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: bioRxiv

Article Title: Epigenetic reader ZMYND11 noncanonical function restricts HNRNPA1-mediated stress granule formation and oncogenic activity

doi: 10.1101/2023.11.28.569112

Figure Lengend Snippet: (A) Schematics of arginine methylation states by PRMT family of enzymes. (B) Co-immunoprecipitation assay of HEK293T cells expressing T7-tagged HNRNPA1 and the indicated GFP-tagged PRMT family member. (C) Co-immunoprecipitation assays using 22Rv1 cell protein extracts co-expressing V5-tagged ZMYND11 and T7-tagged HNRNPA1 while having stable expression of control or shRNAs against PRMT5. (D) Immunoprecipitation-Western blot analysis of an endogenous interaction between ZMYND11 and HNRNPA1 in 22Rv1 cells stably expressing control or ZMYND11-targeting shRNAs. (E) Immunoprecipitation using V5-tag antibodies from 22Rv1 cells ectopically co-expressing ZMYND11 and HNRNPA1 while treated with PRMT1 inhibitors (AM1 or DCLX069, 50μM, 48h) or PRMT5 inhibitors (EPZ015666 or GSK3326595, 10μM, 48h). (F) Co-immunoprecipitation assay of an endogenous interaction between ZMYND11 and HNRNPA1 in 22Rv1 cells treated with the indicated PRMT1/5 inhibitor. (G) Half-inhibitory concentration (IC50) test of two PRMT5 inhibitors (EPZ015666 or GSK3326595, 10μM, 48h) for ZMYND11 knockdown or control cells in 22Rv1. (H) Schematics of in vivo mouse experiments with PRMT5-Selective Inhibitors. (I and J) 22Rv1 cells with stable knockdown of ZMYND11 while expressing luciferase were injected into the tail-vein of male NOD-SCID mice. At 5, 6 and 7 weeks after injection, mice were subject to intraperitoneal treatment with the indicated PRMT5 inhibitors. Shown are the representative images at indicated time points ( I ) and weekly quantification of BLI photon flux of lung metastasis in mice ( J ). Errors bar, ±SD, n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t tests. (K) FACS-analysis of GFP-positive 22Rv1 cells (CTCs) in the peripheral blood of SCID mice (n=4). The scatter plot indicated the number of CTCs recovered from each mouse treated with vesicles or PRMT5 inhibitors. Statistical significance was assessed using two-tailed Student’s t test. * p < 0.05. (L) A model for ZMYND11 recognition of arginine methylation restrains HNRNPA1-mediated tumor progression. Top: higher expression of ZMYND11 in normal cells could specifically recognize R194 methylation of HNRNPA1 catalyzed by PRMT5, thereby compromising the role of HNRNPA1 in stress granule formation and PKM splicing for tumor aggressiveness. Bottom: in tumors with lower expression of ZMYND11, HNRNPA1 ensures an increased formation of stress granule and a higher PKM2/PKM1 ratio contributing to tumor progression. PRMT5 inhibitors are promising in attenuating cancer progression.

Article Snippet: Antibodies used are as follows: Anti-ZMYND11 antibody (CST, 677135); Anti-HNRNPA1 (proteintech, 67844-1-Ig); Anti-V5 tag monoclonal antibody (Thermo Fisher Scientific, R96025), HRP-anti-V5 Tag monoclonal antibody (Thermo Fisher Scientific, R96125); Anti-human GAPDH (Proteintech, 10494-1-AP); HRP-Goat anti-mouse IgG (Proteintech, SA00001-1); HRP-Goat anti-Rabbit IgG (Proteintech, SA00001-2); Anti-Beta Actin antibody (Proteintech, 66009-1-Ig); Anti-PKM1 Polyclonal antibody (Proteintech, 15821-1-AP); Anti-PKM2 polyclonal antibody (Proteintech, 15822-1-AP); Anti-T7 tag monoclonal antibody (Bethyl, A190-117A); Anti-PRMT1 monoclonal antibody (Santa cruz, sc-166963); Anti-PRMT5 monoclonal antibody (Santa cruz, sc-376937); Anti-PRMT7 rabbit polyclonal antibody (Abclonal, A12159); Anti-G3BP1 rabbit polyclonal antibody (Abclonal, A14836); Anti-His-Tag monoclonal antibody (Abclonal, AE003).

Techniques: Methylation, Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Control, Western Blot, Stable Transfection, Concentration Assay, Knockdown, In Vivo, Luciferase, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation of transcription termination by FUS and TDP-43

doi: 10.1101/788778

Figure Lengend Snippet: A . Immunoprecipitation (IP) with the indicated antibodies from HEK293 whole cell lysate (WCL), followed by western blotting with the indicated antibodies. IP with IgG was used as a negative control. The blots indicate that POLR2A, SMN, FUS, TDP-43, PRMT5 and the termination factors SETX and XRN2 interact directly or indirectly with each other . B . A summary of the interactions detected by co-IP experiments. All of these interactions, except the SMN-XRN2 and SMN-TDP-43 interactions, have been shown by various published experiments to be direct ( , , ).

Article Snippet: 8WG16 antibody against unphosphorylated CTD repeats of POLR2A was prepared in the lab. Commercial antibodies were as follows: HA (Sigma, mAb H9658); PRMT5 (Upstate, pAb C7-405, Santa Cruz, mAb sc-22132); SMN (Santa Cruz, pAb H-195); SETX for ChIP and IP (Novus Biologicals, pAb NB100-57543) and for western blots (Bethyl Lab, pAb A301-104A); XRN2 (Santa Cruz, pAb sc-99237); FUS (Santa Cruz, mAb sc-47711); TDP-43 (Bethyl lab, pAb A303-233A); POLR2A N20 (Santa Cruz, pAb sc-899); POLR2A 4H8 (Abcam, mAb ab5408); gammaH2Ax (Millipore, 05-636); H2Ax (Millipore, 07-627); Tubulin (Sigma, mAb T8328); GFP (Abcam, pAb 290); IgG negative controls for ChIP and IP (Millipore, pAb 12-370). α-amanitin was purchased from Sigma (23109-05-9).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Regulation of transcription termination by FUS and TDP-43

doi: 10.1101/788778

Figure Lengend Snippet: A . IP with 8WG16 antibodies for POLR2A from HEK293 WCL upon stably knocking down PRMT5 or SMN, with the knock-down of GFP as negative control. Western blots were performed with the indicated antibodies. The knock-down of PRMT5 or SMN caused a reduction of FUS and TDP-43 interaction with RNAPII. B . IP with the indicated antibodies from HEK293 WCL, upon knocking out of SMN with the CRISPR/Cas9 system or using scrambled guide RNA as negative control. Western blots were performed with the indicated antibodies to show that the SMN knock-out leads to a loss of interaction of TDP-43 and SETX with POLR2A. IP with IgG as negative control. C, D . Quantifications of ChIP in HEK293 cells as FUS/POLR2A ( C ) or TDP-43/POLR2A ( D ) ratio to show the relative effects of knocking down PRMT5 or SMN. Error bars denote biological replicate s.e.m. (n=3).

