Journal: Tumour Virus Research

Article Title: PRMT5–mediated symmetric dimethylation of SHBs at Arg169 stabilizes SHBs and promotes angiogenesis and tumor growth

doi: 10.1016/j.tvr.2026.200340

Figure Lengend Snippet: PRMT interacts with SHBs. (A) Huh7 cells were co–transfected with plasmids encoding SHBs–Strep–Flag (or Strep–Flag control) together with Flag–PRMT9. Strep pull–down was followed by WB with anti–Flag and anti–SHBs to assess co–precipitation. (B) Huh7 and HepG2 cells were co–transfected with plasmids encoding SHBs–Strep–Flag (or Strep–Flag control) together with Flag–PRMT5 and analyzed by Strep pull–down and WB as in (A). (C) Huh7 and HepG2 cells were co–transfected with plasmids encoding Strep–Flag–PRMT5 and SHBs–myc. Strep pull–down was performed and precipitates were immunoblotted for SHBs and Flag to validate the interaction. (D) Direct interaction between SHBs and PRMT5 was tested by GST pull–down. Purified GST or GST–PRMT5 (Coomassie–stained gel, left) was incubated with in vitro–translated SHBs–Flag, and bound SHBs was detected by WB using anti–Flag (right). (E) Confocal microscopy showing subcellular localization of SHBs (red) and PRMT5 (green) with nuclear DAPI staining (blue). Merged images and a representative line–scan fluorescence intensity profile (right) are shown.

Article Snippet: Cells were co–transfected with plasmids encoding Flag–PRMT5 together with SHBs–Strep–Flag or SHBs/R169K–Strep–Flag for 40 h. Cells were then treated with cycloheximide (CHX; 200 μg/mL; MedChemExpress, #HY–12320) to inhibit de novo protein synthesis.

Techniques: Transfection, Control, Purification, Staining, Incubation, In Vitro, Confocal Microscopy, Fluorescence

Journal: Tumour Virus Research

Article Title: PRMT5–mediated symmetric dimethylation of SHBs at Arg169 stabilizes SHBs and promotes angiogenesis and tumor growth

doi: 10.1016/j.tvr.2026.200340

Figure Lengend Snippet: PRMT5 catalyzes symmetric dimethylation of SHBs at arginine 169. (A) Cells were transfected with plasmids encoding SHBs–Strep–Flag together with Flag–PRMT5 or control vector. Strep pull–down was followed by WB for SDMA and SHBs. Densitometric ratios (SDMA/IP–SHBs and SHBs/β–actin) are shown. (B) Cells expressing SHBs–Strep–Flag were transfected with control siRNA (NC) or two independent PRMT5 siRNAs. Strep pull–down/WB was used to assess SDMA on SHBs, and input blots confirmed PRMT5 knockdown and SHBs levels. (C) Cells expressing SHBs–Strep–Flag were treated with the PRMT5 inhibitor GSK591 (10 μM, 48 h) and analyzed by Strep pull–down/WB for SDMA and SHBs; densitometric ratios are shown. (D) Cells were transfected with plasmids encoding SHBs–Strep–Flag together with Flag–PRMT5 or Flag–PRMT5/E444Q. Strep pull–down was followed by WB for SDMA and SHBs. (E) Schematic of the in vitro methylation assay. Strep–Flag–PRMT5 and SHBs–Strep–Flag or SHBs/R169K–Strep–Flag were expressed in 293T cells, affinity–purified using streptavidin/Strep beads, eluted, and incubated ± SAM at 30 °C for 1.5 h prior to WB with SDMA antibody. (F) In vitro methylation reactions using the indicated purified proteins (WT SHBs or R169K mutant) incubated ± SAM. SDMA and total SHBs were detected by WB; SDMA/SHBs ratios are indicated above the blot.

