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: Molecules and cells

Article Title: Recombinant Human HAPLN1 Mitigates Pulmonary Emphysema by Increasing TGF-β Receptor I and Sirtuins Levels in Human Alveolar Epithelial Cells.

doi: 10.14348/molcells.2023.0097

Figure Lengend Snippet: Fig. 5. rhHAPLN1 reduced significantly the length of MLI of alveoli as an indicator of the mouse PPE-induced COPD model. Male C57BL6/N mice aged 6 to 10 weeks were used as the experimental model. Mice were divided into four groups with six mice in each group in each of two independent experiments. In the 1st experiment, the groups were Normal, Control, rhHAPLN1 (0.00033% w/v), and high molecular weight hyaluronan (HMW-HA) groups (0.0057% w/v) (A and B), and in the 2nd experiment, groups were Normal, Control, rhHAPLN1-(a) (0.0001% w/v), and rhHAPLN1-(b) (0.0005% w/v) (C and D). In each experiment, to induce COPD in the three groups except Normal, on the day before the exposure to the test samples, PPE (30 µl/6 Unit/time) was injected into the oral airway with a 0.9 mm × 50 mm-oral zonde as a method of alveolar inhalation, and rhHAPLN1 or HMW-HA solubilized in vehicle saline was administered on the second day for 1 h daily and five times per week for 21 days, and then mice were euthanized after 1 day of the final exposure. The lungs were harvested, parasagittal sectioned, immersed in formalin, and paraffin blocks were made. The lung tissues were cut into 5 µm and stained with H&E. Pictures of representative histological sections of lungs were taken with a 200× optical microscope (A and C). MLI (mean ± SEM) of alveolar septae were measured in the lungs of six Normal mice, Control group of six saline-treated mice, and the rhHAPLN1-treated group of six mice. The Stitcher ImageJ plugin program was used (Preibisch et al., 2009) (B and D). Compared with those of each Control group, the results from 1st experiment showed that rhHAPLN1 significantly reduced the enlargement of alveoli as an indicative of emphysema: 0.00033% rhHAPLN1 group by 95% [(21.2 – 13.8)/21.2 – 14.5) × 100]; 0.0057% HMW-HA by 73.0% [(21.2 – 13.8)/21.2 – 15.8) × 100] (B), while 2nd experiment showed 0.0001% rhHAPLN1-(a) by no significance; 0.0005% rhHAPLN1-(b) by 87% [(45.1 – 26.2)/(45.1 – 23.4) × 100] (D). *P < 0.05, **P < 0.01, ***P < 0.001. ns, no significance. Scale bars = 100 µm. (E) A proposed model showing a cross-talk between HAPLN1, CD44, TGF-β signaling, CARM1, sirtuins (SIRT1/2/6), and p300 for explaining the rhHAPLN1-mediated mitigation of COPD. Arrow lines indicate pathways which have been evidenced by our present data and previous studies; arrow dashed lines indicate pathways which remain elucidated through further studies. rhHAPLN1, recombinant human hyaluronan and proteoglycan link protein 1; MLI, mean linear intercepts; PPE, porcine pancreatic elastase; COPD, chronic obstructive pulmonary disease; HA, hyaluronan; ROS, reactive oxygen species; TGF-β RI, transforming growth factor β receptor 1; p-Smad3, phosphorylated Smad3; Nrf2, nuclear factor erythroid 2-related factor 2; CARM1, co-activator-associated arginine methyltransferase-1; Ac-p300, acetylated p300; p-NF-κB, phosphorylated NF-κB; MMP9, metalloproteinase-9.

Article Snippet: SDS-PAGE was performed with 15 μg protein, which was successively transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA) and incubated overnight at 4°C with specific primary antibodies, namely TGF-β RI, TGF-β RII, CARM1, SIRT6 (Abcam, UK), SIRT1, p53, p21, p16, MMP9, GAPDH (Santa Cruz Biotechnology, USA), SITR2, Samd2/3, p-Smad2, p-Smad3, p300, Ac-p300, Nrf2, NF-κB, p-NF-κB, and CD44 (Cell Signaling Technology, USA) at a dilution of 1:2,000.

Techniques: Control, High Molecular Weight, Injection, Saline, Staining, Microscopy, Recombinant

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: A Western blotting analysis showed the expression of CARM1 in 35 pairs of HCC and adjacent normal liver tissues. B The expression of CARM1 in 66 pairs of HCC and corresponding adjacent normal liver tissues was examined via an IHC assay. C Kaplan-Meier survival analysis of overall survival in HCC patients stratified by CARM1 expression. Patients with low expression ( n = 19) had lower expression values in HCC tissues than in normal tissues, while patients with high expression ( n = 47) had higher expression values in HCC tissues than in normal tissues. D CARM1 mRNA levels in normal and primary HCC tumor tissues from the TCGA database. E Kaplan-Meier survival analysis of HCC patients from the TCGA database was performed according to CARM1 mRNA expression. Patients with high CARM1 expression had expression values in the >3rd quantile, while patients with low CARM1 expression had expression values in the <3rd quartile.

