Journal: Aging (Albany NY)

Article Title: CARM1 promotes non-small cell lung cancer progression through upregulating CCNE2 expression

doi: 10.18632/aging.103280

Figure Lengend Snippet: CARM1 is a positive regulator of CCNE2 gene in NSCLC cells. ( A ) ChIP analysis of human CCNE2 promoter by antibodies against CARM1, H3R17me2a, H3R26me2a or IgG in NC or CARM1-silenced PC9 and HCC827 cells. Relative enrichment of CARM1, H3R17me2a and H3R26me2a marks on the promoter regions was analyzed by real-time PCR assays. The data were presented as means ± SDs of three independent experiments; ** P < 0.01, # P > 0.05. ( B ) The luciferase activity of CCNE2 promoter reporter was significantly increased when CARM1 (100 ng, 200 ng, 500 ng and 1000 ng) was transfected into PC9 and HCC827 cells. The CCNE2 promoter reporter luciferase activity was normalized to beta-galactosidase activity. The data were shown as means ± SDs of three independent experiments; ** P < 0.01. ( C ) The mRNA levels of CCNE2 was downregulated in CARM1-depleted PC9 and HCC827 cells by Real-time PCR assays. β-actin was used as an internal control. The data were presented as means ± SDs of three independent experiments; ** P < 0.01. ( D ) The protein levels of CCNE2 was downregulated in CARM1-depleted PC9 and HCC827 cells by Western blot. GAPDH was used as loading control.

Article Snippet: CARM1 inhibitor-EZM2302 (MCE; HY-111109) was dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of 10 nM for the in vitro experiments according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection, Control, Western Blot

Journal: Aging (Albany NY)

Article Title: CARM1 promotes non-small cell lung cancer progression through upregulating CCNE2 expression

doi: 10.18632/aging.103280

Figure Lengend Snippet: CARM1 promotes NSCLC cell proliferation in vitro. ( A ) Cell proliferation abilities of CARM1-depleted PC9 and HCC827 cells were assessed by CCK-8 assays. The data were presented as means ± SDs of three independent experiments; ** P < 0.01. ( B ) Colony-formative abilities of CARM1-depleted PC9 and HCC827 cells were determined by colony-formation assays. Right panel, the relative colony-formative abilities (% of NC) were quantified. The data were shown as means ± SDs of three independent experiments; ** P < 0.01. ( C ) Overexpression of CARM1 in PC9 and HCC827 cells was examined by Western blot. GAPDH was used as loading control. ( D ) Cell proliferative abilities of CARM1-overexpressed PC9 and HCC827 cells were assessed by CCK-8 assays. The data were presented as means ± SDs of three independent experiments; ** P < 0.01. ( E ) Colony-formative abilities of CARM1-overexpressed PC9 and HCC827 cells were determined by colony-formation assays. Right panel, the relative colony-formative abilities (% of NC) were quantified. The data were shown as means ± SDs of three independent experiments; ** P < 0.01.

Article Snippet: CARM1 inhibitor-EZM2302 (MCE; HY-111109) was dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of 10 nM for the in vitro experiments according to the manufacturer’s instructions.

Techniques: In Vitro, CCK-8 Assay, Over Expression, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: CARM1 promotes non-small cell lung cancer progression through upregulating CCNE2 expression

doi: 10.18632/aging.103280

Figure Lengend Snippet: Inhibition of CARM1 enzymatic activity represses CCNE2 expression in NSCLC cells. ( A ) ChIP analysis of human CCNE2 promoter by antibodies against CARM1, H3R17me2a, H3R26me2a or IgG in DMSO or EZM2302-treated (10 nM) PC9 and HCC827 cells. Relative enrichment of CARM1, H3R17me2a and H3R26me2a marks on the promoter regions was analyzed by real-time PCR assays. The data were presented as means ± SDs of three independent experiments; ** P < 0.01, # P > 0.05. ( B ) The protein levels of CCNE2, H3R17me2a and H3R26me2a were downregulated in EZM2302-treated (10 nM) PC9 and HCC827 cells by Western blot. GAPDH or histone H3 were used as loading controls. ( C ) The mRNA levels of CCNE2 was downregulated in EZM2302-treated (10 nM) PC9 and HCC827 cells by Real-time PCR assays. β-actin was used as an internal control. The data were shown as means ± SDs of three independent experiments; ** P < 0.01. ( D ) Cell proliferation abilities of DMSO or EZM2302-treated (10 nM) PC9 and HCC827 cells were assessed by CCK-8 assays. The data were presented as means ± SDs of three independent experiments; ** P < 0.01. ( E ) Colony-formative abilities of DMSO or EZM2302-treated (10 nM) PC9 and HCC827 cells were determined by colony-formation assays. Right panel, the relative colony-formative abilities (% of NC) were quantified. The data were shown as means ± SDs of three independent experiments; ** P < 0.01.

Article Snippet: CARM1 inhibitor-EZM2302 (MCE; HY-111109) was dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of 10 nM for the in vitro experiments according to the manufacturer’s instructions.

Techniques: Inhibition, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, CCK-8 Assay

Journal: Aging (Albany NY)

Article Title: CARM1 promotes non-small cell lung cancer progression through upregulating CCNE2 expression

doi: 10.18632/aging.103280

Figure Lengend Snippet: Restoration of CCNE2 expression abrogates the proliferation inhibition caused by CARM1 knockdown. ( A ) The restoration of CCNE2 in CARM1-depleted PC9 and HCC827 cells was verified by Western blot. GAPDH was used as loading control. ( B ) Cell proliferation capacities of NC, CARM1 KD (CARM1 shRNA) and CARM1 KD + CCNE2 OE (CCNE2 overexpression)-treated PC9 and HCC827 cells were determined by CCK-8 assays. The data were presented as means ± SDs of three independent experiments; ** P < 0.01. ( C ) Colony-formative abilities of NC, CARM1 KD and CARM1 KD + CCNE2 OE-treated PC9 and HCC827 cells were determined by colony-formation assays. Right panel, the relative colony-formative abilities (% of NC) were quantified. The data were shown as means ± SDs of three independent experiments; ** P < 0.01. ( D ) NC, CARM1 KD and CARM1 KD + CCNE2 OE-treated PC9 cells were subcutaneously injected into the flank of nude mice. Representative images of xenograft tumors excised from mice. ( E ) Tumor growth curves of NC, CARM1 KD and CARM1 KD + CCNE2 OE-treated PC9 cells in nude mice; n =5, ** P < 0.01. ( F ) Tumor weights of NC, CARM1 KD and CARM1 KD + CCNE2 OE-treated PC9 xenograft tumors excised from mice; n =5, ** P < 0.01.

Article Snippet: CARM1 inhibitor-EZM2302 (MCE; HY-111109) was dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of 10 nM for the in vitro experiments according to the manufacturer’s instructions.