Article Snippet: Cells were co–transfected with plasmids encoding Flag–PRMT5 together with SHBs–Strep–Flag or SHBs/R169K–Strep–Flag for 40 h. Cells were then treated with cycloheximide (CHX; 200 μg/mL; MedChemExpress, #HY–12320) to inhibit de novo protein synthesis.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Knockdown, In Vitro, Methylation, Affinity Purification, Incubation, Purification, Mutagenesis

Journal: Tumour Virus Research

Article Title: PRMT5–mediated symmetric dimethylation of SHBs at Arg169 stabilizes SHBs and promotes angiogenesis and tumor growth

doi: 10.1016/j.tvr.2026.200340

Figure Lengend Snippet: PRMT5 stabilizes SHBs protein expression in an Arg169–dependent manner. (A) Huh7 and HepG2 cells were co–transfected with plasmids encoding SHBs–Strep–Flag or SHBs/R169K–Strep–Flag together with increasing amounts of Flag–PRMT5 (0, 1, 3 μg). Whole–cell lysates were immunoblotted for SHBs, Flag, and β–actin; SHBs/β–actin ratios are shown above the blots. (B) Cells expressing SHBs–Strep–Flag or SHBs/R169K–Strep–Flag were transfected with NC or PRMT5 siRNAs (#1, #2). Lysates were immunoblotted for SHBs, PRMT5, and β–actin; SHBs/β–actin ratios are shown. (C–D) Cycloheximide (CHX) chase assays in (C) Huh7 and (D) HepG2 cells. Cells expressing SHBs or SHBs/R169K with vector or Flag–PRMT5 were treated with CHX for the indicated times (0–120 min), followed by WB for SHBs, Flag, and β–actin. Plots show relative SHBs levels normalized to time 0 with fitted linear regression (equations displayed). (E) HepG2 cells were co–transfected with plasmids encoding SHBs–Strep, HA–K48Ub, together with or without Flag–PRMT5, and treated with MG132 (20 μM) for 8 h, the ubiquitination levels of SHBs was evaluated via ubiquitination assay analysis. (F) HepG2 cells were co–transfected with plasmid encoding SHBs–Strep and TRIM21–myc (or control vector) and Flag–PRMT5 (or control vector), the cell lysates were subjected to immunoprecipitation using Strep–Tactin and analyzed by immunoblotting.

Article Snippet: Cells were co–transfected with plasmids encoding Flag–PRMT5 together with SHBs–Strep–Flag or SHBs/R169K–Strep–Flag for 40 h. Cells were then treated with cycloheximide (CHX; 200 μg/mL; MedChemExpress, #HY–12320) to inhibit de novo protein synthesis.

Techniques: Expressing, Transfection, Plasmid Preparation, Ubiquitin Proteomics, Control, Immunoprecipitation, Western Blot

Journal:

Article Title: Methylation of Histone H3 and H4 by PRMT5 Regulates Ribosomal RNA Gene Transcription

doi: 10.1002/jcb.22432

Figure Lengend Snippet: Association of PRMT5 with rRNA promoter is reduced in EBV transformed B-cells. A: PRMT5 level was monitored by Western blot analysis in EBV transformed cells (LCLs), EBV-infected and resting B cells. B: Resting B-cells and exponentially growing EBV transformed cells (LCL1, LCL2) were cross-linked with formaldehyde and chromatin from these cells was immunoprecipitated with anti-PRMT5 antibody. The pulled down DNA was either left uncut (U) or digested with Hpa II (H) or Msp I (M). rRNA promoter was amplified from all three sets of precipitated and digested DNA and analyzed by semi-quantitative PCR. The bar diagram represents association of PRMT5 with unmethylated and methylated rRNA promoter (as described in Fig. 2F).

Article Snippet: PRMT5 siRNA AND ITS TRANSFECTION HeLa cells were transfected at 70% confluency with 100 nM PRMTS siRNA (Santa Cruz, SC37022) or the control scrambled siRNA (Santa Cruz, SC37007) using Lipofectamine 2000.

Techniques: Transformation Assay, Western Blot, Infection, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Methylation

Journal:

Article Title: Methylation of Histone H3 and H4 by PRMT5 Regulates Ribosomal RNA Gene Transcription