Article Snippet: 8WG16 antibody against unphosphorylated CTD repeats of POLR2A was prepared in the lab. Commercial antibodies were as follows: HA (Sigma, mAb H9658); PRMT5 (Upstate, pAb C7-405, Santa Cruz, mAb sc-22132); SMN (Santa Cruz, pAb H-195); SETX for ChIP and IP (Novus Biologicals, pAb NB100-57543) and for western blots (Bethyl Lab, pAb A301-104A); XRN2 (Santa Cruz, pAb sc-99237); FUS (Santa Cruz, mAb sc-47711); TDP-43 (Bethyl lab, pAb A303-233A); POLR2A N20 (Santa Cruz, pAb sc-899); POLR2A 4H8 (Abcam, mAb ab5408); gammaH2Ax (Millipore, 05-636); H2Ax (Millipore, 07-627); Tubulin (Sigma, mAb T8328); GFP (Abcam, pAb 290); IgG negative controls for ChIP and IP (Millipore, pAb 12-370). α-amanitin was purchased from Sigma (23109-05-9).

Techniques: Stable Transfection, Knockdown, Negative Control, Western Blot, CRISPR, Knock-Out

Journal: Nucleic Acids Research

Article Title: The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4

doi: 10.1093/nar/gkq896

Figure Lengend Snippet: Myogenic miRNA expression is compromised in Prmt5 antisense cell lines. ( A ) Immunoblot showing the Prmt5 and MyoD protein levels in vector- and MyoD-infected NIH 3T3 and Prmt5 antisense cell lines C1 and C12 at the onset of differentiation. The same blot was stripped and probed with PI3 Kinase (PI3K) antibody as a loading control. ( B ) qPCR analyses of primary transcripts of miR-1 and miR-133a upon MyoD-mediated differentiation of NIH 3T3 cells and the Prmt5 antisense cell lines, C1 and C12, along with the empty vector (EV) control. Expression levels were monitored 24 h post-differentiation. The expression of each miRNA in NIH 3T3 cells infected and differentiated with the empty vector retrovirus was normalized to 1. Results are the average of three independent experiments ± standard deviation.

Article Snippet: Western blots to detect Prmt5, MyoD and Carm1/Prmt4 were performed on whole cell extracts as described ( ) using rabbit polyclonal antisera against Prmt5 (Santa Cruz sc-22132), MyoD (Santa Cruz sc-32758) and Prmt4 (Millipore 09-818).

Techniques: Expressing, Western Blot, Plasmid Preparation, Infection, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: The expression of myogenic microRNAs indirectly requires protein arginine methyltransferase (Prmt)5 but directly requires Prmt4

doi: 10.1093/nar/gkq896

Figure Lengend Snippet: Expression of myogenin and Mef2D1b complements the loss of myogenic miRNA expression in a Prmt5 AS cell line. ( A and B ) Relative expression of myogenin and Mef2D1b upon differentiation in NIH 3T3 and in the Prmt5 AS cell line, C1, infected with retrovirus encoding myogenin and Mef2D1b. ( C–F ) qPCR analyses of primary transcripts of miR-1 and miR-133a in NIH 3T3 and C1 cells expressing myogenin and Mef2D1b at various times post-differentiation. The data represent the average of three independent experiments ± standard deviation. Expression at Time 0 in the empty vector (EV) control is normalized to 1. h, hours.

Article Snippet: Western blots to detect Prmt5, MyoD and Carm1/Prmt4 were performed on whole cell extracts as described ( ) using rabbit polyclonal antisera against Prmt5 (Santa Cruz sc-22132), MyoD (Santa Cruz sc-32758) and Prmt4 (Millipore 09-818).

Techniques: Expressing, Infection, Standard Deviation, Plasmid Preparation, Control