Techniques: Expressing, Inhibition, Knockdown, Western Blot, Control, shRNA, Over Expression, CCK-8 Assay, Injection

Journal: Aging (Albany NY)

Article Title: CARM1 promotes non-small cell lung cancer progression through upregulating CCNE2 expression

doi: 10.18632/aging.103280

Figure Lengend Snippet: CARM1 is upregulated in NSCLC patients and positively correlated with CCNE2 levels. ( A ) Representative images of IHC staining of CARM1 in 20 cases of NSCLC patients (Tumor) and their adjacent non-tumor tissues (Normal). ( B ) Representative images of IHC staining of CCNE2 in 20 cases of NSCLC patients (Tumor) and their adjacent non-tumor tissues (Normal). ( C ) H score of CARM1 expression in 20 cases of NSCLC tumor tissues and their adjacent non-tumor tissues. ** P < 0.01. ( D ) H score of CCNE2 expression in 20 cases of NSCLC tumor tissues and their adjacent non-tumor tissues. ** P < 0.01. ( E ) Pearson correlation analysis was performed to examine the correlation between CARM1 and CCNE2 expression in 20 cases of NSCLC patients (n = 20; r = 0.6958; P < 0.01). ( F ) Analysis of data from the Kaplan-Meier plotter database suggested that high expression of CARM1 (Cutoff value: 262) was associated with shorter 10-year overall survival of NSCLC (lung adenocarcinoma, n = 461) patients. P < 0.01. ( G ) Analysis of data from the Kaplan-Meier plotter database suggested that high expression of CCNE2 (Cutoff value: 228) was associated with shorter 10-year overall survival of NSCLC (adenocarcinoma) patients. P < 0.01.

Article Snippet: CARM1 inhibitor-EZM2302 (MCE; HY-111109) was dissolved in dimethyl sulfoxide (DMSO) and used at a final concentration of 10 nM for the in vitro experiments according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Expressing

Journal: Scientific Reports

Article Title: Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma

doi: 10.1038/s41598-017-18446-z

Figure Lengend Snippet: Compound structures and biochemical and cell data for CARM1 inhibitors.

Article Snippet: Antibodies: Cell Signaling Technology, CARM1 (C31G9, CS3379) PABP1 (CS4992), PABP1me2a (C60A10, CS3505), aDMA (D10A12E9, CS13076), Histone H3 (96C10, CS3638) EMD Millipore, GAPDH (6C5, MABE374), H3R26me1 (ABE14), Sigma-Aldrich, SmB (12F5, S0698), Abgent, SmB, (AP2959a), Abcam, PABP1 (10E10, ab6125), 21 st Century Biochemicals, H3R17me2a (custom formulation).

Techniques:

Journal: Scientific Reports

Article Title: Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma

doi: 10.1038/s41598-017-18446-z

Figure Lengend Snippet: EZM2302 binds to CARM1 and is a selective inhibitor of CARM1 activity. ( a ) Structure of 2 (top) or EZM2302 (bottom) (yellow) in complex with CARM1 (green). Electron density (2Fo-Fc, 1σ for the compound is shown. Hydrogen bonds are indicated as black dashes; Π interactions are indicated with orange dashes; water molecules are depicted as spheres. ( b ) Ligand affinity maps of EZM2302 across the family trees of human arginine methyltransferases and lysine methyltransferase enzymes show EZM2302 is a selective and potent inhibitor of CARM1. ( c ) Synergy of CARM1 inhibition by EZM2302 with SAH. IC 50 values for EZM2302 were determined at increasing concentrations of SAH. Data were fit using the noncompetitive Cheng-Prusoff and the Yonetani-Theorell equations as shown in the Supplementary Methods. Potency of inhibition by EZM2302 increases with SAH concentrations. ( d ) EZM2302 inhibition of CARM1. CARM1 was preincubated with excess EZM2302 (I) in the presence and absence of SAH and substrates (Subs). The CARM1 complexes were purified by gel filtration using 0.5 mL 7 K MWCO Zeba columns (Peirce) and CARM1 activity was tested.

Article Snippet: Antibodies: Cell Signaling Technology, CARM1 (C31G9, CS3379) PABP1 (CS4992), PABP1me2a (C60A10, CS3505), aDMA (D10A12E9, CS13076), Histone H3 (96C10, CS3638) EMD Millipore, GAPDH (6C5, MABE374), H3R26me1 (ABE14), Sigma-Aldrich, SmB (12F5, S0698), Abgent, SmB, (AP2959a), Abcam, PABP1 (10E10, ab6125), 21 st Century Biochemicals, H3R17me2a (custom formulation).

Techniques: Activity Assay, Inhibition, Purification, Filtration

Journal: Scientific Reports

Article Title: Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma

doi: 10.1038/s41598-017-18446-z

Figure Lengend Snippet: EZM2302 is orally available with potent in vivo CARM1 inhibition. ( a ) Preclinical PK of EZM2302 in ( a ), CD-1 mouse and ( b ) Sprague-Dawley rat with or without jugular- and portal-vein cannulation (JVC and PVC). Data are shown graphically as plasma concentration vs. time profile of mean ± SD (n = 3) following i.v. bolus or oral gavage administration. ( c ) Methyl mark changes induced by twice daily (BID) administration of EZM2302 for 7 days at 150 and 300 mg/kg in untumored CB-17 SCID mice. Compound administration was stopped on day 7, and liver tissue was harvested for PD analysis. ( d ) Each point represents the ratio of PABP1me2a to PAPB1 or SmBme0 to SmB normalized to the vehicle control, measured by western blot. The horizontal lines represent group mean values. Dose groups that showed a statistically significant inhibition are indicated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus vehicle, 1-way ANOVA with a Dunnett’s post-test) P values compared to vehicle were: PABP1me2a: 37.5 mg/kg p = 0.634, 75 mg/kg p = 0.137, 150 mg/kg p = 0.016, 300 mg/kg p = 0.006, SMBme0: 37.5 mg/kg p = 0.092, 75 mg/kg p = 0.052, 150 mg/kg p = 0.0003, 300 mg/kg p = 0.007. ( e ) CARM1 target inhibition in RPMI-8226 xenograft tumor tissue collected from mice euthanized after 2, 4, and 8 days of BID dosing at 150 and 300 mg/kg EZM2302. Images in C and E have been cropped. Uncropped images are presented in Supplementary Figs and .

Article Snippet: Antibodies: Cell Signaling Technology, CARM1 (C31G9, CS3379) PABP1 (CS4992), PABP1me2a (C60A10, CS3505), aDMA (D10A12E9, CS13076), Histone H3 (96C10, CS3638) EMD Millipore, GAPDH (6C5, MABE374), H3R26me1 (ABE14), Sigma-Aldrich, SmB (12F5, S0698), Abgent, SmB, (AP2959a), Abcam, PABP1 (10E10, ab6125), 21 st Century Biochemicals, H3R17me2a (custom formulation).

