Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: A flowchart summarises groups of animals used in different experiments and the schedule of nose-poke operant conditioning. The animals were habituated to the operant chamber (Days 1 to 4) and divided into 5 experiments and 13 groups. Experiment 1: non-conditioned animals (Group 1) were subjected to an operant chamber, but no pellets were dispensed. The conditioned animals (Group 2) were presented with sucrose pellet in response to poking at the active port. The Group 2 rats were trained for sucrose pellet self-administration for 15 min twice/day from the 5th to the 11th day. On the 12th day, one group of rats was subjected to OFT followed by a probe trial, whilst the other group was subjected to LDB followed by a probe trial. These rats were killed 15 min after the probe trial, and one cohort was used for molecular biology, whereas the other was perfused for immunofluorescence analysis. Experiments 2–4: after habituation, animals were operated for cannula implantation and were allowed to recover (5th to 11th day). The cannulated animals were subjected to operant conditioning for 15 min/day twice a day till a steady baseline was achieved (12th to 18th). Experiment 2: the PRMT4 siRNA (Groups 4, 5) and scrambled siRNA (Group 3) were infused in the basolateral amygdala of the conditioned animals on the 18th day after the training session. Experiment 3: PRMT4 (CARM1) inhibitor and aCSF were administered on the 18th day after the training session in rats belonging to Groups 7, 8 and 6, respectively. Animals belonging to Groups 3, 4, 6 and 7 were tested (OFT, LDB and probe trial) on the 19th day (24 h post-infusion) and were killed 15 min after the probe trial. Group 5 and 8 rats were tested for nose poking from the 19th to the 22nd day. On the 23rd day,OFT, LDB and probe trials were conducted, and rats were sacrificed 15 min after the probe trial. Experiment 4: the PRMT4 siRNA/PRMT4 inhibitor/aCSF-treated animals belonging to Groups 9–11 were subsequently treated with NPY peptide on the 19th day, and OFT, LDB, followed by probe trial, were conducted 1 h after NPY infusion. Brains were isolated for the molecular analyses immediately after the probe trial in Groups 1–11. *Separate groups of rats were generated for immunohistochemistry. Experiment 5: cannulated animals were divided into two groups before training. Rats belonging to Group 12 were infused with scrambled siRNA, whereas Group 13 was administered with PRMT4 siRNA on the 11th day and sacrificed on the 12th day

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Immunofluorescence, Isolation, Generated, Immunohistochemistry

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Reward conditioning alters the expression of PRMTs and NPY in the amygdala. A Nose-poke activity of the rats trained for sucrose pellet self-administration and non-conditioned controls for 6 days (from Days 6 to 11 in Experiment 1). B Nose-poke activity of the rats in active port during 5 min probe trial (* p < 0.05; ** p < 0.01; *** p < 0.001 versus non-conditioned). C Schematic representation of the amygdala region used for the analysis. Effect of conditioning on the D mRNA levels of different PRMTs, E , F protein levels of PRMT4 and c-FOS in the amygdala. Graph representing G mRNA and H protein expression of NPY in the conditioned and non-conditioned rats (** p < 0.01; *** p < 0.001 versus non-conditioned). Values ( n = 5/group) are represented as means (± SEM)

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Expressing, Activity Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: PRMT4 and CBP-mediated histone modifications are enriched at the NPY promoter of conditioned rats. A Schematic representation of the NPY promoter. Effect of reward conditioning on B PRMT4 , C H3R17me2a levels at NPY promoter. D Co-immunoprecipitation (Co-IP) and reverse immunoprecipitation assays showing interactions of PRMT4 with CBP. Effect of reward conditioning on E CBP occupancy, F H3K14ac and G PRMT4-CBP co-occupancy at NPY promoter (* p < 0.05, ** p < 0.01, *** p < 0.001 versus non-conditioned). Values ( n = 4–5/group) are represented as means (± SEM)

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Reward conditioning enhances the expression of PRMT4 and NPY in the neurons of the basolateral amygdala (BLA). A Representative confocal images (20 × ) show PRMT4 (green) and NPY-positive cells (red), DAPI (blue), and their colocalization. The merged panels indicate NPY-positive cells co-expressing PRMT4 (arrows). PRMT4 predominantly colocalised with DAPI. The graph shows C PRMT4 immunoreactive area, and D the percentage of NPY-positive area in the BLA of conditioned and non-conditioned rats. The percentage of PRMT4-positive cells increased in the BLA of conditioned rats as compared to non-conditioned controls. Values ( n = 5/group) are represented as means (± SEM) and *** p < 0.001 versus non-conditioned