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: A Representative confocal microscopy image of IF colocalization analysis of CARM1 and PSMD14 in HCC cell lines. Scale bar, 20 μm. B Endogenous PSMD14 proteins were immunoprecipitated with anti-PSMD14 antibodies and then analyzed by immunoblotting (left panel). Endogenous CARM1 proteins were immunoprecipitated with anti-CARM1 antibodies and then analyzed by immunoblotting (right panel). The IgG antibody was used as the control. C Western blotting assays showed the expression of CARM1 in control and PSMD14-knockdown HCC cells. D CARM1 mRNA expression levels in control and PSMD14-knockdown HCC cells were determined by qRT-PCR. E The protein expression levels of CARM1 were assessed by Western blotting analysis in HCC cells with either empty vector or PSMD14 overexpression. F The mRNA expression levels of CARM1 were assessed by qRT-PCR in HCC cells with either control or PSMD14 overexpression. G Control and PSMD14-knockdown HCC cells were treated with CHX (20 µM) for the indicated times, and endogenous CARM1 protein expression was detected by Western blotting analysis (left). The expression levels of CARM1 were determined by densitometry. The level of CARM1 protein in CHX-untreated cells (0 h) was set to 100% ( n = 3; * p < 0.05 and ** p < 0.01).

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: Confocal Microscopy, Immunoprecipitation, Western Blot, Control, Expressing, Knockdown, Quantitative RT-PCR, Plasmid Preparation, Over Expression

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: A Control and PSMD14-knockdown Huh7 cells were treated with MG132 at 10 μM for 6 h. The cell lysates were immunoprecipitated with an anti-CARM1 antibody, and the immunocomplexes were immunoblotted with anti-CARM1 and anti-ubiquitin antibodies. B Huh7 cells were pretreated with 10 μM capzimin for 24 h, and cells treated with equal amounts of DMSO served as controls. All cells were treated with MG132 at 10 μM for 6 h. The cell lysates were then immunoprecipitated with anti-CARM1 antibodies, and the immunocomplexes were immunoblotted with anti-CARM1 and anti-ubiquitin antibodies. C FLAG-CARM1 and HA-Ubi were transiently transferred into HEK293T cells expressing GFP-PSMD14-WT or GFP-PSMD14-MUT. Cells were then treated with MG132 (10 μM) for 6 h. The cell lysates were immunoprecipitated with an anti-FLAG M2 affinity gel. The ubiquitination levels of CARM1 were detected using anti-HA antibodies. D FLAG-CARM1 and HA-Ubi were transiently transferred into HEK293T cells, which were subsequently purified with FLAG antibodies and protein G beads. Moreover, GFP-PSMD14-WT and GFP-PSMD14-MUT were separately transferred into another two sets of HEK293T cells and purified with GFP antibodies and protein G beads. The purified Ubi-FLAG-CARM1 and GFP-PSMD14 proteins were incubated for 1 h. Then, FLAG-CARM1 was immunoprecipitated with anti-FLAG M2 affinity beads and detected with the indicated antibodies. E HEK293T cells were transfected with the indicated plasmids and treated with MG132 (10 μM) for 6 h. The cell lysates were immunoprecipitated with anti-FLAG M2 affinity beads. The ubiquitination levels of CARM1 were detected using anti-HA antibodies. F Schematic illustration of CARM1 truncation plasmids and lysine sites predicted to be modified by ubiquitination. G HEK293T cells expressing GFP-PSMD14 were transfected with FLAG-tagged full-length or truncated CARM1. The cell lysates were immunoprecipitated with anti-GFP antibodies. H HEK293T cells were transfected with the indicated plasmids and treated with MG132 (10 μM) for 6 h. The cell lysates were immunoprecipitated with an anti-FLAG M2 affinity gel. The ubiquitination levels of CARM1 were detected using anti-HA antibodies.