Techniques: In Vivo, Inhibition, Clinical Proteomics, Concentration Assay, Control, Western Blot

Journal: Scientific Reports

Article Title: Identification of a CARM1 Inhibitor with Potent In Vitro and In Vivo Activity in Preclinical Models of Multiple Myeloma

doi: 10.1038/s41598-017-18446-z

Figure Lengend Snippet: EZM2302 shows dose dependent target engagement and tumor growth inhibition in vivo . ( a ) Anti-tumor activity in RPMI-8226 xenograft model induced by twice daily (BID) administration of EZM2302 for 21 days at the indicated doses (N = 8, Mean value ± SEM). Tumor growth rates were significantly reduced at all dose groups (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus vehicle, 2-way ANOVA with a Dunnett’s post-test). P values compared to vehicle were: 37.5 mg/kg p = 0.004, 75 mg/kg p = 0.0008, 150 mg/kg p = 0.007, 300 mg/kg p = 0.0001. Compound administration was stopped on day 21, and tumors were harvested for PD analysis. ( b ) CARM1 target inhibition in RPMI-8226 xenograft tumor tissue collected from mice euthanized after 21 days of BID dosing. ( c ) Each point represents the ratio of PABP1me2a to GAPDH, aDMA to GAPDH, or SmBme0 to SmB normalized to the vehicle control, measured by WB. Dose groups that showed a statistically significant inhibition are indicated (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus vehicle, 1-way ANOVA with a Dunnett’s post-test). P values compared to vehicle were: PABP1me2a: 37.5 mg/kg p = 0.603, 75 mg/kg p = 0.011, 150 mg/kg p = 0.020, 300 mg/kg p = 0.036, SMBme0: 37.5 mg/kg p = 0.0001, 75 mg/kg p = 0.0001, 150 mg/kg p = 0.0004, 300 mg/kg p = 0.005, ADMA: 37.5 mg/kg p = 0.0001, 75 mg/kg p = 0.0001, 150 mg/kg p = 0.0021, 300 mg/kg p = 0.0001. Images in ( b ) have been cropped. Uncropped images are presented in Supplementary Fig. .

Article Snippet: Antibodies: Cell Signaling Technology, CARM1 (C31G9, CS3379) PABP1 (CS4992), PABP1me2a (C60A10, CS3505), aDMA (D10A12E9, CS13076), Histone H3 (96C10, CS3638) EMD Millipore, GAPDH (6C5, MABE374), H3R26me1 (ABE14), Sigma-Aldrich, SmB (12F5, S0698), Abgent, SmB, (AP2959a), Abcam, PABP1 (10E10, ab6125), 21 st Century Biochemicals, H3R17me2a (custom formulation).

Techniques: Drug discovery, Inhibition, In Vivo, Activity Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAc Transferase Regulates Mitotic Chromatin Dynamics *

doi: 10.1074/jbc.M110.158170

Figure Lengend Snippet: CARM1 is affected by OGT overexpression. A, left panels, extracts from synchronized cells were probed for CARM1 to demonstrate that it becomes mitotically phosphorylated (indicated by arrows). Upper right panel, phosphorylation was confirmed by immunoprecipitating CARM1 from asynchronous (A) and mitotic (M) extracts and treating with phosphatase. CARM1 was immunoprecipitated from asynchronous and mitotic extracts and probed for total O-GlcNAc levels. IgG was included as a specificity control. Lower right panels, blots were stripped and reprobed for total CARM1 in the immunoprecipitates (IP). λ, λ-phosphatase. B, total cell extract from asynchronous or mitotic cells overexpressing either GFP or OGT were probed using antibodies directed against OGT, CARM1, GFP, or tubulin. Overexpression of OGT prevents CARM1 mitotic phosphorylation as indicated with asterisks. CARM1 was immunoprecipitated from asynchronous and mitotic cells overexpressing either GFP or OGT and probed for CARM1 and OGT. OGT does not associate with CARM1 during mitosis in control cells, whereas in OGT-overexpressing cells, OGT associates with CARM1 regardless of cell cycle state. dark exp, dark exposure. C, treatment of cells with the O-GlcNAcase inhibitor Thiamet G (TMG) does not exhibit the same effects as OGT overexpression. Left panel, acid-extracted histones were probed for the CARM1-specific target H3R17me2. Middle and lower left panels, total cell extract was probed using antibodies directed against O-GlcNAc (middle panel) and CARM1 and actin (lower left panels). Right panels, inhibition of O-GlcNAcase using NAG-thiazoline dramatically increases CARM1 O-GlcNAcylation without affecting mitotic phosphorylation. CARM1 was immunoprecipitated from asynchronous and mitotic extracts treated with or without NAG-thiazoline and probed for O-GlcNAc. Blots were stripped and reprobed for total CARM1 in the immunoprecipitates. GT, NAG-thiazoline.

Article Snippet: Antibody to coactivator-associated arginine methyltransferase 1 (CARM1) was from Bethyl Laboratories.

Techniques: Over Expression, Immunoprecipitation, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAc Transferase Regulates Mitotic Chromatin Dynamics *

doi: 10.1074/jbc.M110.158170

Figure Lengend Snippet: CARM1 localizes to the pericentriolar region during mitosis. HeLa cells were stained for tubulin (green), DNA (blue), and CARM1 (red) from cells in prometaphase (A), metaphase (B), and anaphase (C). CARM1 is found concentrated near the centrosomes. A′, B′, and C′ are enlargements of CARM1 (black and white) and merged images from A, B, and C to show the ring CARM1 forms around the centrosome.

Article Snippet: Antibody to coactivator-associated arginine methyltransferase 1 (CARM1) was from Bethyl Laboratories.

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAc Transferase Regulates Mitotic Chromatin Dynamics *

doi: 10.1074/jbc.M110.158170

Figure Lengend Snippet: OGT overexpression causes mislocalization of CARM1 and DNA abnormalities. OGT-overexpressing cells were stained for tubulin (green), DNA (blue), and CARM1 (red) from cells in prometaphase (A), metaphase (B), and anaphase (C). CARM1 does not concentrate near the centrosome in panels A–C. In panels D–F, CARM1 is found in the pericentriolar region; however, there is centrosome amplification in panel D, the degree of DNA condensation in panel E is less than in control cells, and the formation of a chromosomal bridge is shown in panel F; all are characteristics of cells overexpressing OGT.

Article Snippet: Antibody to coactivator-associated arginine methyltransferase 1 (CARM1) was from Bethyl Laboratories.