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Expressing

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: PRMT4-NPY axis in the basolateral amygdala is essential for the nose-poke activity of conditioned rats. A Schematic representation (left) and photomicrograph (right) of the coronal section of rat brain showing the position of the cannula targeted towards Basolateral Amygdala (BLA) at coordinates AP: − 3.14 mm, ML: ± 5 mm, DV: 4 mm (Paxinos and Watson ). Effect of intra-BLA infusion of B PRMT4 siRNA, C PRMT4 inhibitor on nose-poke activity of the reward conditioned rats (*** p < 0.001 versus non-conditioned; ### p < 0.001 versus conditioned + scrambled siRNA-24 h or conditioned + aCSF-24 h; $$$ p < 0.001 versus conditioned + PRMT4 siRNA or conditioned + PRMT4 inhibitor), D effect of NPY administration on PRMT4 siRNA/inhibitor-treated rats ( $$$ p < 0.001 versus conditioned + PRMT4 siRNA-24 h or conditioned + PRMT4 inhibitor-24 h, the number of nose pokes shown in the figure was recorded on the 19th day, 1 h after NPY administration during a 5-min probe trial session). Graphs representing the effect of intra-BLA infusion of scrambled siRNA, aCSF, PRMT4 siRNA, PRMT4 inhibitor, NPY peptide, and PRMT4 siRNA + NPY and PRMT4 inhibitor + NPY on E anxiety-like behaviours; L time spent in light compartment and D time spent in dark compartment, and F locomotion of conditioned rats (*** p < 0.001 versus conditioned + scrambled siRNA-24 h or conditioned + aCSF-24 h). Values ( n = 5–8/group) are represented as means (± SEM)

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Activity Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: NPY expression in the amygdala of conditioned rats declined with the reduction in PRMT4 function by siRNA or inhibitor. Effect of A PRMT4 siRNA, B PRMT4 inhibitor and C PRMT4 siRNA/inhibitor co-administered with NPY peptide on PRMT4 mRNA expression in the amygdala. D Representative photomicrograph of western blot and E relative quantification of PRMT4 protein levels in conditioned rats infused with scrambled siRNA and PRMT4 siRNA. Graph representing the effect of F PRMT4 siRNA, G PRMT4 inhibitor and H PRMT4 siRNA/inhibitor co-administered with NPY peptide on NPY mRNA levels in the amygdala. I Graph representing NPY protein levels in PRMT4 siRNA-treated animals as estimated by ELISA. The intra-BLA infusion of PRMT4 siRNA/PRMT4 inhibitor caused a reduction in the PRMT4 and NPY expression, which was recovered in 5 days (** p < 0.01, *** p < 0.001 versus non-conditioned; ## p < 0.01, ### p < 0.001 versus Conditioned + aCSF/scrambled siRNA-24 h; $$$ p < 0.001 versus Conditioned + NPYp)

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Expressing, Western Blot, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Effect of PRMT4 siRNA and PRMT4 inhibitor on the PRMT4 immunoreactivity in the BLA. Representative images (× 20) with PRMT4 (green) and NeuN-positive cells (red), and their colocalization (yellow). The merged panels indicate NeuN-positive cells co-expressing PRMT4 (arrows). PRMT4 predominantly colocalised with NeuN (C) Graph represents the intensity of PRMT4 immunofluorescence in different treatment groups (** p < 0.01 versus non-conditioned; ### p < 0.001 versus conditioned + scrambled siRNA-24 h)

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Expressing, Immunofluorescence

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: PRMT4-mediated histone arginine methylation regulates the NPY promoter. Effect of intra-BLA administration of scrambled siRNA, PRMT4 siRNA, NPY peptide and PRMT4 siRNA + NPY peptide on levels of A PRMT4, B H3R17me2a and C CBP occupancy at the NPY promoters. Values ( n = 5/group) are represented as means (± SEM) and (* p < 0.05, *** p < 0.001 versus non-conditioned; ### p < 0.001 versus conditioned + scrambled siRNA-24 h)