doi: 10.1002/jcb.22432

Figure Lengend Snippet: PRMT5 expression remains unaltered in serum starved and serum replenished HeLa cells. A: Exponentially growing HeLa cells were serum starved (0%) for 72 h (−FBS) and then allowed to grow in presence of 10% serum for 3 h. 47S rRNA synthesis in these cells was compared by real time PCR and normalized to β-actin. B: Whole cell extracts from HeLa cells (−FBS and +FBS) were subjected to Western blot analysis with anti-PRMT5 antibody. The blot was reprobed with anti-GAPDH antibody for protein normalization. C: PRMT5 co-localizes with nucleolin in HeLa cell nucleolus. HeLa cells were stained with TRITC tagged mouse monoclonal antibody against nucleolin and with FITC-tagged rabbit polyclonal antibody against PRMT5. The cells were also stained with DAPI and visualized under fluorescence microscope. D: PRMT5 preferentially associates with unmethylated rRNA promoter in serum starved HeLa cells. Formaldehyde crosslinked chromatin prepared from serum starved (−FBS) and serum replenished (+FBS) HeLa cells was immunoprecipitated with anti-PRMT5 antibody (PRMT5) or preimmune sera (Preimn), and rRNA promoter was amplified from immunoprecipitated DNA and input DNA (Input). E: The immunoprecipitated DNA was either left undigested, or digested with Hpa II or Msp I. rRNA promoter was amplified from uncut, Hpa II and Msp I digested DNA by semi-quantitative PCR. The bar diagram represents PRMT5 binding to unmethylated and methylated rRNA promoter (as described in Fig. 2F).

Article Snippet: PRMT5 siRNA AND ITS TRANSFECTION HeLa cells were transfected at 70% confluency with 100 nM PRMTS siRNA (Santa Cruz, SC37022) or the control scrambled siRNA (Santa Cruz, SC37007) using Lipofectamine 2000.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Fluorescence, Microscopy, Immunoprecipitation, Amplification, Binding Assay, Methylation

Journal:

Article Title: Methylation of Histone H3 and H4 by PRMT5 Regulates Ribosomal RNA Gene Transcription

doi: 10.1002/jcb.22432

Figure Lengend Snippet: Ectopic expression of PRMT5 in HeLa cells inhibits the activity of both unmethylated and methylated rRNA promoter. A: Schematic representation of the rRNA promoter/luciferase reporter plasmid (pHrD-IRES-Luc) used in the transfection study. B: HeLa cells were transiently transfected with Flag tagged PRMT5 expression vector. The expression of PRMT5 protein was monitored by Western blot analysis using anti-Flag M2-monoclonal antibody. C: HeLa cells were transiently transfected with empty vector (0) or 2 and 4 µg of PRMT5 expression vector along with pHrD-IRES-Luc and the internal control pRL-TK. Cells harvested 48 h after transfection were analyzed for luciferase activity and normalized to pRL-TK. D: HeLa cells were transiently transfected with empty vector (0) and 4 µg of PRMT5 expression vector along with unmethylated or methylated pHrD-IRES-Luc and pRL-TK. Cells harvested 48 h after transfection were analyzed for luciferase activity and normalized to pRL-TK. E: HeLa cells were transfected with PRMT5 siRNA or control siRNA and expression of PRMT5 was monitored by western blot analysis at 24, 48, and 72 h after transfection. F: HeLa cells transfected with PRMT5 siRNA or control siRNA were harvested 24 h posttransfection and rRNA synthesis was monitored by real time PCR. Error bars represents standard deviation of triplicate measurements.

Article Snippet: PRMT5 siRNA AND ITS TRANSFECTION HeLa cells were transfected at 70% confluency with 100 nM PRMTS siRNA (Santa Cruz, SC37022) or the control scrambled siRNA (Santa Cruz, SC37007) using Lipofectamine 2000.

Techniques: Expressing, Activity Assay, Methylation, Luciferase, Plasmid Preparation, Transfection, Western Blot, Control, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Advanced Science

Article Title: Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues

doi: 10.1002/advs.202002602

Figure Lengend Snippet: Adipocyte‐specific knockout of Prmt5 (Prmt5 AKO ) leads to loss of fat mass in white adipose tissues. a) Targeting strategy for conditional knockout of Prmt5. Upper: Prmt5 gene structure showing exons (blue boxes), LoxP insertions (red triangles) and 5′ and 3′ untranslated regions (orange boxes). Middle: PRMT5 protein domain structure with amino acids numbers labeled. MTase is the catalytic domain. Lower: Cre‐mediated excision of LoxP‐flanked exon results in reading frame shift and a premature translational stop codon, generating a truncated protein containing only partial of the TM barrel domain. b) Efficient reduction of PRMT5 protein levels in epididymal White Adipose Tissue (eWAT) and inguinal White Adipose Tissue (iWAT), and brown adipose tissue (BAT) of the 2 month old male Prmt5 AKO (AKO) mice. c) Representative images of male WT (Prmt5 flox/flox ) and Prmt5 AKO mice at 10 month old (left panel), showing progressive reduction in body weight of the Prmt5 AKO mice relative to WT mice (right panel). d) Prmt5 AKO leads to progressive loss of total body fat, relative to fat mass in WT mice. c,d) n = 8, 6, and 4 pairs of male mice for 3‐, 6‐, and 10‐month‐old, respectively. e) Representative images of BAT and WAT depots from male mice showing progressive mass reduction of Prmt5 KO WAT with age. f,h,j) Weights of various BAT and WAT (iWAT, eWAT and anterior subcutaneous White Adipose Tissue, asWAT) depots from male mice at different ages, as shown in (e). g,i,k) H&E staining of eWAT sections from WT (Prmt5 flox/flox ) and Prmt5 AKO male mice at 3, 6, and 10month old. Scale bar: 50 µm. Data represent mean ± s.e.m. ( t ‐test: ** p < 0.01).