Techniques: Over Expression, Staining, Amplification

Journal: The Journal of Biological Chemistry

Article Title: O -GlcNAc Transferase Regulates Mitotic Chromatin Dynamics *

doi: 10.1074/jbc.M110.158170

Figure Lengend Snippet: Summary of immunofluorescence data DNA segregation errors are defined as DNA caught within the plane of division.

Article Snippet: Antibody to coactivator-associated arginine methyltransferase 1 (CARM1) was from Bethyl Laboratories.

Techniques: Immunofluorescence

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: The primary antibodies used were as follows: PRMT4 (Proteintech, #55246-1-AP, 1:1000), AKT(Proteintech, #10176-2-AP, 1:1000), p-AKT (Proteintech, #66444-1-lg, 1:2000), mTOR(Proteintech, #66888-1-lg, 1:2000), p-mTOR (Proteintech, #67778-1-lg, 1:2000), 4E-BP1(Cell Signaling, #9644, 1:1000), p-4E-BP1(Cell Signaling, #2855, 1:1000), RPS6 (Proteintech, #14823-1-AP, 1:500), p-RPS6 (Proteintech, #29223-1-AP, 1:1000) and GAPDH (Proteintech, #10494-1-AP, 1:5000).

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: The primary antibodies used were as follows: PRMT4 (Proteintech, #55246-1-AP, 1:1000), AKT(Proteintech, #10176-2-AP, 1:1000), p-AKT (Proteintech, #66444-1-lg, 1:2000), mTOR(Proteintech, #66888-1-lg, 1:2000), p-mTOR (Proteintech, #67778-1-lg, 1:2000), 4E-BP1(Cell Signaling, #9644, 1:1000), p-4E-BP1(Cell Signaling, #2855, 1:1000), RPS6 (Proteintech, #14823-1-AP, 1:500), p-RPS6 (Proteintech, #29223-1-AP, 1:1000) and GAPDH (Proteintech, #10494-1-AP, 1:5000).

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: The primary antibodies used were as follows: PRMT4 (Proteintech, #55246-1-AP, 1:1000), AKT(Proteintech, #10176-2-AP, 1:1000), p-AKT (Proteintech, #66444-1-lg, 1:2000), mTOR(Proteintech, #66888-1-lg, 1:2000), p-mTOR (Proteintech, #67778-1-lg, 1:2000), 4E-BP1(Cell Signaling, #9644, 1:1000), p-4E-BP1(Cell Signaling, #2855, 1:1000), RPS6 (Proteintech, #14823-1-AP, 1:500), p-RPS6 (Proteintech, #29223-1-AP, 1:1000) and GAPDH (Proteintech, #10494-1-AP, 1:5000).

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: The primary antibodies used were as follows: PRMT4 (Proteintech, #55246-1-AP, 1:1000), AKT(Proteintech, #10176-2-AP, 1:1000), p-AKT (Proteintech, #66444-1-lg, 1:2000), mTOR(Proteintech, #66888-1-lg, 1:2000), p-mTOR (Proteintech, #67778-1-lg, 1:2000), 4E-BP1(Cell Signaling, #9644, 1:1000), p-4E-BP1(Cell Signaling, #2855, 1:1000), RPS6 (Proteintech, #14823-1-AP, 1:500), p-RPS6 (Proteintech, #29223-1-AP, 1:1000) and GAPDH (Proteintech, #10494-1-AP, 1:5000).

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: The primary antibodies used were as follows: PRMT4 (Proteintech, #55246-1-AP, 1:1000), AKT(Proteintech, #10176-2-AP, 1:1000), p-AKT (Proteintech, #66444-1-lg, 1:2000), mTOR(Proteintech, #66888-1-lg, 1:2000), p-mTOR (Proteintech, #67778-1-lg, 1:2000), 4E-BP1(Cell Signaling, #9644, 1:1000), p-4E-BP1(Cell Signaling, #2855, 1:1000), RPS6 (Proteintech, #14823-1-AP, 1:500), p-RPS6 (Proteintech, #29223-1-AP, 1:1000) and GAPDH (Proteintech, #10494-1-AP, 1:5000).

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: The primary antibodies used were as follows: PRMT4 (Proteintech, #55246-1-AP, 1:1000), AKT(Proteintech, #10176-2-AP, 1:1000), p-AKT (Proteintech, #66444-1-lg, 1:2000), mTOR(Proteintech, #66888-1-lg, 1:2000), p-mTOR (Proteintech, #67778-1-lg, 1:2000), 4E-BP1(Cell Signaling, #9644, 1:1000), p-4E-BP1(Cell Signaling, #2855, 1:1000), RPS6 (Proteintech, #14823-1-AP, 1:500), p-RPS6 (Proteintech, #29223-1-AP, 1:1000) and GAPDH (Proteintech, #10494-1-AP, 1:5000).

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

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: Fig. 1 CARM1 is upregulated in HCC tissues and indicates poor clinical outcomes. 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: For immunoprecipitation, CARM1 antibodies (Novus), H3R17me2 antibodies (Active Motif), or IgG antibodies (Millipore) were mixed with the nuclear lysates.

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: Fig. 2 The stability of CARM1 is regulated by PSMD14. 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: For immunoprecipitation, CARM1 antibodies (Novus), H3R17me2 antibodies (Active Motif), or IgG antibodies (Millipore) were mixed with the nuclear lysates.

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: Fig. 4 CARM1 is a downstream effector of PSMD14 that affects the prognosis of HCC patients. 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: For immunoprecipitation, CARM1 antibodies (Novus), H3R17me2 antibodies (Active Motif), or IgG antibodies (Millipore) were mixed with the nuclear lysates.

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: Fig. 5 CARM1 promotes the proliferation of HCC cells. 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: For immunoprecipitation, CARM1 antibodies (Novus), H3R17me2 antibodies (Active Motif), or IgG antibodies (Millipore) were mixed with the nuclear lysates.

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: Fig. 6 CARM1 enhances the metastatic ability of HCC cells. 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: For immunoprecipitation, CARM1 antibodies (Novus), H3R17me2 antibodies (Active Motif), or IgG antibodies (Millipore) were mixed with the nuclear lysates.

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: Fig. 7 FERMT1 functions downstream of CARM1 via histone modification. 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: For immunoprecipitation, CARM1 antibodies (Novus), H3R17me2 antibodies (Active Motif), or IgG antibodies (Millipore) were mixed with the nuclear lysates.

