group; ( H ) ≥ 530 cells in each group; ( I ) n = 3 biological replicates. Bars represent medians ± 95% CI in ( G ) or means ± SD in ( I ). Hypergeometric test was used in ( C ). Kruskal–Wallis test followed by Dunn’s multiple comparisons test was used in ( G ), Chi-square test in ( H ), and Student’s t test (two-sided) in ( I ). .

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

Techniques: RNA Sequencing Assay, Expressing, Transfection, Luciferase, Construct

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: ( A ) H&E histology of Yap cKO and YapTaz cKO muscles at 5 dpi (Con ( Pax7 CreERT2/+ ) histology not included); quantifications of regenerated myofiber cross-sectional area to the right. ( B ) Venn diagram shows overlapping DEGs between the YapTaz cKO and the Mpp7 cKO. ( C , D ) IF images of TAZ ( C ) and YAP ( D ) in FACS-isolated and cultured Con ( Pax7 CreERT2/+ ), Mpp7 cKO, and Amot cKO MuSCs at 48 h; relative fluorescent signals (in AU) to the right. ( E ) Co-IP of V5-TAZ and HA-AMOT by FLAG-MPP7 expressed in 293 T cells; tagged epitopes used for IP and Western blotting are indicated; (−), empty expression construct. Quantification of relative levels of co-IPed V5-TAZ is to the right. ( F ) Relative TEAD-reporter (8XGTIIC-luciferase, depicted at top) activities when co-transfected with V5-Taz, Flag-Mpp7, and/or Ha-Amot constructs in 293T cells; (−), empty expression construct. ( G ) Relative activities of WT and TEAD-binding site mutated (MUT) Carm1-reporters co-transfected with V5-Taz or Flag-Mpp7 constructs; (−), empty expression construct. ( H ) Relative IF signals (AU) of CARM1 in Mpp7 cKO MuSCs transfected with empty vector (−), V5-Taz WT and V5-Taz S89A constructs (all with IRES-mGFP) ( I ) Relative cell fate fractions among Con ( Pax7 CreERT2/+ ), Mpp7 cKO, and Mpp7 cKO MuSCs transfected with V5-Taz S89A and V5-Taz WWm construct in single myofiber culture; Con (−) and Mpp7 cKO (−) from Fig. ; keys at the top. Data information: Scale bar = 50 µm in ( A ) and 25 µm in ( C , D ). ( A ) N = 5 mice in each group; ( C , D , H ) 200 cells from 2 to 3 mice in each group; ( E – G ) n = 3 biological experiments; ( I ) ≥ 150 cells in each group. Bars represent medians ± 95% CI ( C , D , H ) or means ± SD ( A , E , F , G ). Hypergeometric test was used in ( B ), one-way ANOVA with Tukey’s post hoc test performed in ( A , E , F , G ), Kruskal–Wallis test followed by Dunn’s multiple comparisons test in ( C , D , H) , and Chi-square test used in ( I ). .

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

Techniques: Muscles, Isolation, Cell Culture, Co-Immunoprecipitation Assay, Western Blot, Expressing, Construct, Luciferase, Transfection, Binding Assay, Plasmid Preparation

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: ( A ) Promoters of 4 common DEGs among Mpp7, Amot, YapTaz , and Yy1 cKO (Chen et al, ) data sets. Putative YY1 binding sites are red blocks and TEAD binding sites, blue blocks. ( B ) Location and sequence of the TEAD and YY1 binding sites are indicated in the Carm1 promoter. Carm1-reporter was co-transfected with constructs in x -axis to assess transcriptional activity; (−), empty expression construct. ( C ) Same as in ( B ), except that the YY1 binding site is mutated (top). ( D ) Same as in ( B ), except that the TEAD binding site was mutated (top). ( E ) Co-IP between TAZ and YY1 is enhanced by fusing MPP7’s L27 domain to TAZ in 293T cells. Constructs and tagged epitopes for detection are indicated; quantification of co-IPed HA-YY1 to the right. ( F ) Co-IP assay to determine the relative contributions of L27 and PDZ domains of MPP7 to its interaction with YY1 in 293T cells. Constructs and tagged epitopes for detection are indicated; quantification of co-IPed HA-YY1 to the right. ( G ) A model for TAZ-YY1 cooperation mediated by AMOT and MPP7. S175 of AMOT is subjected to phosphorylation and regulation by actin dynamics. Data information: ( B – F ) n = 3 biological replicates. Error bars represent means ± SD. One-way ANOVA with Tukey’s post hoc test was performed in ( B – F ). .