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Loop formation requires Prmt5. ( A ) Western blot time course of Prmt5 protein levels over the time course of C3H10T1/2 cells differentiation. PI3K levels were monitored as a control. ( B – E ) C3H10T1/2 cells were infected with retroviruses containing either a control vector, wild-type Prmt5 or antisense Prmt5. (B) Western blots showing levels of Prmt5 in cells expressing empty vector, antisense and overexpressed Prmt5 96 h post-differentiation. GAPDH levels were monitored as a control. (C) Oil red O staining for cells 4 days post-differentiation. (D) Control vector and antisense Prmt5 expressing cells either collected just prior to differentiation (0 h) or 96 h later were tested by 3C for interactions between the PPARγ2 promoter and the −10 kb enhancer. (E) Control or cells overexpressing Prmt5 were differentiated for the specified times. Relative interaction frequencies for the interaction between the PPARγ2 promoter and the −10 kb enhancer were set to the value at the 0 h time point. All results are from three independent samples performed in triplicate. Error bars: Mean + SD (** P < 0.01, *** P < 0.001 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Western Blot, Control, Infection, Plasmid Preparation, Expressing, Staining, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Prmt5 binding at the PPARγ2 enhancer versus the promoter. ( A ) Samples were collected for ChIP from differentiated C3H10T1/2 cells infected with a retrovirus containing the control vector. ( B ) Samples were collected for ChIP from differentiated C3H10T1/2 cells infected with a retrovirus for Prmt5 to generate Prmt5 overexpression. ( C ) Overexpression of Prmt5 resulted in precocious PPARγ2 expression. RNA samples were taken at the specified time points. The level of PPARγ2 expression in cells expressing the control empty vector at time 0 was set to 1. Experiments from three independent samples were performed in triplicate and significance is based on values relative to the 0 h time point. (* P < 0.05, ** P < 0.01, by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Binding Assay, Infection, Control, Plasmid Preparation, Over Expression, Expressing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Knockdown of Prmt5 blocked binding of Brg1 and MED1 to the PPARγ2 locus. C3H10T1/2 cells were treated with one of two independent siRNAs for Prmt5 or a scrambled sequence control. ( A – C ) Oil Red O staining of cells treated with scrambled, Prmt5 siRNA Oligo 3 or Prmt5 siRNA oligo 5. ( D ) Western demonstrating Prmt5 knockdown. ( E – G ) Chromatin immunoprecipitation (ChIP) experiments were performed on C3H10T1/2 cells differentiated for 0, 12 and 96 h. DNA was immunoprecipitated with antibodies against (E) MED1, (F) Brg1 or (G) Prmt5. Top panels indicate binding at the PPARγ2 promoter while bottom panels indicate binding at the −10 kb enhancer. Data are presented as fold enrichment over the amount of DNA amplified after immunoprecipitation by the control IgG. Analysis of three independent samples were performed in triplicate. Error bars: Mean + SD (* P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Knockdown, Binding Assay, Sequencing, Control, Staining, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Overexpression of Prmt5 results in earlier recruitment of MED1 to the PPARγ2 promoter. C3H10T1/2 cells were infected with retrovirus containing either empty vector or Prmt5. ChIP experiments for MED1 were performed on cells differentiated for the indicated times. DNA immunoprecipitated by the MED1 antibody was amplified and quantified by real-time PCR for the ( A ) PPARγ2 promoter or ( B ) the −10 kb enhancer. Data are presented as fold enrichment over the amount of DNA amplified after immunoprecipitation by the control IgG. Experiments from three independent samples were performed in triplicate. Error bars: Mean + SD (* P < 0.05, *** P < 0.001 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Over Expression, Infection, Plasmid Preparation, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Knockdown of MED1 blocked binding of Brg1 and Prmt5 to the PPARγ2 locus. C3H10T1/2 cells were treated with one of two independent siRNAs for MED1 or a scrambled control siRNA. ChIP experiments were performed on cells differentiated for the indicated times. DNA immunoprecipitated by antibodies against ( A ) MED1, ( B ) Brg1 or ( C ) Prmt5 was amplified and quantified by real-time PCR for the PPARγ2 promoter (top) and the PPARγ2 −10 kb enhancer (bottom). Data are presented as fold enrichment over the amount of DNA amplified after immunoprecipitation by the control IgG. Experiments from three independent samples were performed in triplicate. Error bars: Mean + SD (* P < 0.05, ** P < 0.01, *** P < 0.001 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Knockdown, Binding Assay, Control, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: MED1 and Prmt5 are co-localized on the same regulatory sequences. Re-ChIP experiments were performed on C3H10T1/2 cells differentiated for 6 or 96 h. ( A ) Experiments analyzing binding to PPARγ2 promoter or enhancer sequences using a MED1 antibody followed by either IgG or a Prmt5 antibody for the second ChIP. ( B ) Experiments analyzing binding to PPAPγ2 promoter or enhancer sequences using a Prmt5 antibody followed by either IgG or a MED1 antibody for the second ChIP. Results are shown as % input rather than relative enhancement to allow better presentation of the IgG control. Experiments from three independent samples were performed in triplicate. Error bars: Mean + SD (* P < 0.05 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Binding Assay, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Promoter–enhancer looping at the PPARγ2 locus during adipogenic differentiation requires the Prmt5 methyltransferase

doi: 10.1093/nar/gkw129

Figure Lengend Snippet: Prmt5 mediates the formation of other DNA loops and inter-chromosomal interactions during adipogenic differentiation. ( A ) Time course of relative interaction frequency between the Ucp2 and Ucp3 loci. Samples of C3H10T1/2 cells were taken at the 0 h timepoint and at 3, 6, 12, 24 and 96 h following differentiation. ( B ) Control vector and antisense Prmt5 expressing C3H10T1/2 cells either collected prior to differentiation (0 h) or 96 h later were tested by 3C for interactions between the Ucp2 and Ucp3 loci. ( C ) Control vector and antisense Prmt5 expressing C3H10T1/2 cells either collected prior to differentiation (0 h) or 96 h later were used to determine expression levels of Ucp3 and Ucp2, normalized to levels of GAPDH, by quantitative PCR. Data are presented as fold induction relative to the expression at time 0, which was set to 1. ( D and E ) Interaction frequencies between the PPARγ2 promoter and the promoters of the adiponectin (AdipoQ) or perilipin 2 (Plin2) genes in C3H10T1/2 cells treated with scramble sequence (scr) siRNA or siRNAs targeting Prmt5 that were differentiated for the indicated times. All results are from three independent samples performed in triplicate. Error bars: Mean + SD (* P < 0.05, ** P < 0.01, *** P < 0.001 by Student's two tailed t -test).

Article Snippet: Either polyclonal rabbit antisera against Prmt5 ( ) and Brg1 ( ) and purified antibodies against MED1 (Bethyl Labs, Montgomery, TX, USA) or normal rabbit IgG (Millipore Corp., Bedford, MA, USA) were used.

Techniques: Control, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Sequencing, Two Tailed Test

Journal: Cell reports

Article Title: Actin R256 Mono-methylation Is a Conserved Post-translational Modification Involved in Transcription

doi: 10.1016/j.celrep.2020.108172

Figure Lengend Snippet: (A) Amino acid sequence alignment of actin homologs from different species showing high conservation of the R256 residue and sequence around it.. (B and C) Western blot analysis of the whole-cell extract, purified cytoplasmic actin, nuclear-actin-containing INO80 complex, cytosolic and nuclear fractions of NIH 3T3 cells using actin R256me1 antibodies, actin (C4) (loading control), and histone H3Ac (nuclear fraction marker), as indicated. (D) Immunofluorescence confocal images of the NIH 3T3 cells, stained with actin (C4) and actin R256me1 antibodies, showing the preferential staining of nuclear actin over cytoplasmic actin by actin R256me1 antibodies. DAPI was used to stain the nucleus. Scale bar: 50 μm. (E) Western blot analyses of the hINO80 complex purified from HEK293 cells after FLAG tagging hIno80 in the genome using antibodies as indicated. (F and G) Western blot analyses of whole-cell extract of HEK293 cells transfected with siRNA against individual PRMTs as mentioned at the top, using actin (C4) (loading control) antibodies and actin R256me1 antibodies as indicated (F). Results shows that PRMT5 knockdown substantially affects actin the R256me1 level compared to the knockdown of other known PRMTs after adding the PRMT5 inhibitor (CMP5), using antibodies as indicated (G). (H) Western blot analyses after in vitro methylation assay using purified recombinant PRMT5 and rabbit muscle actin (unmethylated actin substrate) using antibodies as indicated at the right. Actin (C4) is a loading control, and histone H4 methylation by PRMT5 was used as a positive control in the assay. (I) Western blot analyses of hINO80 complex purified from HEK293 cells after FLAG tagging hIno80 in the genome using antibodies as indicated. Data presented are the representative of three independent biological repeats with similar results.

Article Snippet: Rabbit anti-PRMT5 , Abcam , Cat# ab109451.