Techniques: RNA Sequencing, 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: Fig. 8 Antitumor effects of the CARM1 inhibitor SGC2085 in HCC. A The cytotoxicity of various concentrations of SCG2085 to MHCC-97h and PLC/PRF/5 cells was examined by a CCK-8 assay. B MHCC-97h cells were pretreated with the indicated concentrations of SGC2085 for 48 h. Transwell assays were conducted to evaluate the migratory and invasive potential of the cells. The accompanying figures depict representative images (left) and the corresponding statistical findings derived from the transwell assay (right). Nude mice with subcutaneous tumors formed by MHCC-97h cells were treated with vehicle control or SGC2085 (10 mg/kg; intraperitoneally (i.p.), three times each week) for 24 days (C). The tumor volume was measured every 3 days, and the data were presented as a line graph (D). The tumor weights of the xenografts from the different groups were calculated (E). (n = 3; *p < 0.05, **p < 0.01, and ***p < 0.001).

Article Snippet: For immunoprecipitation, CARM1 antibodies (Novus), H3R17me2 antibodies (Active Motif), or IgG antibodies (Millipore) were mixed with the nuclear lysates.

Techniques: CCK-8 Assay, Derivative Assay, Transwell Assay, Control

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: Fig. 9 Schematic diagram of the PSMD14-CARM1-FERMT1 axis in HCC. 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: For immunoprecipitation, CARM1 antibodies (Novus), H3R17me2 antibodies (Active Motif), or IgG antibodies (Millipore) were mixed with the nuclear lysates.

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: 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: 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: PRMT4 (cat# 12495), cleaved caspase 3 (cat# 9661) and cleaved caspase 9 (cat# 7237) antibodies were from Cell Signalling (Danvers, Massachusetts, 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: PRMT4 (cat# 12495), cleaved caspase 3 (cat# 9661) and cleaved caspase 9 (cat# 7237) antibodies were from Cell Signalling (Danvers, Massachusetts, 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: PRMT4 (cat# 12495), cleaved caspase 3 (cat# 9661) and cleaved caspase 9 (cat# 7237) antibodies were from Cell Signalling (Danvers, Massachusetts, 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: PRMT4 (cat# 12495), cleaved caspase 3 (cat# 9661) and cleaved caspase 9 (cat# 7237) antibodies were from Cell Signalling (Danvers, Massachusetts, 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: PRMT4 (cat# 12495), cleaved caspase 3 (cat# 9661) and cleaved caspase 9 (cat# 7237) antibodies were from Cell Signalling (Danvers, Massachusetts, 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: PRMT4 (cat# 12495), cleaved caspase 3 (cat# 9661) and cleaved caspase 9 (cat# 7237) antibodies were from Cell Signalling (Danvers, Massachusetts, 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: PRMT4 (cat# 12495), cleaved caspase 3 (cat# 9661) and cleaved caspase 9 (cat# 7237) antibodies were from Cell Signalling (Danvers, Massachusetts, USA).

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

Journal: Chembiochem

Article Title: Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors

doi: 10.1002/cbic.202100506

Figure Lengend Snippet: A) CARM1 active site with key active residues interacting with cofactor SAM and the target arginine of a peptide substrate. The double E‐loop consists of glutamate residues Glu 258 and Glu 267 . His 415 is involved in substrate recognition as part of the THW‐loop and interacts with Asp 166 for the deprotonation of the guanidine moiety facilitating methyl group transfer. B) Design strategy used in preparing bi‐substrate analogues for structural studies and peptidic inhibitors of CARM1.

Article Snippet: The commercially available PRMT1 and CARM1 chemiluminescent assay kits (BPS Bioscience, Dan Diego, CA, USA) were used for evaluation of methyltransferase inhibition as previously described.

Techniques:

Journal: Chembiochem

Article Title: Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors

doi: 10.1002/cbic.202100506

Figure Lengend Snippet: General synthetic scheme for the preparation of transition state peptidomimetics with the adenosine moiety covalently linked to the side chain of the CARM1 target arginine. Also indicated is the neighbouring lysine residue in either acetylated or nonacetylated state. Details of the synthesis of the specific H3 peptidomimetics prepared as well as the preparation of the Pbf‐protected adenosine thiourea building block are provided in the Supporting Information.

Article Snippet: The commercially available PRMT1 and CARM1 chemiluminescent assay kits (BPS Bioscience, Dan Diego, CA, USA) were used for evaluation of methyltransferase inhibition as previously described.

Techniques: Blocking Assay

Journal: Chembiochem

Article Title: Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors

doi: 10.1002/cbic.202100506

Figure Lengend Snippet: Electron density (2 F obs‐ F calc) weighted maps for subunit A of mmCARM1 bound to: A) peptidomimetic 3 (H3 13–31 Lys 18 −NH 2 ), PDB code 7OS4 and B) peptidomimetic 4 (H3 13–31 Lys 18 Ac), PDB code 7OKP. CARM1 is represented as cartoon and H3 peptidomimetics are represented as stick. Maps are represented as a mesh contouring level set to 1σ.

Article Snippet: The commercially available PRMT1 and CARM1 chemiluminescent assay kits (BPS Bioscience, Dan Diego, CA, USA) were used for evaluation of methyltransferase inhibition as previously described.

Techniques:

Journal: Chembiochem

Article Title: Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors

doi: 10.1002/cbic.202100506

Figure Lengend Snippet: IC 50 values and for compounds 5–14 against CARM1 and PRMT1. [a]

Article Snippet: The commercially available PRMT1 and CARM1 chemiluminescent assay kits (BPS Bioscience, Dan Diego, CA, USA) were used for evaluation of methyltransferase inhibition as previously described.