Article Snippet: 5 μg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody (Epigentek, A‐3005‐100) or with anti‐IgG as a negative control at 4 °C overnight.

Techniques: Knock-Out, Labeling, Staining

Journal: Advanced Science

Article Title: Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues

doi: 10.1002/advs.202002602

Figure Lengend Snippet: Prmt5 AKO mice are protected from high‐fat diet (HFD)‐induced fat accumulation. a) Growth curve of male (left) and female (right) WT (Prmt5 flox/flox ) and Prmt5 AKO mice during 10 week of HFD feeding. b) Weight gain of male (left) and female (right) WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 10 week of HFD feeding. a,b) n = 8 and 9 pairs of mice for male and female mice, respectively. c) Body weight, fat mass, and muscle mass of male (upper) and female (lower) WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 12 week of HFD feeding. n = 8 and 4 pairs of mice for male and female mice, respectively. d) WAT and BAT absolute weights from male (upper panel) and female (lower panel) WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 12 week of HFD, n = 5 pairs of mice. e) H&E staining of eWAT sections from male WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 12 week of HFD. Scale bar: 50 µm. Data represent mean ± s.e.m. ( t ‐test: * p < 0.05, ** p < 0.01).

Article Snippet: 5 μg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody (Epigentek, A‐3005‐100) or with anti‐IgG as a negative control at 4 °C overnight.

Techniques: Staining

Journal: Advanced Science

Article Title: Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues

doi: 10.1002/advs.202002602

Figure Lengend Snippet: Impaired glucose tolerance and insulin sensitivity in Prmt5 AKO mice. a–c) Blood glucose concentrations during glucose tolerance tests (GTT) performed on a) 3, b) 6, and c) 10–12 months old male WT (Prmt5 flox/flox ) and Prmt5 AKO mice (left). Area under curve (AUC) calculated based on data in left panel (right). n = 8, 8, and 7 pairs of mice for 3, 6, and 10–12 month old, respectively. d–f) Percentage changes of blood glucose concentrations during insulin tolerance tests (ITT) performed on d) 3‐, e) 6‐, and f) 10–12 month old male WT (Prmt5 flox/flox ) and Prmt5 AKO mice (left). Area above curve (AAC) calculated based on data in left panel (right). n = 6, 6, and 7 pairs of mice for 3, 6, and 10–12 month old, respectively. g) Blood glucose concentrations during glucose tolerance tests (GTT) performed on male WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 8 week of HFD (left). AUC calculated based on data in left panel (right), n = 8 pairs of male mice. h) Percentage changes of blood glucose concentrations during insulin tolerance tests (ITT) performed on male WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 14 week of HFD (left). AAC calculated based on data in left panel (right), n = 6 pairs of male mice. i,j) Blood insulin level of 4–6 months old male mice i)before and j) after 14 week HFD feeding, n = 4 pairs of male mice. Data represent mean ± s.e.m. ( t ‐test: * p < 0.05, ** p < 0.01).

Article Snippet: 5 μg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody (Epigentek, A‐3005‐100) or with anti‐IgG as a negative control at 4 °C overnight.