Techniques: Sequencing, Western Blot, Purification, Marker, Immunofluorescence, Staining, Transfection, In Vitro, Methylation, Recombinant, Positive Control

Journal: Cell reports

Article Title: Actin R256 Mono-methylation Is a Conserved Post-translational Modification Involved in Transcription

doi: 10.1016/j.celrep.2020.108172

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-PRMT5 , Abcam , Cat# ab109451.

Techniques: Recombinant, Transfection, Fractionation, Software

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 2. Knockdown of PRMT5 potentiates TRAIL-induced apoptosis in cancer cells. A. HeLa cells were transfected with control, PRMT5-1, or PRMT5-3 siRNA. Cell lysates were immunoblotted with the indicated antibodies. B. HeLa cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL in the presence or absence of 10 μmol/L Z-VAD for 24 h. Cell viability was evaluated using the MTS assay in a percentage of the control siRNA-transfected cells without TRAIL treatment. *, P = 0.0016; **, P < 0.00005, compared with TRAIL-untreated and the same siRNA-transfected cells; #, P > 0.05, compared with TRAIL-untreated and the same siRNA- and Z-VAD-treated cells. C. HeLa, A549, HCT116, and HT1080/DR4 cells were transfected with the indicated siRNA and then treated with the indicated concentrations of TRAIL. Cell viability was evaluated using the MTS assay in a percentage of the corresponding cells without TRAIL treatment. *, P < 0.05; **, P < 0.01, compared with control siRNA-transfected and the same concen- tration of TRAIL-treated cells. D. HeLa cells were transfected with control (left) or PRMT5-3 (right) siRNA and then treated with a vehicle (open areas) or 100 ng/mL TRAIL (closed areas) for 24 h. Cells were stained with Annexin V-phycoerythrin, and the fluorescence was analyzed using a flow cytometer. E. WI- 38 and TIG3 fibroblast cells were transfected with the indicated siRNA. Cell lysates were immunoblotted with the indicated antibodies. F. WI-38 (left) and TIG3 (right) cells were transfected with the indicated siRNA and then treated with the indicated concentrations of TRAIL. Cell viability was evaluated using MTS assay. #, P > 0.05, compared with TRAIL-untreated and PRMT5-3 siRNA-transfected cells. Bars, SD of triplicate experiments (B, C, and F).

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Knockdown, Transfection, Control, MTS Assay, Staining, Fluorescence, Flow Cytometry

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 3. PRMT5 confers TRAIL resistance independent of its methyltransferase activity. A, D, and F. HCT116 clones stably transfected with pcDNA3- Myc (Mock1, Mock3, and Mock4), pcDNA3-Myc-WT-PRMT5 (WT2, WT24, and WT42), pcDNA3-Myc-MD-PRMT5 (MD6, MD28, and MD32), or pcDNA3-Myc- ΔC325-PRMT5 (ΔC9, ΔC16, and ΔC25) were treated with the indicated concentrations of TRAIL (A) or 30 ng/mL TRAIL (D and F). Cell viability was evaluated using the MTS assay in a percentage of the corresponding cells without TRAIL treatment. Bars, SD of multiple experiments [A and F (n = 3) and D (n = 5)]. B. HeLa cells were transiently transfected with pcDNA3-Myc vectors encoding none (-), WT-PRMT5 (WT), or MD-PRMT5 (MD). Myc-tagged proteins were immunoprecipitated and subjected to an in vitro methyltransferase assay using recombinant H2A (rH2A) as a substrate (top). Immunopreci- pitated proteins were immunoblotted with an antibody to Myc tag (bottom). C, E, and G. Cell lysates of the stable transfectants were immunoblotted with the indicated antibodies.

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Activity Assay, Clone Assay, Stable Transfection, Transfection, MTS Assay, Immunoprecipitation, In Vitro, Recombinant

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 4. Involvement of PRMT5 in TRAIL-induced IKK activation and IκB degradation. A. HeLa cells were transfected with the indicated siRNA and then incubated with control mouse IgG or antibodies to DR4 (left) or DR5 (right). Fluorescence was analyzed using a flow cytometer. Broken lines, fluores- cence of control IgG-treated and control siRNA-treated cells; open and closed areas, control and PRMT5-3 siRNA-treated cells, respectively. B. HT1080/DR4 cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL for 30 min. V5-tagged DR4 proteins were immunoprecipitated with anti-V5 agarose. Immunoprecipitated proteins (IP) and cell lysates (Input) were immunoblotted with the indicated antibodies. C. HT1080/DR4 cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL or 20 ng/mL TNF-α for the indicated times. Cell lysates were immunoblotted with the indicated antibodies (left). Relative intensities of the phospho-IκBα bands normalized with total IκBα bands were quantified (right). D. HT1080/DR4 cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL for 30 min. Endogenous IKK complexes were immunoprecipitated with anti-NEMO agarose and incubated with GST-tagged recombinant IκBα (rIκBα) as described in Materials and Methods. Control experiments were done with control rabbit IgG agarose. Reactions (in vitro kinase assay) and cell lysates were immunoblotted with the indicated antibodies. Relative intensities of the phospho-IκBα bands were quantified. E. HT1080/DR4 cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL for 30 min. Cell lysates were immunoblotted with the indicated antibodies.

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Activation Assay, Transfection, Incubation, Control, Fluorescence, Flow Cytometry, Immunoprecipitation, Recombinant, In Vitro, Kinase Assay

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 5. TRAIL-induced NF-κB activation is dependent on PRMT5. HT1080/DR4 (A, left, and B) or HCT116 (A, right) cells were trans- fected with the indicated siRNA. After transfection for 24 h, cells were then transfected with pNF-κB-Luc and phRL-TK plasmids. After a further 24 h incubation, cells were treated with 100 ng/mL TRAIL (A) or 20 ng/mL TNF-α (B) for 4 h. Luciferase activities were measured as described in Materi- als and Methods. C. HT1080/DR4 cells were transfected with pNF-κB-Luc and phRL-TK plasmids. Four hours later, cells were treated with the 50 μmol/L methyltransferase inhibitor periodate- oxidized adenosine (AdOx) for 44 h. Then, cells were treated with 100 ng/ mL TRAIL for 4 h. Luciferase activities were measured. Bars, SD of triplicate experiments (A-C).

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Activation Assay, Transfection, Incubation, Luciferase

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 6. PRMT5-mediated transcription of NF-κB target genes. A to C. HCT116 (left) or HT1080/DR4 (right) cells were transfected with the indicated siRNA and then treated with 100 ng/mL TRAIL for 3 h. Total RNA was separa- ted and analyzed using quantitative RT-PCR (A and B) as described in Materials and Methods. Levels of cIAP1 (A) and cIAP2 (B) mRNA were normalized to the level of GAPDH mRNA. *, P < 0.005; **, P < 0.0005, compared with control siRNA-transfected and TRAIL-treated cells. Bars, SD of triplicate experiments (A and B). Cell lysates were immunoblotted with the indicated antibodies (C).