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

Techniques: Binding Assay, Sequencing, Transfection, Construct, Activity Assay, Expressing, Co-Immunoprecipitation Assay

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: ( A ) ChIP-qPCR shows MPP7, YY1, and TAZ binding to Carm1 promoter (diagramed a top) at site 1 (red) harboring both TEAD and YY1 binding sites, but not at a distal site 2 (blue) without either binding site; IgG, negative control. Bar graph shows fold-enrichment (by Bio-Rad CFX Maestro); keys to the left. ( B ) Same as in ( A ) with 6 more common DEG promoters (x-axis) with YY1 and TEAD binding sites; keys and quantification as in ( A ). ( C ) Representative PLA images for MPP7 and YAP/TAZ in 3D reconstruction and single plane, countered stained with DAPI; quantification to the right. ( D ) Same as in ( C ) for MPP7 and YY1.( E ) Same as in ( C ) for YY1 and YAP/TAZ in Con ( Pax7 CreERT2/+ ) and Mpp7 cKO. ( F ) Same as in ( E ) for CARM1-PAX7; ( G ) Restoring CARM1-PAX7 PLA signal by L27-TAZ but not by empty vector (EV, with IRES-mGFP, same as (−) in previous figures). Each data point in ( C – G ) represents PLA dots in a cell quantified by 3D imaging. Single Ab controls are shown in Appendix Fig. S . Data information: Scale bars = 5 µm ( C – G ). ( A , B ) n = 3 biological replicates. ( C ) 5 MuSCs for each group; ( D ) 5 MuSCs for MPP7 or YY1 and 8 MuSCs for MPP7-YY1; ( E ) 5 MuSCs for Con ( Pax7 CreERT2/+ ) and 7 MuSCs for Mpp7 cKO; ( F ) 15 MuSCs for Con and 16 MuSCs for Mpp7 cKO; (G) 11 MuSCs for EV and 19 MuSCs for L27-Taz. Error bars represent means ± SD. One-way ANOVA with Tukey’s post hoc test in ( A – D ). Student’s t test (two-sided) in ( E – G) . .

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

Techniques: Binding Assay, Negative Control, Staining, Plasmid Preparation, Imaging

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: International Journal of Medical Sciences

Article Title: PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis

doi: 10.7150/ijms.62467

Figure Lengend Snippet: Relationship between the PRMT4 expression and the clinicopathologic features of HCC.

Article Snippet: Lentiviral short hairpin RNA (shRNA) targeting PRMT4 (GeneChem, Inc., Shanghai) was inserted into the pLKO.1-TRC vector.

Techniques: Expressing

Journal: International Journal of Medical Sciences

Article Title: PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis

doi: 10.7150/ijms.62467

Figure Lengend Snippet: Univariate and multivariate cox regression analyses of overall survival.

Article Snippet: Lentiviral short hairpin RNA (shRNA) targeting PRMT4 (GeneChem, Inc., Shanghai) was inserted into the pLKO.1-TRC vector.

Techniques: Expressing

Journal: International Journal of Medical Sciences

Article Title: PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis

doi: 10.7150/ijms.62467

Figure Lengend Snippet: Univariate and multivariate cox regression analyses of disease-free survival.

Article Snippet: Lentiviral short hairpin RNA (shRNA) targeting PRMT4 (GeneChem, Inc., Shanghai) was inserted into the pLKO.1-TRC vector.