Techniques:

Journal: Advanced Science

Article Title: Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues

doi: 10.1002/advs.202002602

Figure Lengend Snippet: Prmt5 AKO mice exhibit lower metabolic rate and lower basal respiration in Prmt5 deficient adipocytes. a) O2 consumption measured by an indirect calorimetry and normalized to lean mass is shown for a 48 h cycle of 10‐month‐old male WT (Prmt5 flox/flox ) and Prmt5 AKO mice. b–d) Average day and night O2 consumption (b) VO2, CO2 production c) VCO2 and d) RER. a–d) n = 5 pairs of 10‐month‐old male mice. e) O2 consumption measured same as above on male WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 12 week of HFD. f–h) Average day and night O2 consumption f) VO2, CO2 production g) VCO2 and h) RER. e–h) n = 6 pairs of male mice after 12 week of HFD. i,j) Food intake of WT (Prmt5 flox/flox ) and Prmt5 AKO mice during i) control‐diet or j) HFD feeding. n = 3 groups of male mice for control‐diet (12‐month‐old) and HFD feeding, respectively. Each dot represents the average food consumption per day of two mice. k,l) Seahorse‐based measurement of oxygen consumption rate (OCR) on white adipocytes differentiated from preadipocytes isolated from iWAT of 8 week old WT (Prmt5 flox/flox ) and Prmt5 AKO mice. k) OCR was measured at basal state and after sequential addition of Oligomycin, FCCP, and Antimycin A/Rotenone to determine the l) Basal Respiration (BR), Maximal Respiration (MR), Spare Respiratory Capacity (SRC), Non‐Mitochondrial Oxygen Consumption (NMOC), and ATP Production (AP), respectively, cells were isolate from the iWAT of n = 4 pairs of male WT (Prmt5 flox/flox ) and Prmt5 AKO mice, measured in five technical replicates. Data represent mean ± s.e.m. ( t ‐test: * p < 0.05, ** p < 0.01).

Article Snippet: 5 μg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody (Epigentek, A‐3005‐100) or with anti‐IgG as a negative control at 4 °C overnight.

Techniques: Control, Isolation

Journal: Advanced Science

Article Title: Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues

doi: 10.1002/advs.202002602

Figure Lengend Snippet: Prmt5 AKO leads to abnormal lipid droplet biogenesis due to downregulating Bscl2 (Bscl2) expression. a) Prediction of Bscl2 as a PRMT5 target gene based on gene expression and phenotypic correlations. b) Role of seipin in lipid droplet biosynthesis at the endoplasmic reticulum (ER). c) Prmt5 and Bscl2 expression level in SVF preadipocytes from iWAT of WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 10 days of differentiation in vitro, n = 3 pairs of male mice at 8 week old. d) Expression levels of Bscl2 in the eWAT from 3‐, 6‐, and 10‐month‐old WT (Prmt5 flox/flox ) and Prmt5 AKO mice, n = 3 pairs of male mice. e) Pharmacological inhibition of PRMT5 (BLL3.3, 20 µ m ) reduces Bscl2 expression in 3T3‐L1 cells. DMSO is vehicle control, n = 3. f) Luciferase assay of HEK‐293A cells co‐transfected with Prmt5 and pGL‐Bscl2 1kb promoter (pGL3‐Bscl2‐1kb), n = 4 biological replicates for each independent experiment. g) Bscl2 mRNA levels in control and Prmt5 ‐overexpressed 3T3‐L1 cell lines, n = 3 biological repeats, and each sample was detected in three technical repeats. h) Oil red staining of SVF preadipocytes from male WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 10 days of differentiation in vitro (4 days of induction medium followed by 6 days of differentiation medium). Arrows and arrowheads indicate abnormally large and small lipid droplets, respectively. Scale bar: 50 µm. i,j) Representative Transmission Electron Microscopy (TEM) images of eWAT from i) 10‐month‐old and j) 12‐week HFD fed WT (Prmt5 flox/flox ) and Prmt5 AKO mice, n = 3 pairs of male mice. k) Representative images of SVF preadipocytes from iWAT of male Prmt5 AKO mice after 10 day of differentiation treated with GFP‐expressing adenovirus (control) or Bscl2/GFP co‐expressing adenovirus, white arrowheads indicate the supersized lipid droplets, scale bar: 50 µm. l) Size distribution lipid droplets in GFP adenovirus or Bscl2/GFP adenovirus treated SVF preadipocytes from Prmt5 AKO mice after 10 day of differentiation. In total, 535 LDs and 495 LDs from six different Prmt5 AKO mice were counted. Five random microscopic images were captured and counted from either Ad‐GFP or Ad‐Bscl2/GFP groups. m) Average lipid droplet composition of different diameters in one cell and percentage of cells containing lipid droplet with diameter of >50 pixels from GFP adenovirus or Bscl2‐GFP adenovirus treated SVF preadipocytes from Prmt5 AKO mice after 10 days of differentiation. Data represent mean ± s.e.m. ( t ‐test: * p < 0.05, ** p < 0.01). Diameters in l&m are in pixels, 1 pixel = 0.3223 µm.