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Transfection, Quantitative RT-PCR, Control

Journal: Molecular Cancer Research

Article Title: PRMT5, a Novel TRAIL Receptor-Binding Protein, Inhibits TRAIL-Induced Apoptosis via Nuclear Factor-κB Activation

doi: 10.1158/1541-7786.mcr-08-0197

Figure Lengend Snippet: FIGURE 7. PRMT5 contributes to TRAIL resistance via NF-κB activation. A. HCT116 (left) or HT1080/DR4 (right) cells were treated with 10 ng/mL TRAIL or 20 ng/mL TNF-α in the presence or absence of the 10 μmol/L IKK inhibitor BMS-345541. Cell viability was evaluated using the MTS assay. **, P < 0.001, compared with TRAIL-untreated or TNF-untreated and BMS-345541-treated cells. B. HCT116 (left) or A549 (right) cells were transfected with the indicated siRNA and then treated with 10 ng/mL TRAIL or 100 ng/mL TNF-α. Cell viability was evaluated using the MTS assay. *, P < 0.05; **, P < 0.001; #, P > 0.05, compared with TRAIL or TNF-untreated and PRMT5-3 siRNA-transfected cells. C. HT1080 cells were transfected with control (-) or PRMT5-3 (+) siRNA. After transfection for 24 h, cells were also transfected with pEGFP-C1 (-) or pEGFP-C1-CA-IKKβ (+). After additional incubation for 24 h, cells were treated with 100 ng/mL TRAIL for 6 h. Cells were fixed and stained with Hoechst 33342. Cellular images were visualized using a fluorescence microscope. The rate that apoptotic cells showed condensed and fragmented nuclei was determined by counting >100 GFP-positive cells (left). Green, GFP proteins; blue, nuclei (right). *, P = 0.037; **, P = 0.00061, compared with control siRNA-transfected and TRAIL-treated cells, respectively; †, P = 0.0054, compared with IKKβ- untransfected, PRMT5-3 siRNA-transfected and TRAIL-treated cells. Bars, SD of triplicate experiments (A-C).

Article Snippet: We used primary antibodies to FLAG tag (M2; Sigma); Myc tag (9E10; Roche); PRMT2 (Aviva Systems Biology); PRMT3 or PRMT5 (Upstate); DR4 or DR5 (CT; ProSci); PRMT6 (Imgenex); α-tubulin (YL1/2; AbD Serotec); PRMT5 Regulates TRAIL Signaling Mol Cancer Res 2009;7(4).

Techniques: Activation Assay, MTS Assay, Transfection, Control, Incubation, Staining, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27 Kip1

doi: 10.1371/journal.pone.0074710

Figure Lengend Snippet: The arginine methyltransferase PRMT5 is significantly overexpressed in human melanocytic nevi, malignant, and metastatic tissues compared to normal epidermis.

Article Snippet: The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 (Abcam # ab31751; Cambridge, MA), PRMT5 (Cell Signaling Technology #2252; Danvers, MA), β-actin (Sigma # A5441-2; St. Louis, MO); GAPDH (Sigma # G8795); lamin B (Santa Cruz # sc-6216; Santa Cruz, CA), MITF (Thermo-Scientific # MS-772-PO; Lafayette, CO), MITF (Spring Bioscience # E17904; Pleasanton, CA), cyclin D1 (Abcam # ab6152), cyclin D1 (Cell Signaling Technology #2978), Mep50 (Bethyl Laboratories # A301561A; Montgomery, TX), Mep50 (Abnova #79804; Taipei, Taiwan), STAT3 (Cell Signaling Technology #9132), and p27 Kip1 (Cell Signaling Technology #2552).

Techniques:

Journal: PLoS ONE

Article Title: PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27 Kip1

doi: 10.1371/journal.pone.0074710

Figure Lengend Snippet: A, Brightfield image of PRMT5 staining in a Stage 2/3 tumor and its adjacent normal epidermis. PRMT5 protein was visualized with Vulcan FastRed label (pink); samples were counterstained with hematoxylin to delineate nuclei (arrows) and connective tissue (blue). B, malignant skin tumor of pathologic stage 4, showing predominantly nuclear PRMT5. C, malignant skin tumor of pathologic stage 4, showing predominantly cytoplasmic PRMT5 (arrows indicate hematoxylin-stained nuclei). D, representative images of PRMT5 staining in patient tumors of pathologic stages I–IV.

Article Snippet: The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 (Abcam # ab31751; Cambridge, MA), PRMT5 (Cell Signaling Technology #2252; Danvers, MA), β-actin (Sigma # A5441-2; St. Louis, MO); GAPDH (Sigma # G8795); lamin B (Santa Cruz # sc-6216; Santa Cruz, CA), MITF (Thermo-Scientific # MS-772-PO; Lafayette, CO), MITF (Spring Bioscience # E17904; Pleasanton, CA), cyclin D1 (Abcam # ab6152), cyclin D1 (Cell Signaling Technology #2978), Mep50 (Bethyl Laboratories # A301561A; Montgomery, TX), Mep50 (Abnova #79804; Taipei, Taiwan), STAT3 (Cell Signaling Technology #9132), and p27 Kip1 (Cell Signaling Technology #2552).

Techniques: Staining

Journal: PLoS ONE

Article Title: PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27 Kip1

doi: 10.1371/journal.pone.0074710

Figure Lengend Snippet: The distribution of nuclear PRMT5 protein differs significantly among normal epidermis, benign nevi, and malignant and metastatic tumors.

Article Snippet: The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 (Abcam # ab31751; Cambridge, MA), PRMT5 (Cell Signaling Technology #2252; Danvers, MA), β-actin (Sigma # A5441-2; St. Louis, MO); GAPDH (Sigma # G8795); lamin B (Santa Cruz # sc-6216; Santa Cruz, CA), MITF (Thermo-Scientific # MS-772-PO; Lafayette, CO), MITF (Spring Bioscience # E17904; Pleasanton, CA), cyclin D1 (Abcam # ab6152), cyclin D1 (Cell Signaling Technology #2978), Mep50 (Bethyl Laboratories # A301561A; Montgomery, TX), Mep50 (Abnova #79804; Taipei, Taiwan), STAT3 (Cell Signaling Technology #9132), and p27 Kip1 (Cell Signaling Technology #2552).

Techniques:

Journal: PLoS ONE

Article Title: PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27 Kip1

doi: 10.1371/journal.pone.0074710

Figure Lengend Snippet: The distribution of cytoplasmic PRMT5 protein differs significantly among normal epidermis, benign nevi, and malignant and metastatic tumors.