Techniques: Expressing

Journal: International Journal of Medical Sciences

Article Title: PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis

doi: 10.7150/ijms.62467

Figure Lengend Snippet: PRMT4 overexpression promotes proliferation, migration, and invasion of HCC cells in vitro . (A) Western blots showing the overexpression of PRMT4 in YY-8103 cells. (B) The effect of PRMT4 overexpression on the proliferation of YY-8103 was measured by CCK-8 assays. (C) The effect of PRMT4 overexpression on the proliferation of YY-8103 cells was detected by crystal violet assays. (D) The OD value of crystal violet assays in YY-8103 cells. (E) The effect of PRMT4 overexpression on the migration and invasion of YY-8103 cells was detected by Transwell assays (×400 magnification). (F) Calculation of cells that migrated and invaded through the filter in YY-8103 cells. Measurement data were expressed as mean ± standard deviation (SD), and the experiments were repeated at least 3 times. ** P < 0.01, *** P < 0.001 vs Vector group.

Article Snippet: Lentiviral short hairpin RNA (shRNA) targeting PRMT4 (GeneChem, Inc., Shanghai) was inserted into the pLKO.1-TRC vector.

Techniques: Over Expression, Migration, In Vitro, Western Blot, CCK-8 Assay, Standard Deviation, Plasmid Preparation

Journal: International Journal of Medical Sciences

Article Title: PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis

doi: 10.7150/ijms.62467

Figure Lengend Snippet: PRMT4 knockdown inhibits proliferation, migration, and invasion of HCC cells in vitro . (A) Western blots showing the downregulation of PRMT4 in Huh7 cells. (B) The effect of PRMT4 knockdown on the proliferation of Huh7 was measured by CCK-8 assays. (C) The effect of PRMT4 knockdown on the proliferation of Huh7 cells was detected by crystal violet assays. (D) The OD value of crystal violet assays in Huh7 cells. (E) The effect of PRMT4 knockdown on the migration and invasion of Huh7 cells was detected by Transwell assays (×400 magnification). (F) Calculation of cells that migrated and invaded through the filter in Huh7 cells. Measurement data were expressed as mean ± SD, and the experiments were repeated at least 3 times. ** P < 0.01, *** P < 0.001 vs SCR group. SCR, scrambled; shRNA, short hairpin RNA.

Article Snippet: Lentiviral short hairpin RNA (shRNA) targeting PRMT4 (GeneChem, Inc., Shanghai) was inserted into the pLKO.1-TRC vector.

Techniques: Knockdown, Migration, In Vitro, Western Blot, CCK-8 Assay, shRNA

Journal: International Journal of Medical Sciences

Article Title: PRMT4 promotes hepatocellular carcinoma progression by activating AKT/mTOR signaling and indicates poor prognosis

doi: 10.7150/ijms.62467

Figure Lengend Snippet: PRMT4 positively regulates the AKT/mTOR signaling pathway . (A) The expression levels of AKT, p-AKT, mTOR, p-mTOR, RPS6, p-RPS6, 4E-BP1 and p-4E-BP1 were detected by western blot in stable cell lines. (B) The inhibition of the AKT/mTOR pathway abolished the cell proliferation mediated by PRMT4 overexpression, as indicated by CCK-8 assays. (C) The inhibition of the AKT/mTOR pathway abolished the cell migration and invasion mediated by PRMT4 overexpression, as indicated by Transwell assays (×400 magnification). (D) Calculation of cells that migrated and invaded through the filter in YY-8103 cells. Measurement data were expressed as mean ± SD, and the experiments were repeated at least 3 times. *P < 0.05, **P < 0.01vs PRMT4 group.

Article Snippet: Lentiviral short hairpin RNA (shRNA) targeting PRMT4 (GeneChem, Inc., Shanghai) was inserted into the pLKO.1-TRC vector.

Techniques: Expressing, Western Blot, Stable Transfection, Inhibition, Over Expression, CCK-8 Assay, Migration