Article Snippet: 5 μg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody (Epigentek, A‐3005‐100) or with anti‐IgG as a negative control at 4 °C overnight.

Techniques: Expressing, Gene Expression, In Vitro, Inhibition, Control, Luciferase, Transfection, Staining, Transmission Assay, Electron Microscopy

Journal: Advanced Science

Article Title: Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues

doi: 10.1002/advs.202002602

Figure Lengend Snippet: PRMT5 interacts with and methylates the transcriptional pausing factor SPT5 to affect its binding to the Bscl2 TSS. a) ChIP‐sequencing results showing co‐occupancy of PRMT5, SPT5, and RNA polymerase II (POL2) at the same TSS region (boxed region) of Bscl2 genome (shown in the bottom panel). Note that PRMT5 only occupies the region in differentiated (D1) but not undifferentiated (D0) 3T3‐L1 cell. SPT5 ChIP‐seq is based on results from mouse embryonic stem cell (mES) and POL2 ChIP‐seq is based on results from eWAT. [ ²⁷ , ²⁸ ] b,c) ChIP‐qPCR analysis of b) PRMT5 and c) SPT5 binding to the boxed region shown in a in undifferentiated (D0) and differentiated 3T3‐L1 cells (D2). d) SPT5 ChIP‐qPCR results on Prmt5 overexpressing 3T3‐L1 cells and control cells. e) SPT5 ChIP‐qPCR of Bscl2 promoter in SVF cells from WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 6 day of differentiation in vitro. f) Lysates from undifferentiated (D0) and differentiated 3T3‐L1 cells (D6) were immunoprecipitated (IP) with SPT5 antibody and blotted with SPT5 and PRMT5 antibodies showing association with PRMT5 only in differentiated cells. g) Proximity ligation assay (PLA) using PRMT5 and SPT5 antibodies in WT undifferentiated (D0) and differentiated SVF preadipocytes (D4) showing PRMT5 interacts with SPT5 in nucleus only in differentiated cells. h) Lysates from eWAT of WT (Prmt5 flox/flox ) and Prmt5 AKO mice were immunoprecipitated (IP) with SYM10 antibody and blotted with SPT5 antibody. ( t ‐test: ** p < 0.01).

Article Snippet: 5 μg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody (Epigentek, A‐3005‐100) or with anti‐IgG as a negative control at 4 °C overnight.

Techniques: Binding Assay, ChIP-sequencing, ChIP-qPCR, Control, In Vitro, Immunoprecipitation, Proximity Ligation Assay