Article Snippet: The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 (Abcam # ab31751; Cambridge, MA), PRMT5 (Cell Signaling Technology #2252; Danvers, MA), β-actin (Sigma # A5441-2; St. Louis, MO); GAPDH (Sigma # G8795); lamin B (Santa Cruz # sc-6216; Santa Cruz, CA), MITF (Thermo-Scientific # MS-772-PO; Lafayette, CO), MITF (Spring Bioscience # E17904; Pleasanton, CA), cyclin D1 (Abcam # ab6152), cyclin D1 (Cell Signaling Technology #2978), Mep50 (Bethyl Laboratories # A301561A; Montgomery, TX), Mep50 (Abnova #79804; Taipei, Taiwan), STAT3 (Cell Signaling Technology #9132), and p27 Kip1 (Cell Signaling Technology #2552).

Techniques:

Journal: PLoS ONE

Article Title: PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27 Kip1

doi: 10.1371/journal.pone.0074710

Figure Lengend Snippet: A, Detection of PRMT5 in cell lysates from immortalized human epidermal melanocytes (HEM) or a panel of human metastatic melanoma cell lines. β-actin was used as loading control. B, HEM and melanoma cell lines underwent subcellular fractionation and immunoblot analysis for PRMT5, Mep50, or markers for nuclear (lamin B) and cytoplasmic (GAPDH) fractions. C, 1×10 6 cells from the A375 and Hs294T human melanoma cell lines were implanted into the flank of immunocompromised mice and grown for 21 days. Tumors were removed, fixed in formalin, and analyzed via IHC for PRMT5. Data represent total percentage of cells in each tumor positive for PRMT5 in nucleus, cytoplasm, or both. n≥2 tumors were analyzed.

Article Snippet: The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 (Abcam # ab31751; Cambridge, MA), PRMT5 (Cell Signaling Technology #2252; Danvers, MA), β-actin (Sigma # A5441-2; St. Louis, MO); GAPDH (Sigma # G8795); lamin B (Santa Cruz # sc-6216; Santa Cruz, CA), MITF (Thermo-Scientific # MS-772-PO; Lafayette, CO), MITF (Spring Bioscience # E17904; Pleasanton, CA), cyclin D1 (Abcam # ab6152), cyclin D1 (Cell Signaling Technology #2978), Mep50 (Bethyl Laboratories # A301561A; Montgomery, TX), Mep50 (Abnova #79804; Taipei, Taiwan), STAT3 (Cell Signaling Technology #9132), and p27 Kip1 (Cell Signaling Technology #2552).

Techniques: Control, Fractionation, Western Blot

Journal: PLoS ONE

Article Title: PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27 Kip1

doi: 10.1371/journal.pone.0074710

Figure Lengend Snippet: A, HEM and representative human melanoma cell lines were harvested and subjected to total cell lysis followed by immunoprecipitation. 10% of input protein, as well as protein from IgG control and IP directed against Mep50 and cyclin D1, underwent immunoblot analysis for IP target proteins as well as PRMT5. B, IP against STAT3 was performed on the same cell line panel, and subjected to immunoblot analysis using antibodies against STAT3 or PRMT5.

Article Snippet: The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 (Abcam # ab31751; Cambridge, MA), PRMT5 (Cell Signaling Technology #2252; Danvers, MA), β-actin (Sigma # A5441-2; St. Louis, MO); GAPDH (Sigma # G8795); lamin B (Santa Cruz # sc-6216; Santa Cruz, CA), MITF (Thermo-Scientific # MS-772-PO; Lafayette, CO), MITF (Spring Bioscience # E17904; Pleasanton, CA), cyclin D1 (Abcam # ab6152), cyclin D1 (Cell Signaling Technology #2978), Mep50 (Bethyl Laboratories # A301561A; Montgomery, TX), Mep50 (Abnova #79804; Taipei, Taiwan), STAT3 (Cell Signaling Technology #9132), and p27 Kip1 (Cell Signaling Technology #2552).

Techniques: Lysis, Immunoprecipitation, Control, Western Blot

Journal: PLoS ONE

Article Title: PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27 Kip1

doi: 10.1371/journal.pone.0074710

Figure Lengend Snippet: A, Melanoma cell lines were transiently transfected with scrambled siRNA or siRNA targeting PRMT5. Cell growth was measured by MTT assay at 48-transfection. Data represent percent of growth inhibition ± SEM (n≥3). B, Analysis of PRMT5, MITF, p27 KIP1 , and β-actin protein expression at 48 hours post-transfection. n≥2 transfections were performed per cell line; representative immunoblots are shown here. The MITF antibody used here for IB predominantly recognizes one of the two known MITF isoforms in human melanocytes.

Article Snippet: The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 (Abcam # ab31751; Cambridge, MA), PRMT5 (Cell Signaling Technology #2252; Danvers, MA), β-actin (Sigma # A5441-2; St. Louis, MO); GAPDH (Sigma # G8795); lamin B (Santa Cruz # sc-6216; Santa Cruz, CA), MITF (Thermo-Scientific # MS-772-PO; Lafayette, CO), MITF (Spring Bioscience # E17904; Pleasanton, CA), cyclin D1 (Abcam # ab6152), cyclin D1 (Cell Signaling Technology #2978), Mep50 (Bethyl Laboratories # A301561A; Montgomery, TX), Mep50 (Abnova #79804; Taipei, Taiwan), STAT3 (Cell Signaling Technology #9132), and p27 Kip1 (Cell Signaling Technology #2552).

Techniques: Transfection, MTT Assay, Inhibition, Expressing, Western Blot

Journal: PLoS ONE

Article Title: PRMT5 Is Upregulated in Malignant and Metastatic Melanoma and Regulates Expression of MITF and p27 Kip1

doi: 10.1371/journal.pone.0074710

Figure Lengend Snippet: Effects of PRMT5 siRNA on microRNA (miR) expression in human melanoma cell lines.

Article Snippet: The following antibodies were used for immunohistochemistry (IHC), immunoblot (IB), or immunoprecipitation (IP): PRMT5 (Abcam # ab31751; Cambridge, MA), PRMT5 (Cell Signaling Technology #2252; Danvers, MA), β-actin (Sigma # A5441-2; St. Louis, MO); GAPDH (Sigma # G8795); lamin B (Santa Cruz # sc-6216; Santa Cruz, CA), MITF (Thermo-Scientific # MS-772-PO; Lafayette, CO), MITF (Spring Bioscience # E17904; Pleasanton, CA), cyclin D1 (Abcam # ab6152), cyclin D1 (Cell Signaling Technology #2978), Mep50 (Bethyl Laboratories # A301561A; Montgomery, TX), Mep50 (Abnova #79804; Taipei, Taiwan), STAT3 (Cell Signaling Technology #9132), and p27 Kip1 (Cell Signaling Technology #2552).