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Methylation

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Effect of intra-BLA administration of PRMT4 siRNA in the naïve control rats on the levels of NPY mRNA ( A ), PRMT4 mRNA ( B ) and protein ( C and D ), and H3R17me2a ( E ) and PRMT4 ( F ) occupancy at the NPY promoter. Naïve rats infused with PRMT4 siRNA demonstrated a reduction in levels of PRMT4 and H3R17me2a at the NPY promoter, resulting in low NPY expression

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Control, Expressing

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Schematic representation of NPY gene regulation in the amygdala and its effect on reward and reinforcement. Reward conditioning upregulates PRMT4, which in turn methylates 17th arginine in histone 3 (H3R17me2a) at the NPY promoter. PRMT4 cooperates with CBP and augments histone acetylation (H3K14ac) at the NPY promoter. These events remodel the NPY promoter to facilitate the NPY expression and promote reward-seeking activity. The PRMT4 siRNA infusion reversed the NPY promoter remodelling due to reward conditioning, attenuated NPY levels, and reduced nose-poke activity. NPY peptide administration following the siRNA infusion normalised the positive reinforcement. Therefore, PRMT4 in association with CBP seems to play an essential role in NPY expression within the framework of basolateral amygdala and reward processing

Article Snippet: The stock solution for PRMT4 inhibitor (217531, Merck) was prepared in DMSO to make 1.8 mM and was further diluted in aCSF to 100 μM and infused at the dosage of 25 μM/0.5 μl (IC 50 = 50.7 μM, Supplementary Fig. S2).

Techniques: Expressing, Activity Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: A flowchart summarises groups of animals used in different experiments and the schedule of nose-poke operant conditioning. The animals were habituated to the operant chamber (Days 1 to 4) and divided into 5 experiments and 13 groups. Experiment 1: non-conditioned animals (Group 1) were subjected to an operant chamber, but no pellets were dispensed. The conditioned animals (Group 2) were presented with sucrose pellet in response to poking at the active port. The Group 2 rats were trained for sucrose pellet self-administration for 15 min twice/day from the 5th to the 11th day. On the 12th day, one group of rats was subjected to OFT followed by a probe trial, whilst the other group was subjected to LDB followed by a probe trial. These rats were killed 15 min after the probe trial, and one cohort was used for molecular biology, whereas the other was perfused for immunofluorescence analysis. Experiments 2–4: after habituation, animals were operated for cannula implantation and were allowed to recover (5th to 11th day). The cannulated animals were subjected to operant conditioning for 15 min/day twice a day till a steady baseline was achieved (12th to 18th). Experiment 2: the PRMT4 siRNA (Groups 4, 5) and scrambled siRNA (Group 3) were infused in the basolateral amygdala of the conditioned animals on the 18th day after the training session. Experiment 3: PRMT4 (CARM1) inhibitor and aCSF were administered on the 18th day after the training session in rats belonging to Groups 7, 8 and 6, respectively. Animals belonging to Groups 3, 4, 6 and 7 were tested (OFT, LDB and probe trial) on the 19th day (24 h post-infusion) and were killed 15 min after the probe trial. Group 5 and 8 rats were tested for nose poking from the 19th to the 22nd day. On the 23rd day,OFT, LDB and probe trials were conducted, and rats were sacrificed 15 min after the probe trial. Experiment 4: the PRMT4 siRNA/PRMT4 inhibitor/aCSF-treated animals belonging to Groups 9–11 were subsequently treated with NPY peptide on the 19th day, and OFT, LDB, followed by probe trial, were conducted 1 h after NPY infusion. Brains were isolated for the molecular analyses immediately after the probe trial in Groups 1–11. *Separate groups of rats were generated for immunohistochemistry. Experiment 5: cannulated animals were divided into two groups before training. Rats belonging to Group 12 were infused with scrambled siRNA, whereas Group 13 was administered with PRMT4 siRNA on the 11th day and sacrificed on the 12th day

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Immunofluorescence, Isolation, Generated, Immunohistochemistry