Journal: Advanced Science

Article Title: Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues

doi: 10.1002/advs.202002602

Figure Lengend Snippet: Prmt5 regulates fatty acid metabolic pathways through interacting with SREBP1a. a) Heatmap of top 25 significant changed TAGs from eWAT of WT (Prmt5 flox/flox ) and Prmt5 AKO mice (upper) and list of TAGs that were decrease by >2‐fold in Prmt5 AKO compared to WT (Prmt5 flox/flox ) eWAT (lower), n = 5 pairs male mice of 6‐month‐old. b) Concentration of FFA, cholesterol, HDL, LDL, and TAG from the serum of WT (Prmt5 flox/flox ) and Prmt5 AKO mice, n = 4 pairs male mice of 6‐month‐old. c) Heatmap of top 25 significant changed TAGs from WT (Prmt5 flox/flox ) and Prmt5 AKO mice (upper) and list of TAGs that were decreased by >1.6‐fold in Prmt5 AKO compared to WT (Prmt5 flox/flox ) eWAT (lower), after 12‐week of HFD, n = 4 pairs of male mice. d) Concentration of FFA, cholesterol, HDL, LDL, and TAG from the serum of WT (Prmt5 flox/flox ) and Prmt5 AKO mice, n = 3 pairs male mice after 12‐week HFD feeding. e) Fold change of Free Fatty Acids (FFA) in eWAT of WT (Prmt5 flox/flox ) and Prmt5 AKO mice. f) Ratio of 18:1/16:0 FFA indicating de novo lipogenesis. e,f) n = 5 pairs of 6‐month‐old male mice. g,h) Expression of g) adipogenic and h) lipogenic genes in eWAT from WT (Prmt5 flox/flox ) and Prmt5 AKO mice, n = 3 pairs of male mice at 6‐month‐old. i) Representative Western blots showing relative protein levels of PPAR γ , ATGL, CEBP α , FABP4, and β ‐Tubulin from eWAT of male WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 12‐week of HFD. j) Heatmap from RNA‐sequencing data showing relative expression of genes related to Fatty acid transport, TAG synthesis and LD biogenesis in eWAT of WT (Prmt5 flox/flox ) and Prmt5 AKO mice, n = 3 pairs male mice of 6‐month‐old. k) A diagramatic pathway showing enzymes involved in triacylglycerol (TAG) synthesis, red arrows represent genes down‐regulated in Prmt5 AKO eWAT. i) Lysates from 293T cells overexpressing Flag‐SRBEP1a or Flag‐SRBEP1a and Myc‐PRMT were immunoprecipitated (IP) with Flag or PRMT5 antibody and blotted with Flag, PRMT5, SYM10, and GAPDH antibodies. m) Representative Western blots showing relative protein levels of SREBP1, PRMT5, and β ‐Tubulin from eWAT of male WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 12 week of HFD. n) Representative Western blots showing relative protein levels of SREBP1, PRMT5, GAPDH, and β ‐Tubulin from control and PRMT5‐overexpressed 3T3‐L1 cell line. o) Expression of genes related to TAG synthesis after PRMT5 overexpression, samples were collected from three different wells with three technical repeats for each. p) Representative Western blots showing the overexpression of Flag‐SRBEP1a in SVF preadipocytes from iWAT of 8‐week‐old male WT (Prmt5 flox/flox ) and Prmt5 AKO mice after 8 day of differentiation in vitro. q) Expression of Bscl2 , Fasn , and Dgat1 in SVF preadipocytes from iWAT of 8‐week‐old male WT (Prmt5 flox/flox ) and Prmt5 AKO mice with/without Flag‐SRBEP1a overexpression after 8 day of differentiation in vitro, n = 3 pairs of mice and cells from each mouse received three individual transfections. Data represent mean ± s.e.m. ( t ‐test: * p < 0.05, ** p < 0.01).

Article Snippet: 5 μg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody (Epigentek, A‐3005‐100) or with anti‐IgG as a negative control at 4 °C overnight.

Techniques: Concentration Assay, Expressing, Western Blot, RNA Sequencing, Immunoprecipitation, Control, Over Expression, In Vitro, Transfection

Journal: Advanced Science

Article Title: Protein Arginine Methyltransferase PRMT5 Regulates Fatty Acid Metabolism and Lipid Droplet Biogenesis in White Adipose Tissues

doi: 10.1002/advs.202002602

Figure Lengend Snippet: A model depicting PRMT5's role in regulating lipogenesis. PRMT5 regulates Bscl2 expression by methylating SPT5 during Lipid droplet formation. Prmt5 also methylates SREBP1a to promote TAG synthesis. X: unknown factors connecting PRMT5 to Bscl2 TSS.

Article Snippet: 5 μg chromatin was subjected to immunoprecipitation with rabbit polyclonal Prmt5 antibody (Epigentek, A‐3005‐100) or with anti‐IgG as a negative control at 4 °C overnight.

Techniques: Expressing

Journal: Oncogene

Article Title: PRMT5 methylating SMAD4 activates TGF-β signaling and promotes colorectal cancer metastasis.

doi: 10.1038/s41388-023-02674-x

Figure Lengend Snippet: Fig. 5 Targeting the methylation of SMAD4 at R361 inhibits CRC metastasis in vivo. A, B 2 × 106 luciferase-expressing LoVo cells of different genotypes, shNC, PRMT5 sh#1 and sh#2, were injected intrasplenic to BALB/c nude mice. After 6 weeks, bioluminescence was detected, then livers were dissected, and metastatic tumors were weighed. Representative images (A) and quantitative analysis of metastatic tumor weight (B) (*p < 0.05, **p < 0.01, t-test). C IHC staining of PRMT5 and SMAD4 R361me2s in tumor tissue. D, E 2 × 106 luciferase-expressing cells of different genotypes, WT, R361K mutant, PRMT5 overexpressing and PRMT5 overexpressing R361K mutant, were injected intrasplenic into BALB/c nude mice. After 3 weeks of tumor cell injection, half of the mice injected with WT and R361K cells were treated with GSK3326595 (40 mg/kg) every 3 days. After another 3 weeks, bioluminescence was detected, then livers were dissected, and metastatic tumors were weighed. Representative images (D) and quantitative analysis of metastatic tumor weight (E) (*p < 0.05, **p < 0.01, t-test).