Techniques: Sequencing

Journal: Cancer Discovery

Article Title: BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma

doi: 10.1158/2159-8290.CD-21-1491

Figure Lengend Snippet: BAF complex ATPase inhibition and degradation are novel therapeutic strategies in pediatric H3K27M-glioma. A, Heat map of IC 50 values comparing small-molecule inhibitors and a degrader targeting BAF complex members, and its regulators (BRG1/BRM inhibitors: Compounds 11, 12, 14, PFI-3; CARM1 inhibitors: CARM1 inhibitor, TP064; BRD9 inhibitor: I-BRD9; and BRD9 degrader: dBRD9-13) in H3.3K27M ( n = 5), H3.1K27M ( n = 1), and H3WT ( n = 3) pediatric glioma neurosphere models and nonmalignant cell lines ( n = 2, NHA-hTERT: immortalized normal human astrocytes, and Oli Neu: immortalized normal mouse OPCs). B, PRISM analysis of 694 cancer cell lines representing 23 lineages (Broad Institute), treated with a BRG1/BRM inhibitor (Compound 11) at an 8-point dose curve (3-fold dilution, with a maximum of 10 μmol/L) for 5 days. The black dashed line represents the mean AUC computed over cell lines of all lineages. Cancer lineages below this line represent those sensitive to BRG1/BRM inhibition by Compound 11. C, Chemical structures of BRG1/BRM inhibitors (Compounds 11, 12, and 14) and a novel BRG1/BRM degrader (JQ-dS-4). D, Log 2 fold change (FC) of differential proteins (left) as assessed by SILAC of DMSO control (light isotope labeled) and 1 μmol/L JQ-dS-4 (heavy isotope labeled)–treated BT869 H3.3K27M-glioma neurospheres (2 days of treatment). Heat map (right) of BAF complex proteins (with encoding genes shown in parentheses) depleted upon JQ-dS-4 treatment in BT869 neurospheres. E, Immunoblot for BRG1 and BRM protein levels in BT869, HSJD-DIPG007, and SU-DIPGXIIIP* neurospheres treated with novel BRG1/BRM degraders (AU-15330 and JQ-dS-4) at indicated doses and time points. Cleaved PARP was used as a marker for apoptosis. Total H3 and GAPDH served as loading controls. F, Heat map of IC 50 values comparing two BRG1/BRM degraders (JQ-dS-4 and AU-15330) in H3.3K27M ( n = 5), H3.1K27M ( n = 1), and H3WT ( n = 3) pediatric glioma neurosphere models and nonmalignant cell lines ( n = 2, NHA-hTERT: immortalized normal human astrocytes, and Oli Neu: immortalized normal mouse OPCs). G, Dose–response curves for BRG1/BRM degraders (AU-15330 and JQ-dS-4) in H3.3K27M ( n = 5), H3.1K27M ( n = 1), and H3WT ( n = 3) pediatric glioma neurosphere models and nonmalignant cell lines ( n = 2, NHA-hTERT: immortalized normal human astrocytes, and Oli Neu: immortalized normal mouse OPCs).

Article Snippet: CARM1 inhibitors (CARM1 inhibitor and TP064) were purchased from Sigma and Tocris Biosciences, respectively.

Techniques: Inhibition, Multiplex sample analysis, Control, Labeling, Western Blot, Marker

Journal: Cancer Discovery

Article Title: CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells

doi: 10.1158/2159-8290.cd-20-1144

Figure Lengend Snippet: Figure 1. CARM1 is an epigenetic inhibitor in tumor-specific T cells. A, Experimental design for in vivo discovery of epigenetic regulators that inhibit CD8 T-cell accumulation in tumors. B, In vivo CRISPR screen with epigenetic gRNA library in tumor-specific CD8 T cells. gRNA quantification in CD8 T cells was compared in tumors (experimental site) and spleens (control organ; log2 fold change). Major experimental genes and positive control genes are highlighted in red and blue, respectively. C, MAGeCK analysis of in vivo CRISPR screen data; MAGeCK score provides integrated readout for strength of gene effects. D, T-cell cytotoxicity assay with Carm1-KO and control-KO OT-I CD8 T cells. CD8 T cells were edited by electroporation with Cas9 protein and bound gRNA, and cells were grown in IL15 + IL7 containing T-cell media for 5 days. T cells were cocultured with B16F10-OVA-ZsGreen tumor cells at indicated E/T ratios (n = 8–10/replicates per condition); 24 hours later, live GFP-positive tumor cells were counted using a Celigo image cytometer. Data are representative of three experiments and shown as mean ± SEM. ****, P < 0.0001, by unpaired two-sided Mann–Whitney test. E, Antitumor activity of adoptively transferred Carm1-KO or control-KO OT-I CD45.1 CD8 T cells. B16-OVA-ZsGreen tumor cells (0.1 × 106) were implanted subcutaneously. On day 7 after tumor cell inoculation, edited CD8 T cells (1 × 106) were transferred via tail-vein injection. Tumor size was recorded; n = 8–10 mice per group. F, Tumor weights 7 days following adoptive T-cell transfer for experiment shown in E. G, Flow cytometry analysis of tumor-infiltrating Carm1-KO or control-KO CD8 T cells following adoptive transfer of edited OT-I CD45.1 CD8 T cells (n = 10 mice/group) with gating on CD45.1 and CD8 T-cell markers. Quantification of CD8 T-cell infiltration and expression of effector (granzyme B, IFNγ) as well as proliferation (Ki-67) markers.

Article Snippet: Doxycycline-Inducible Expression of Carm1 In Vivo C57BL/6 mice bearing Carm1-KO B16F10 tumor cells (transduced with Dox–CARM1 or empty vector constructs) were fed a doxycycline-containing diet (625 ppm; Envigo Teclad) until the experimental endpoint (18 days).

Techniques: In Vivo, CRISPR, Control, Positive Control, Cytotoxicity Assay, Electroporation, Cytometry, MANN-WHITNEY, Activity Assay, Injection, Flow Cytometry, Adoptive Transfer Assay, Expressing

Journal: Cancer Discovery

Article Title: CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells

doi: 10.1158/2159-8290.cd-20-1144

Figure Lengend Snippet: Figure 2. Carm1-KO in CD8 T cells enhances their antitumor function. A, RNA-seq analysis of differentially expressed genes in Carm1-KO or control- KO OT-I CD8 T cells cocultured for 24 hours with B16F10-OVA tumor cells (four biological replicates per condition). Color code represents z scores for differential gene expression. B, Volcano plot of all differentially expressed genes between Carm1-KO and control-KO OT-I CD8 T cells. Statistical signifi- cance (log10 adjusted P value) was plotted against log2 fold change of gene expression levels (Carm1-KO/control-KO cells). C, RT-qPCR analysis of Tcf7, Myb, Bcl6, Btg2, Itgae, Havcr2, and Klrg1 mRNA levels in Carm1-KO and control-KO CD8 T cells (targeting of Carm1 with two different gRNAs, triplicate measurements). D, Gene Ontology (GO) analysis of significantly upregulated/downregulated pathways in Carm1-KO versus control-KO T cells. E, Tumor-infiltrating Carm1-KO or control-KO CD8 T cells following adoptive transfer of edited OT-I CD45.1 CD8 T cells (n = 10 mice/group) with gating on CD45.1 and CD8 T-cell markers. Quantification of TCF7+ T cells with high BCL2 protein levels and TCF7+ CD8 T cells. F, Quantification of BCL-high tumor-infiltrating Carm1-KO or control-KO CD8 T cells. G–I, Tumor-infiltrating Carm1-KO or control-KO CD8 T cells were analyzed 16 or 24 days following adoptive transfer of edited OT-I CD45.1 CD8 T cells (n = 8 mice/group) with gating on CD45.1 and CD8 T-cell markers. Quantification of CD8 T activation marker (CD69; G) and markers of T-cell exhaustion (H–I). Data shown are representative of two experiments. Two-way ANOVA was used to determine statistical significance for time points when all mice were viable for tumor measurement. Graphs shown represent data summarized as mean ± SD and were analyzed by unpaired two-sided Mann–Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Doxycycline-Inducible Expression of Carm1 In Vivo C57BL/6 mice bearing Carm1-KO B16F10 tumor cells (transduced with Dox–CARM1 or empty vector constructs) were fed a doxycycline-containing diet (625 ppm; Envigo Teclad) until the experimental endpoint (18 days).