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Reward conditioning alters the expression of PRMTs and NPY in the amygdala. A Nose-poke activity of the rats trained for sucrose pellet self-administration and non-conditioned controls for 6 days (from Days 6 to 11 in Experiment 1). B Nose-poke activity of the rats in active port during 5 min probe trial (* p < 0.05; ** p < 0.01; *** p < 0.001 versus non-conditioned). C Schematic representation of the amygdala region used for the analysis. Effect of conditioning on the D mRNA levels of different PRMTs, E , F protein levels of PRMT4 and c-FOS in the amygdala. Graph representing G mRNA and H protein expression of NPY in the conditioned and non-conditioned rats (** p < 0.01; *** p < 0.001 versus non-conditioned). Values ( n = 5/group) are represented as means (± SEM)

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Expressing, Activity Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: PRMT4 and CBP-mediated histone modifications are enriched at the NPY promoter of conditioned rats. A Schematic representation of the NPY promoter. Effect of reward conditioning on B PRMT4 , C H3R17me2a levels at NPY promoter. D Co-immunoprecipitation (Co-IP) and reverse immunoprecipitation assays showing interactions of PRMT4 with CBP. Effect of reward conditioning on E CBP occupancy, F H3K14ac and G PRMT4-CBP co-occupancy at NPY promoter (* p < 0.05, ** p < 0.01, *** p < 0.001 versus non-conditioned). Values ( n = 4–5/group) are represented as means (± SEM)

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Reward conditioning enhances the expression of PRMT4 and NPY in the neurons of the basolateral amygdala (BLA). A Representative confocal images (20 × ) show PRMT4 (green) and NPY-positive cells (red), DAPI (blue), and their colocalization. The merged panels indicate NPY-positive cells co-expressing PRMT4 (arrows). PRMT4 predominantly colocalised with DAPI. The graph shows C PRMT4 immunoreactive area, and D the percentage of NPY-positive area in the BLA of conditioned and non-conditioned rats. The percentage of PRMT4-positive cells increased in the BLA of conditioned rats as compared to non-conditioned controls. Values ( n = 5/group) are represented as means (± SEM) and *** p < 0.001 versus non-conditioned

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Expressing

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: PRMT4-NPY axis in the basolateral amygdala is essential for the nose-poke activity of conditioned rats. A Schematic representation (left) and photomicrograph (right) of the coronal section of rat brain showing the position of the cannula targeted towards Basolateral Amygdala (BLA) at coordinates AP: − 3.14 mm, ML: ± 5 mm, DV: 4 mm (Paxinos and Watson ). Effect of intra-BLA infusion of B PRMT4 siRNA, C PRMT4 inhibitor on nose-poke activity of the reward conditioned rats (*** p < 0.001 versus non-conditioned; ### p < 0.001 versus conditioned + scrambled siRNA-24 h or conditioned + aCSF-24 h; $$$ p < 0.001 versus conditioned + PRMT4 siRNA or conditioned + PRMT4 inhibitor), D effect of NPY administration on PRMT4 siRNA/inhibitor-treated rats ( $$$ p < 0.001 versus conditioned + PRMT4 siRNA-24 h or conditioned + PRMT4 inhibitor-24 h, the number of nose pokes shown in the figure was recorded on the 19th day, 1 h after NPY administration during a 5-min probe trial session). Graphs representing the effect of intra-BLA infusion of scrambled siRNA, aCSF, PRMT4 siRNA, PRMT4 inhibitor, NPY peptide, and PRMT4 siRNA + NPY and PRMT4 inhibitor + NPY on E anxiety-like behaviours; L time spent in light compartment and D time spent in dark compartment, and F locomotion of conditioned rats (*** p < 0.001 versus conditioned + scrambled siRNA-24 h or conditioned + aCSF-24 h). Values ( n = 5–8/group) are represented as means (± SEM)

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Activity Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: NPY expression in the amygdala of conditioned rats declined with the reduction in PRMT4 function by siRNA or inhibitor. Effect of A PRMT4 siRNA, B PRMT4 inhibitor and C PRMT4 siRNA/inhibitor co-administered with NPY peptide on PRMT4 mRNA expression in the amygdala. D Representative photomicrograph of western blot and E relative quantification of PRMT4 protein levels in conditioned rats infused with scrambled siRNA and PRMT4 siRNA. Graph representing the effect of F PRMT4 siRNA, G PRMT4 inhibitor and H PRMT4 siRNA/inhibitor co-administered with NPY peptide on NPY mRNA levels in the amygdala. I Graph representing NPY protein levels in PRMT4 siRNA-treated animals as estimated by ELISA. The intra-BLA infusion of PRMT4 siRNA/PRMT4 inhibitor caused a reduction in the PRMT4 and NPY expression, which was recovered in 5 days (** p < 0.01, *** p < 0.001 versus non-conditioned; ## p < 0.01, ### p < 0.001 versus Conditioned + aCSF/scrambled siRNA-24 h; $$$ p < 0.001 versus Conditioned + NPYp)