Article Snippet: Smad4 Rabbit mAb (A19116), Smad4 Rabbit pAb (A5657), Smad3 Rabbit mAb (A19115), Smad2 Rabbit mAb (A19114), SMAD2/SMAD3 Rabbit pAb (A18674), Histone H3 Rabbit pAb (A2348), PRMT5 Rabbit mAb (A19533), PRMT5 Rabbit pAb (A1520), Symmetric Di-Methyl-Histone H4-R3 Rabbit pAb (A3159), Snail Rabbit pAb (A11794), Rhodamine Phalloidin (RM02835), Slug Rabbit pAb (A13352), were bought from Abclonal, China.

Techniques: Methylation, In Vivo, Luciferase, Expressing, Injection, Immunohistochemistry, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties

doi: 10.1128/mcb.26.3.843-851.2006

Figure Lengend Snippet: FIG. 3. PRMT5 interacts with and methylates the N-terminal RG-rich repeat of MBD2. (A) Purified TAP-MBD2 and TAP-MBD3 complexes were analyzed by Western blotting using a PRMT5 antibody. (B) Sequence of MBD2 with the RG repeats being underlined. (C) In vitro methylation of purified MBD2 complex upon incubation with S-[14C]adenosylmethionine. Methylated protein is indicated with an arrow. Free label is indicated with an asterisk. MBD2 complex was purified using IgG beads. The left panel depicts a Western blot analysis of the purified MBD2 complex or a 293 control purification using anti-ProtA-horseradish peroxidase antibody. The arrow shows TAP-tagged MBD2. (D) (Left panels) In vitro methylation of recombinant GST-RG(n) and GST-MBD2 in the presence of S-[14C]adenosylmethionine and a purified PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). GST-PAH2 was used as a negative control. (Right panels) In vitro methylation of recombinant GST-RG(n), GST-MBD2 lacking the RG stretch, and GST-MBD3 in the presence of S-[14C]adenosylmethionine and a purified PRMT5/MEP50 fraction (middle panel) or the purified MBD2 complex (top panel). Free label is indicated with an asterisk. Loading controls for GST-PAH2, GST-RG(n), GST-MBD2, GST MBD2 lacking the RG stretch, and GST-MBD3 are shown in the bottom panels. (E) Silver-stained gel of purified N-terminally truncated MBD2 complex from HEK 293 cells. The table shows the FT-MS/MS analysis with all identified proteins and their respective peptide numbers and percent sequence coverage.

Article Snippet: Four micrograms of the following antibodies was used for immunoprecipitations: PRMT5 12-303 (Upstate Biotechnology), MBD2 IMG-147 (Imgenex), MBD3 (IBL, Japan), MTA2 PC656 (Oncogene), and anti-dimethyl-histone H4 (Arg3) (07-213) (Upstate Biotechnology).

Techniques: Western Blot, Sequencing, In Vitro, Methylation, Incubation, Control, Recombinant, Negative Control, Staining, Tandem Mass Spectroscopy

Journal: Molecular and Cellular Biology

Article Title: MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties

doi: 10.1128/mcb.26.3.843-851.2006

Figure Lengend Snippet: FIG. 4. MBD2 recruits PRMT5 to chromatin. (A) Schematic representation of the primer sets used in the ChIP experiments. Exons are indicated with black rectangles. CpG islands are indicated in gray. Primer pairs are indicated with arrows. (B) ChIP analysis of MBD2, MBD3, PRMT5, and MTA2 in MCF7 cells. Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three independent chromatin isolations. (C) ChIP analysis of MCF7 cells treated with 5-azacytidine. Immunoprecipitations were performed with antibodies against MBD2, MBD3, MTA2, PRMT5, and anti-dimethyl-histone H4 (Arg3). Relative occupancy over a control BMX region is shown. Values are the means with standard deviations of the results from ChIP experiments from three independent chromatin isolations.

Article Snippet: Four micrograms of the following antibodies was used for immunoprecipitations: PRMT5 12-303 (Upstate Biotechnology), MBD2 IMG-147 (Imgenex), MBD3 (IBL, Japan), MTA2 PC656 (Oncogene), and anti-dimethyl-histone H4 (Arg3) (07-213) (Upstate Biotechnology).

Techniques: Control