Techniques: Expressing

Journal: Nature Communications

Article Title: PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis

doi: 10.1038/s41467-021-23833-2

Figure Lengend Snippet: a , b Immunoblot (IB) analysis of whole cell lysates (WCLs) derived from MCF7 cells infected with lentiCRISPR virus ( a ) or shRNAs virus targeting PRMT5 ( b ). The cells were selected with 2 μg/ml puromycin for 4 days to eliminate the non-infected cells. c IB analysis of WCLs derived from MCF7 cells treated with GSK591 for 24 h. d AKT in vitro kinase assays were performed using endogenous AKT1 (IgG as a negative control) immunopurified (IP) from control cells or sg PRMT5 cells as the kinase source and recombinant GST-GSK-3β purified from bacteria as the substrate. e IB analysis of WCLs derived from control cells or PRMT5 -depleted MCF7 cells. Cells were serum-starved for 16 h and then treated with 100 nM insulin for 0, 30, and 60 min before collecting. Similar results were obtained in n ≥ 2 independent experiments in a – e . Uncropped immunoblots are provided in Source Data file.

Article Snippet: Anti-AKT-pT308 antibody (13038, 1:1000), anti-AKT-pS473 antibody (4060, 1:3000), anti-AKT1 antibody (2938, 1:3000), anti-AKT pan antibody (4685, 1:3000), anti-pGSK-3β antibody (5558, 1:5000), anti-GSK-3β antibody (12456, 1:5000), anti-pFOXO antibody (2599, 1:1000), anti-PRMT1 (2449, 1:1000), anti-PRMT4 antibody (12495, 1:1000), anti-PRMT5 antibody (79998, 1:1000), anti-S6K1 antibody (9202, 1:2000), anti-E-cadherin antibody (3195, 1:3000), anti-PDK1 antibody (13037, 1:1000), anti-Ki-67 antibody (9027, 1:800 for IHC), anti-Myc-tag rabbit antibody (2278, 1:1000), anti-Glutathione S-transferase (GST) rabbit antibody (2625, 1:2000), anti-HA rabbit antibody (3724, 1:2000), and anti-Sdme-RG antibody (13222, 1:1000) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Derivative Assay, Infection, Virus, In Vitro, Negative Control, Control, Recombinant, Purification, Bacteria

Journal: Nature Communications

Article Title: PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis

doi: 10.1038/s41467-021-23833-2

Figure Lengend Snippet: a MCF7 cells stably expressing myr-AKT1 were infected with shRNA targeting PRMT5 and subjected to IB analysis. Similar results were obtained in n = 3 independent experiments. b , c Cells generated in a were subjected to colony formation and soft agar assays. Representative images are shown in b and relative colony numbers are plotted in c . Data are shown as the mean ± SD of n = 3 or 6 independent experiments. d DLD-1 cells ectopically expressing myr-AKT1 and/or depleting PRMT5 were injected into nude mice. Tumor growth was monitored for the indicated time period. Data are shown as the mean ± SEM for n = 7 mice. e Weight of the tumors. Data are shown as the mean ± SD for n = 7 tumors. f Representative images of IHC staining of Ki-67 in n = 4 xenograft tumors. Scale bar, 100 μm. g Representative images of TUNEL assays in xenograft tumors. Scale bar, 50 μm. h Quantification of TUNEL-positive cells in xenograft tumors. Data are shown as the mean ± SD for n = 4 tumors. Statistical significance was determined by two-tailed Student’s t -test in c , e , h and by two-way ANOVA in d . Uncropped immunoblots and statistical source data are provided in Source Data files.

Article Snippet: Anti-AKT-pT308 antibody (13038, 1:1000), anti-AKT-pS473 antibody (4060, 1:3000), anti-AKT1 antibody (2938, 1:3000), anti-AKT pan antibody (4685, 1:3000), anti-pGSK-3β antibody (5558, 1:5000), anti-GSK-3β antibody (12456, 1:5000), anti-pFOXO antibody (2599, 1:1000), anti-PRMT1 (2449, 1:1000), anti-PRMT4 antibody (12495, 1:1000), anti-PRMT5 antibody (79998, 1:1000), anti-S6K1 antibody (9202, 1:2000), anti-E-cadherin antibody (3195, 1:3000), anti-PDK1 antibody (13037, 1:1000), anti-Ki-67 antibody (9027, 1:800 for IHC), anti-Myc-tag rabbit antibody (2278, 1:1000), anti-Glutathione S-transferase (GST) rabbit antibody (2625, 1:2000), anti-HA rabbit antibody (3724, 1:2000), and anti-Sdme-RG antibody (13222, 1:1000) were purchased from Cell Signaling Technology.

Techniques: Stable Transfection, Expressing, Infection, shRNA, Generated, Injection, Immunohistochemistry, TUNEL Assay, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis

doi: 10.1038/s41467-021-23833-2

Figure Lengend Snippet: a , b IB analysis of WCLs and immunoprecipitation (IP) products derived from MCF7 cells. IgG was used as a negative control. c In vitro methylation of AKT1 in the presence of 3 H-SAM. GST-AKT1, PRMT5-WT, and PRMT5-E444Q proteins were purified from HEK293 cells. d Sequence of the evolutionarily conserved residue R391 (red) in AKT. e In vitro methylation of AKT1-KD-WT and AKT1-KD-R391K in the presence of 3 H-SAM. Recombinant GST-AKT1-WT and AKT1-KD-R391K proteins were purified from bacteria and HA-PRMT5/MEP50 proteins were immunopurified from HEK293 cells. f IB analysis of WCLs and IP products derived from MCF7 cells stably expressing AKT1-WT or R391K. Flag-PRMT5/MEP50 was transiently transfected into these cells. g Knockout of PRMT5 abrogates AKT1-R391 methylation. IB analysis of WCLs and IP products derived from PRMT5 -knockout cells or control cells transfected with HA-AKT1. h Representative images of IHC staining for PRMT5 and R391-me2s in a breast cancer tissue array ( n = 100 tissue specimens). Scale bar, 50 μm. i Quantification of cases with PRMT5 and R391-me2s staining ( n = 100 tissue specimens). P -values were calculated using two-tailed χ 2 -test. Similar results were obtained in n ≥ 2 independent experiments in a – c and e – g . Uncropped immunoblots and statistical source data are provided in Source Data files.