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Expressing, Western Blot, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Effect of PRMT4 siRNA and PRMT4 inhibitor on the PRMT4 immunoreactivity in the BLA. Representative images (× 20) with PRMT4 (green) and NeuN-positive cells (red), and their colocalization (yellow). The merged panels indicate NeuN-positive cells co-expressing PRMT4 (arrows). PRMT4 predominantly colocalised with NeuN (C) Graph represents the intensity of PRMT4 immunofluorescence in different treatment groups (** p < 0.01 versus non-conditioned; ### p < 0.001 versus conditioned + scrambled siRNA-24 h)

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Expressing, Immunofluorescence

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: PRMT4-mediated histone arginine methylation regulates the NPY promoter. Effect of intra-BLA administration of scrambled siRNA, PRMT4 siRNA, NPY peptide and PRMT4 siRNA + NPY peptide on levels of A PRMT4, B H3R17me2a and C CBP occupancy at the NPY promoters. Values ( n = 5/group) are represented as means (± SEM) and (* p < 0.05, *** p < 0.001 versus non-conditioned; ### p < 0.001 versus conditioned + scrambled siRNA-24 h)

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Methylation

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Effect of intra-BLA administration of PRMT4 siRNA in the naïve control rats on the levels of NPY mRNA ( A ), PRMT4 mRNA ( B ) and protein ( C and D ), and H3R17me2a ( E ) and PRMT4 ( F ) occupancy at the NPY promoter. Naïve rats infused with PRMT4 siRNA demonstrated a reduction in levels of PRMT4 and H3R17me2a at the NPY promoter, resulting in low NPY expression

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Control, Expressing

Journal: Cellular and Molecular Neurobiology

Article Title: Histone Arginine Methylation Regulates Neuropeptide Y Expression in the Basolateral Amygdala to Promote Reward-Seeking Behaviour

doi: 10.1007/s10571-025-01614-5

Figure Lengend Snippet: Schematic representation of NPY gene regulation in the amygdala and its effect on reward and reinforcement. Reward conditioning upregulates PRMT4, which in turn methylates 17th arginine in histone 3 (H3R17me2a) at the NPY promoter. PRMT4 cooperates with CBP and augments histone acetylation (H3K14ac) at the NPY promoter. These events remodel the NPY promoter to facilitate the NPY expression and promote reward-seeking activity. The PRMT4 siRNA infusion reversed the NPY promoter remodelling due to reward conditioning, attenuated NPY levels, and reduced nose-poke activity. NPY peptide administration following the siRNA infusion normalised the positive reinforcement. Therefore, PRMT4 in association with CBP seems to play an essential role in NPY expression within the framework of basolateral amygdala and reward processing

Article Snippet: PRMT4 siRNA 5′ (CUU-GAG-CAG-GGU-UAU-UGC-C) TT3′ and scrambled siRNA (with anti-sense and sense dTdT 3′ overhangs) were procured from Eurogentec (Liege, Belgium) and reconstituted in RNase-free water to create a 10 μg/μl stock solution.