Techniques: RNA Sequencing, Control, Gene Expression, Quantitative RT-PCR, Adoptive Transfer Assay, Activation Assay, Marker, MANN-WHITNEY

Journal: Cancer Discovery

Article Title: CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells

doi: 10.1158/2159-8290.cd-20-1144

Figure Lengend Snippet: Figure 3. Inactivation of Carm1 gene in tumor cells elicits tumor immunity. A, CARM1 mRNA levels in a diverse panel of 1,208 cancer cell lines from the CCLE. Tumor cell lines were grouped based on cancer type. B, Strategy for targeting Carm1 in tumor cells to study impact on T cell–mediated tumor immunity. C, Western blot analysis of CARM1 protein in B16F10 melanoma cells following electroporation with RNPs composed of Cas9 protein and bound gRNAs (control, Carm1); two different control and Carm1 gRNAs were evaluated. D, Growth of Carm1-KO and control-KO B16F10 tumors (left) and survival of tumor bearing mice (right). Mice (n = 8–10/group) were treated with CD8 depleting or isotype control antibodies. This in vivo phenotype was confirmed with a second Carm1 gRNA (Supplementary Fig. S3A). E, Growth of Carm1-KO tumors in T cell–deficient mice. Carm1-KO and control-KO B16F10 tumor cells (0.2 × 106) were implanted into immunocompetent or immunodeficient (Tcra-KO) mice (n = 8–10 mice/group); tumor growth (left) and survival (right) were recorded. F, Growth of Carm1-KO or control-KO 4T1 tumor cells following implantation into the mammary fat pad (n = 8–10 mice/ group); tumor growth (left) and survival (right) were recorded. G, Quantification of spontaneous lung metastases formed by Carm1-KO or control-KO 4T1 tumors in immunocompetent mice. Representative images of lung metastases (V, ventral; D, dorsal; right). H, Tumor growth (left) and survival (right) following implantation of Carm1-KO and control-KO MC38 tumor cells (n = 8–10 mice/group). I, T-cell cytotoxicity assay with Carm1-KO or control- KO B16F10-OVA-ZsGreen tumor cells. Tumor cells were cocultured for 24 hours with OT-I CD8 T cells at indicated E/T ratios (n = 8–10 replicates per condition). J, Induction of tumor cell apoptosis (Carm1-KO or control-KO B16F10-OVA-ZsGreen cells) by CD8 T cells (as described in I), measured with a caspase 3/7 dye at different E/T ratios (n = 8–10 replicates/group). K, Sensitization of tumor cells to T cells with a CARM1 inhibitor. B16F10-OVA- ZsGreen tumor cells were pretreated with CARM1 inhibitor (EZM2302, 0.1 μmol/L) for 24 hours. Vehicle or inhibitor-treated tumor cells were cocultured with OT-I CD8 T cells at indicated E/T ratios (n = 7–8 replicates/condition). L, T-cell cytotoxicity assay with human BT549 TNBC and human CD8 T cells that expressed a NY-ESO-1 TCR. Tumor cells were pretreated with CARM1 inhibitor (EZM2302, 0.1 μmol/L) for 24 hours (n = 7–10 replicates/group); numbers of surviving tumor cells were quantified after 24 hours of coculture. Two-way ANOVA was used to determine statistical significance for tumor measurements at time points when all mice were alive. Statistical significance for survival of mice in each treatment group were calculated by log-rank (Mantel–Cox) test. Bar graphs represent data summarized as mean ± SEM and were analyzed by unpaired two-sided Mann–Whitney test. Data shown are representative of three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Doxycycline-Inducible Expression of Carm1 In Vivo C57BL/6 mice bearing Carm1-KO B16F10 tumor cells (transduced with Dox–CARM1 or empty vector constructs) were fed a doxycycline-containing diet (625 ppm; Envigo Teclad) until the experimental endpoint (18 days).

Techniques: Western Blot, Electroporation, Control, In Vivo, Cytotoxicity Assay, MANN-WHITNEY

Journal: Cancer Discovery

Article Title: CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells

doi: 10.1158/2159-8290.cd-20-1144

Figure Lengend Snippet: Figure 4. Innate immune activation in Carm1-deficient tumor cells. A, RNA-seq analysis of differentially expressed genes in Carm1-KO and control-KO B16F10 tumor cells (n = 3/group). Data are representative of two independent experiments. B, Gene ontology (GO) analysis of significantly upregulated/ downregulated genes in Carm1-KO compared with control-KO B16F10 tumor cells. C, Venn diagram representing number of overlapping differentially expressed genes in Carm1-KO tumor and CD8 T cells. D, Validation of ISGs by RT-qPCR in Carm1-KO compared with control-KO B16F10 cells (n = 3/group). E, RT-qPCR analysis of ISG mRNA levels following treatment of B16F10 cells with CARM1 inhibitor EZM2302 (0.1–1 μmol/L) or solvent control for 7 days (n = 3/group). F, Expression of selected ISGs in control-KO, Carm1-KO, cGAS-KO, and Carm1/cGAS double-KO (dKO) B16F10 cells analyzed by RT-qPCR (n = 3/group). G, T-cell cytotoxicity assay with control-KO, Carm1-KO, cGAS-KO, and Carm1/cGAS dKO B16F10 cells. Tumor cells were cocultured with OT-I CD8 T cells at indicated E/T ratios for 24 hours (n = 7–10 replicates/condition); live GFP-positive tumor cells were counted using a Celigo image cytometer. Data are shown as mean ± SEM. H and I, dsDNA damage in Carm1-KO versus control-KO B16F10 tumor cells based on labeling with γH2AX (H) and RAD51 (I) Abs. Representative immunofluorescence images (left) of γH2AX or RAD51 antibody labeling (red); nuclei labeled with DAPI. Quantifica- tion of number of γH2AX or RAD51 foci/nucleus (right). Data are shown as mean ± SEM. Scale bar, 10 μm. J, Detection of micronuclei in Carm1-KO and control-KO B16F10 tumor cells. DNA was labeled with DAPI; representative images (left) and quantification of cells positive for micronuclei (right). Data are shown as mean ± SEM. Scale bar, 10 μm. K, Analysis of Carm1-KO versus control-KO CD8+ T cells for dsDNA damage. OT-I CD8 T cells were plated 7 days post editing using CRIPSR/Cas9 as previously described and stained for CD8α, γH2AX, and DAPI. Representative immunofluorescence images of CD8α antibody labeling (red) and γH2AX antibody labeling (green); nuclei stained with DAPI (blue). Scale bar, 20 μm. Data shown in D to J are representative of three independent experiments. Bar graphs represent data summarized as mean ± SEM and were analyzed by unpaired two-sided Mann–Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant. Error bars for all qPCR data represent SD with three replicates per group.