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: Control, Knockdown, Immunoprecipitation, Expressing, Purification, Incubation, Transfection, Modification

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: A CARM1 was transfected into HCC cells with PSMD14 knockdown. A CCK-8 assay was performed to detect proliferation. B CARM1 was transfected into HCC cells with PSMD14 knockdown. Then, a colony formation assay was conducted. C CARM1 was transfected into HCC cells with PSMD14 knockdown. Then, a transwell assay was performed to detect migration and invasion. Representative images of the transwell assay are shown. The cells in five randomly selected fields were counted under a microscope. D Representative images of immunohistochemical staining of PSMD14 and CARM1 in the same HCC and corresponding adjacent normal liver tissues are shown. E Correlation analysis of PSMD14 and CARM1 in HCC tissues. The data were statistically analyzed by the Chi-square test. R indicates the Pearson correlation coefficient. F Scatter diagram showing a positive correlation between PSMD14 and CARM1 in HCC tissues by IHC. G Survival analysis of HCC patients was conducted using Kaplan-Meier plots and log-rank tests. The patients were categorized into high and low PSMD14 and CARM1 expression groups based on IHC staining. ( n = 3; * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: Transfection, Knockdown, CCK-8 Assay, Colony Assay, Transwell Assay, Migration, Microscopy, Immunohistochemical staining, Staining, Expressing, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: A Western blotting analysis showed the knockdown efficacy of CARM1 in Huh7 and PLC/PRF/5 cells infected with lentiviral particles expressing shRNAs targeting CARM1. B Proliferation of control and CARM1-knockdown Huh7 cells was detected by CCK-8 assays on the indicated days. C Colony formation assays were performed to detect the proliferation of control and CARM1-knockdown HCC cells. The data are presented in a bar chart. D Control or CARM1-knockdown Huh7 cells were subcutaneously injected into nude mice for observation of tumor growth. E The tumor volume was measured every three days and is presented as a line graph. F The tumor weights of the xenografts from the different groups were calculated. G Immunohistochemical analysis of mouse subcutaneous tumors was performed with anti-CARM1 and anti-Ki-67 antibodies. ( n = 3; * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: Western Blot, Knockdown, Infection, Expressing, Control, CCK-8 Assay, Injection, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: A Representative images of the transwell assay results for control and CARM1-knockdown Huh7 and PLC/PRF/5 cells showing their migration and invasion ability (left). The cells in five randomly selected fields were counted under a microscope, and the data were presented as a bar chart (right). B Representative microscopy images of pulmonary metastatic lesions 8 weeks after the injection of the indicated Huh7 cells into the tail vein of nude mice. C The number of lung metastatic tumors in each group was determined. ( n = 3; * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: Transwell Assay, Control, Knockdown, Migration, Microscopy, Injection

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: A RNA-seq analysis revealed genes whose expression was upregulated (red) or downregulated (blue) in control and CARM1-knockdown Huh7 cells. B Schematic illustration of the peaks identified by ChIP-seq analysis with an anti-CARM1 antibody in Huh7 cells. C Integration of ChIP-seq and RNA-seq data. The Venn diagram shows the overlap between targets and differentially expressed genes. D Detection of the mRNA levels of CARM1 and FERMT1 in control and CARM1-knockdown Huh7 cells by qRT-PCR. E Schematic representation of the four segments near the TSS of FERMT1. ChIP primers were designed for each of the four sequences. F A ChIP assay was performed to detect CARM1 enrichment in the FERMT1 promoter region using an anti-CARM1 antibody. G Western blotting analysis was performed to show the expression level of H3R17me2 in control and CARM1-knockdown cells. H ChIP assay was performed to detect H3R17me2 enrichment in the FERMT1 promoter region using an anti-H3R17me2 antibody. The IgG antibody was used as the negative control. The inhibitory effect of CARM1 depletion on Huh7 cells, as demonstrated by rescue experiments, was effectively counteracted by the overexpression of FERMT1, as shown by both the CCK-8 ( I ) and transwell ( J ) assays. (n = 3; * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: RNA Sequencing Assay, Expressing, Control, Knockdown, ChIP-sequencing, Quantitative RT-PCR, Western Blot, Negative Control, Over Expression, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: PSMD14-mediated deubiquitination upregulates CARM1 expression, which in turn transcriptionally activates its downstream target FERMT1 through histone H3R17me2. This PSMD14-CARM1-FERMT1 signaling axis significantly promotes HCC growth and metastasis. Pharmacological inhibition of CARM1 using SGC2085 effectively suppresses the malignant phenotypes of HCC cells, suggesting a potential therapeutic strategy for HCC treatment.

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: Expressing, Inhibition

Journal: Cell Death & Disease

Article Title: PSMD14-mediated deubiquitination of CARM1 facilitates the proliferation and metastasis of hepatocellular carcinoma by inducing the transcriptional activation of FERMT1

doi: 10.1038/s41419-025-07416-3

Figure Lengend Snippet: Correlations between CARM1 expression and the clinicopathological features of patients with HCC.

Article Snippet: Serial sections were deparaffinized, hydrated, and incubated in 3% H 2 O 2 for 20 min at room temperature and then stained with anti-PSMD14 (1:3200, A9608, ABclonal) and anti-CARM1 (1:1000, NB200-342, Novus) at 4 °C overnight.

Techniques: Expressing