Techniques: Expressing, Activity Assay

Journal: Advanced Science

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

doi: 10.1002/advs.202416809

Figure Lengend Snippet: CARM1 is highly expressed in ccRCC and promotes proliferation. A,B) CARM1 is highly expressed in ccRCC patients and is correlated with poor prognosis on the UALCAN database. C) CARM1 and H3R17me2a are highly expressed in ccRCC cells such as 786‐O, A498, and Caki‐1. D,E) Immunofluorescence staining using the tissue microarray revealed that CARM1 is highly expressed in ccRCC tissue samples. Scale bar = 100 µm. (Tumor n = 79, Normal n = 79) F) CcRCC patients with advanced T stage have higher CARM1 expression, which may be associated with poor prognosis. (T1 n = 53, T2 n = 23, T3 n = 3) G,H) Immunofluorescence staining using the tissue microarray revealed that H3R17me2a level is higher in ccRCC tissue samples. Scale bar = 100 µm. (Tumor n = 79, Normal n = 79) I) CcRCC patients with advanced T stage have higher H3R17me2a level, which may be associated with poor prognosis. (T1 n = 53, T2 n = 23, T3 n = 3) J) The siRNA targeting CARM1 mRNA can effectively interfere with CARM1 and decreases H3R17me2a in ccRCC cells. K–N) Reduction of CARM1 and H3R17me2a significantly inhibited the proliferation of ccRCC cells, as demonstrated by CCK‐8 (K), colony formation (L) and EdU proliferation assays (M‐N). Scale bar = 100 µm. Data are shown as mean ± SD. ** p < 0.01, *** p < 0.001.

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

Techniques: Immunofluorescence, Staining, Microarray, Expressing, CCK-8 Assay

Journal: Advanced Science

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

doi: 10.1002/advs.202416809

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

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

Techniques: Biomarker Discovery, Knock-Out, Knockdown

Journal: Advanced Science

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

doi: 10.1002/advs.202416809

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

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

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

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: PRMT4/CARM1 Is a Novel Factor Promoting DNA Double-Strand Break Repair.

doi: 10.1111/gtc.70031

Figure Lengend Snippet: FIGURE 3 | Catalytic activity of PRMT4 was indispensable for releasing PRMT4 from the DSB sites. (A) U2OS cells expressing GFP-PRMT4 (wild-type or R168A mutant) were subjected to laser micro-irradiation and live-cell imaging. The track of laser micro-irradiation was indicated by white triangles. Scale bar: 10 μm. (B) Relative GFP signal intensity at DSB sites in (A) was quantified. The signal intensity 1 min after laser micro- irradiation when the accumulation of PRMT4 reached maximum was set to 1 and plotted (mean ± SEM, n = 10). *p < 0.05.

Article Snippet: Soluble fraction was subjected to immunoprecipitation with an anti- GFP antibody coupled with magnetic beads (GFP- Trap_MA, ChromoTek, PlaneggMartinsried, Germany) or anti- PRMT4 antibody (Cell Signaling Technology, 12495T, 1 μg/100 μg cell extract) by rotating overnight at 4°C.

Techniques: Activity Assay, Expressing, Mutagenesis, Irradiation, Live Cell Imaging

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: PRMT4/CARM1 Is a Novel Factor Promoting DNA Double-Strand Break Repair.

doi: 10.1111/gtc.70031

Figure Lengend Snippet: FIGURE 4 | PRMT4 was a novel factor promoting DSB repair. (A) Genotyping of a PRMT4 KO cell line. The target site of gRNA and primers used for genotyping are indicated with the dashed red line and black arrows, respectively (upper). The result of the agarose gel electrophoresis was shown (lower). Amplification of β-actin gene was the loading control. (B) Cell extracts prepared from U2OS (wild type) and PRMT4 KO cell lines were ex- amined by immunoblotting with the indicated antibodies. (C) Neutral comet assay was carried out with U2OS (wild type) and PRMT4 KO cell line. Relative tail moments that were obtained by normalizing with the tail moment of the undamaged sample were plotted for damaged and repaired samples. Mean and standard error are indicated with the horizontal bars. ***p < 0.0001. (D) U2OS (wild type) and PRMT4 KO cell line were treated with 150 μg/mL phleomycin for 2 h. After washing out phleomycin, cells were further cultured for the indicated periods. Immunoblotting analysis with the indicated antibodies was carried out. H2AX and α-tubulin were loading controls for γH2AX and PRMT4, respectively.

Article Snippet: Soluble fraction was subjected to immunoprecipitation with an anti- GFP antibody coupled with magnetic beads (GFP- Trap_MA, ChromoTek, PlaneggMartinsried, Germany) or anti- PRMT4 antibody (Cell Signaling Technology, 12495T, 1 μg/100 μg cell extract) by rotating overnight at 4°C.

Techniques: Agarose Gel Electrophoresis, Amplification, Control, Western Blot, Neutral Comet Assay, Cell Culture