Article Snippet: Doxycycline-Inducible Expression of Carm1 In Vivo C57BL/6 mice bearing Carm1-KO B16F10 tumor cells (transduced with Dox–CARM1 or empty vector constructs) were fed a doxycycline-containing diet (625 ppm; Envigo Teclad) until the experimental endpoint (18 days).

Techniques: Activation Assay, RNA Sequencing, Control, Biomarker Discovery, Quantitative RT-PCR, Solvent, Expressing, Cytotoxicity Assay, Cytometry, Labeling, Immunofluorescence, Antibody Labeling, Staining, MANN-WHITNEY

Journal: Cancer Discovery

Article Title: CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells

doi: 10.1158/2159-8290.cd-20-1144

Figure Lengend Snippet: Figure 5. CARM1 inhibition overcomes resistance to checkpoint blockade. A, Treatment of Carm1-KO or control-KO B16F10 tumors with CTLA4 or isotype control antibodies (n = 8 mice/group). Tumor growth (left) and survival of tumor-bearing mice (right) are shown. Mice bearing comparable tumor volume (∼50 mm3) were randomized into different treatment groups. B, Treatment of B16F10 tumors with CARM1 inhibitor (EZM2302) or vehicle control in combination with CTLA4 or isotype control antibodies (n = 8 mice/group). EZM2302 (150 mg/kg twice a day) was orally administered for 2 weeks (days 7–21). C, Treatment of Carm1-KO or control-KO 4T1 tumors with anti-CTLA4 or isotype control antibodies (n = 8 mice/group). D, Change in number of spontaneous lung metastases (left) and representative images of lung metastases formed by 4T1 tumors treated as described in C (right; n = 8 mice/group). E, Quantifica- tion of tumor-infiltrating CD8 T cells in Carm1-KO and control-KO B16F10 tumors (n = 8 mice/group) following treatment with CTLA4 or isotype control anti- bodies (day 18 after tumor cell implantation). Representative flow plots (left) and quantification of CD8 T cells as percentage of CD3+ cells and per gram of tumor (middle and right, respectively). F and G, Quantification of PD-1–positive and PD-1/TIM3 double-positive tumor-infiltrating CD8+ T cells for experiment described in E. H and I, Quantification of CD8 T cells expressing effector markers (GZMB and IFNγ), migratory cross-presenting cDC1 (CD45+/CD3−/F4/80-/ CD11c+/MHC-IIhigh/CD103+/CD11b−), and NK cells (CD45+/CD3−/CD49b+) per gram of tumor for the experiment described in E. Data shown are representative of two experiments. Two-way ANOVA was used to determine statistical significance for time points when all mice were viable for tumor measurement. Log- rank (Mantel–Cox) test was used to determine statistical significance for survival of mice. Bar graphs represent data summarized as mean ± SEM and were analyzed by unpaired two-sided Mann–Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Doxycycline-Inducible Expression of Carm1 In Vivo C57BL/6 mice bearing Carm1-KO B16F10 tumor cells (transduced with Dox–CARM1 or empty vector constructs) were fed a doxycycline-containing diet (625 ppm; Envigo Teclad) until the experimental endpoint (18 days).

Techniques: Inhibition, Control, Expressing, MANN-WHITNEY

Journal: Cancer Discovery

Article Title: CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells

doi: 10.1158/2159-8290.cd-20-1144

Figure Lengend Snippet: Figure 6. TDRD3 and MED12 are downstream effectors of CARM1. A, Comparison of ISG expression in control-KO, Tdrd3-KO, cGAS-KO, and Tdrd3- cGAS dKO B16F10 cells by RT-qPCR (n = 3/group). B, Comparison of ISG expression in control-KO, Med12-KO, cGAS-KO, and Med12-cGAS dKO B16F10 cells by RT-qPCR (n = 3/group). C, Immunofluorescence analysis of dsDNA damage by γH2AX antibody staining (red foci; nuclei labeled with DAPI) in control-KO, Carm1-KO, Tdrd3-KO, and Med12-KO B16F10 tumor cells (left). Images for control-KO and Carm1-KO reshown from Fig. 4 to illustrate com- parison to other KO tumor cell lines. Quantification of number of γH2AX foci/nucleus for each cell line (right). Scale bar, 10 μm. D, Analysis of micronuclei in control-KO, Carm1-KO, Tdrd3-KO, and Med12-KO B16F10 tumor cells using DAPI as a DNA stain. Representative images (left) and quantification of percentage of cells with micronuclei (right). Scale bar, 10 μm. E, Tumor growth and survival of mice bearing control-KO and Tdrd3-KO B16F10 tumors. Mice were treated with CD8 T cell–depleting or isotype control antibodies (n = 8–10 mice/group). F, Tumor growth and survival of control-KO and Tdrd3- KO B16F10 tumors in immunocompetent and T cell–deficient (Tcra-KO) mice (n = 8–10 mice/group). G, Tumor growth and survival of anti-CTLA4 or isotype control antibody treated mice bearing Tdrd3-KO or control-KO B16F10 tumors (n = 8 mice/group). H, Arginine methylation of Med12 by CARM1. Immunoprecipitation of MED12 protein from nuclear extracts of control-KO, Carm1-KO, or Tdrd3-KO B16F10 tumor cells, followed by Western blot detection with an antibody specific for asymmetric dimethylation of arginine residues (ADMA; left). Western blot analysis of nuclear extracts from the same cell lines with antibodies for the indicated proteins (right). I, Effect of Carm1 on interaction of MED12 with histone H3. Immunoprecipitation of MED12 protein from control-KO or Carm1-KO B16F10 tumor cells, followed by Western blot detection with histone H3 antibody (top). Input levels of histone H3 in immunoprecipitated samples are shown (middle); quantification of histone H3 bound to MED12 normalized to total histone H3 (bottom). Graph shows proposed biochemical interactions (right). Two-way ANOVA was used to determine statistical significance for time points when all mice were viable for tumor measurement. Statistical significance for survival of mice in each treatment group was calculated by log-rank (Mantel–Cox) test. Bar graphs represent data summarized as mean ± SEM and were analyzed by unpaired two-sided Mann–Whitney test. Data are representative of three (A–H) and two (I) experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Doxycycline-Inducible Expression of Carm1 In Vivo C57BL/6 mice bearing Carm1-KO B16F10 tumor cells (transduced with Dox–CARM1 or empty vector constructs) were fed a doxycycline-containing diet (625 ppm; Envigo Teclad) until the experimental endpoint (18 days).

Techniques: Comparison, Expressing, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Labeling, Methylation, Immunoprecipitation, Western Blot, MANN-WHITNEY