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: 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

Journal: Medicine

Article Title: The protein arginine methyltransferases (PRMTs) PRMT1 and CARM1 as candidate epigenetic drivers in prostate cancer progression

doi: 10.1097/md.0000000000027094

Figure Lengend Snippet: Figure 1. Graphical representation of the nuclear and cytoplasmic expression of PRMT1 and CARM1 across the spectrum of PCa progression. PCa=prostate cancer, PRMTs=protein arginine methyltransferases.

Article Snippet: Antigen Dilution Source AR 1:50 Dako, Carpentaria, CA, USA CARM1 1:750 Novus Biologicalis.

Techniques: Expressing

Journal: Medicine

Article Title: The protein arginine methyltransferases (PRMTs) PRMT1 and CARM1 as candidate epigenetic drivers in prostate cancer progression

doi: 10.1097/md.0000000000027094

Figure Lengend Snippet: Figure 3. CARM1 expression increases from non-neoplastic to neoplastic cells and from low grade, to high grade to treated cases (original magnification 400). PCa=prostate cancer, PRMTs=protein arginine methyltransferases.

Article Snippet: Antigen Dilution Source AR 1:50 Dako, Carpentaria, CA, USA CARM1 1:750 Novus Biologicalis.

Techniques: Expressing

Journal: Medicine

Article Title: The protein arginine methyltransferases (PRMTs) PRMT1 and CARM1 as candidate epigenetic drivers in prostate cancer progression

doi: 10.1097/md.0000000000027094

Figure Lengend Snippet: Figure 4. PRMT1, CARM1, ZEB1, and TWIST1 expression correlated with one-another in PCa (original magnification 400). A case with low expression of all markers is shown in the upper panel and a case with high expression of all markers in the lower panel. PCa=prostate cancer, PRMTs=protein arginine methyltransferases.

Article Snippet: Antigen Dilution Source AR 1:50 Dako, Carpentaria, CA, USA CARM1 1:750 Novus Biologicalis.

Techniques: Expressing