Article Snippet: Anti-AKT-pT308 antibody (13038, 1:1000), anti-AKT-pS473 antibody (4060, 1:3000), anti-AKT1 antibody (2938, 1:3000), anti-AKT pan antibody (4685, 1:3000), anti-pGSK-3β antibody (5558, 1:5000), anti-GSK-3β antibody (12456, 1:5000), anti-pFOXO antibody (2599, 1:1000), anti-PRMT1 (2449, 1:1000), anti-PRMT4 antibody (12495, 1:1000), anti-PRMT5 antibody (79998, 1:1000), anti-S6K1 antibody (9202, 1:2000), anti-E-cadherin antibody (3195, 1:3000), anti-PDK1 antibody (13037, 1:1000), anti-Ki-67 antibody (9027, 1:800 for IHC), anti-Myc-tag rabbit antibody (2278, 1:1000), anti-Glutathione S-transferase (GST) rabbit antibody (2625, 1:2000), anti-HA rabbit antibody (3724, 1:2000), and anti-Sdme-RG antibody (13222, 1:1000) were purchased from Cell Signaling Technology.

Techniques: Immunoprecipitation, Derivative Assay, Negative Control, In Vitro, Methylation, Purification, Sequencing, Residue, Recombinant, Bacteria, Stable Transfection, Expressing, Transfection, Knock-Out, Control, Immunohistochemistry, Staining, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis

doi: 10.1038/s41467-021-23833-2

Figure Lengend Snippet: a , b IB analysis of WCLs and IP products derived from MCF7 cells. The cells were serum-starved for 16 h and then treated with 100 nM insulin for indicated time periods before collection. c IB analysis of WCLs and IP products derived from MCF7 cells. The cells were serum-starved for 24 h and treated with PI3K inhibitors for 12 h followed by insulin for 30 min before collection. LY294002 (20 μM), Wortmannin (10 μM), and Buparlisib (10 μM). d , e IB analysis of WCLs and IP products derived from HEK293T cells transfected with indicated constructs. f In vitro binding assays were performed with recombinant GST-AKT1 protein purified from mammalian cells and Myc beads bound with PRMT5/MEP50. The binding was performed at 4 °C for 4 h incubated with or without PIP3 (20 μM) and subjected to IB analysis. Similar results were obtained in n ≥ 2 independent experiments in a – f . Uncropped immunoblots are provided in Source Data file.

Article Snippet: Anti-AKT-pT308 antibody (13038, 1:1000), anti-AKT-pS473 antibody (4060, 1:3000), anti-AKT1 antibody (2938, 1:3000), anti-AKT pan antibody (4685, 1:3000), anti-pGSK-3β antibody (5558, 1:5000), anti-GSK-3β antibody (12456, 1:5000), anti-pFOXO antibody (2599, 1:1000), anti-PRMT1 (2449, 1:1000), anti-PRMT4 antibody (12495, 1:1000), anti-PRMT5 antibody (79998, 1:1000), anti-S6K1 antibody (9202, 1:2000), anti-E-cadherin antibody (3195, 1:3000), anti-PDK1 antibody (13037, 1:1000), anti-Ki-67 antibody (9027, 1:800 for IHC), anti-Myc-tag rabbit antibody (2278, 1:1000), anti-Glutathione S-transferase (GST) rabbit antibody (2625, 1:2000), anti-HA rabbit antibody (3724, 1:2000), and anti-Sdme-RG antibody (13222, 1:1000) were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Transfection, Construct, In Vitro, Binding Assay, Recombinant, Purification, Incubation, Western Blot

Journal: Nature Communications

Article Title: PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis

doi: 10.1038/s41467-021-23833-2

Figure Lengend Snippet: a IB analysis of WCLs and GST pulldown products derived from HEK293 cells transfected with indicated constructs. b Biotin-conjugated R391 peptides with (me2s) or without (me0) dimethylation were incubated with recombinant GST-AKT1-PH and precipitated with streptavidin. Peptide (4 μg) was used for each sample. c IB analyses of PIP3 pulldown products. HA-AKT1 proteins were purified from HEK293T cells transfected with HA-AKT1-WT or HA-AKT1-R391K. Empty beads (ctr) serve as a negative control. d IB analyses of PIP3 pulldown products and WCLs derived from PRMT5 -knockout MCF7 cells. e , f IB analysis of cell fractionations derived from PRMT5 -depleted MCF7 cells ( e ) or from DLD-1- AKT1/2 −/− cells expressing AKT1-WT or R391K mutant ( f ). g IB analysis of WCLs and IP products derived from PRMT5 -knockdown MCF7 cells. h IB analysis of WCLs and IP products derived from DLD-1- AKT1/2 −/− cells expressing AKT1-WT or R391K mutant. Cells were serum-starved for 16 h and then treated with 100 nM insulin for 60 min before collection. Similar results were obtained in n ≥ 2 independent experiments in a – h . Uncropped immunoblots are provided in Source Data file.

Article Snippet: Anti-AKT-pT308 antibody (13038, 1:1000), anti-AKT-pS473 antibody (4060, 1:3000), anti-AKT1 antibody (2938, 1:3000), anti-AKT pan antibody (4685, 1:3000), anti-pGSK-3β antibody (5558, 1:5000), anti-GSK-3β antibody (12456, 1:5000), anti-pFOXO antibody (2599, 1:1000), anti-PRMT1 (2449, 1:1000), anti-PRMT4 antibody (12495, 1:1000), anti-PRMT5 antibody (79998, 1:1000), anti-S6K1 antibody (9202, 1:2000), anti-E-cadherin antibody (3195, 1:3000), anti-PDK1 antibody (13037, 1:1000), anti-Ki-67 antibody (9027, 1:800 for IHC), anti-Myc-tag rabbit antibody (2278, 1:1000), anti-Glutathione S-transferase (GST) rabbit antibody (2625, 1:2000), anti-HA rabbit antibody (3724, 1:2000), and anti-Sdme-RG antibody (13222, 1:1000) were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Transfection, Construct, Incubation, Recombinant, Purification, Negative Control, Knock-Out, Expressing, Mutagenesis, Knockdown, Western Blot

Journal: bioRxiv

Article Title: Epigallocatechin-3-gallate inhibit the protein arginine methyltransferase 5 and Enhancer of Zeste homolog 2 in breast cancer both in vitro and in vivo

doi: 10.1101/2024.02.18.580855

Figure Lengend Snippet: (a-b) Cancer types summary of PRMT5 and EZH2 in all cancer types, and red color indicates the amplification which is more in breast cancer datasets for PRMT5 & EZH2. (c-d) The amplification frequency data of PRMT5 & EZH2 in breast, lung, pancreatic and urothelial bladder cancers. Breast cancer has highest amplification count for both PRMT5 & EZH2. (e-f) Oncoprints visualization of PRMT5 & EZH2, revealed that both major type of alterations in cancer patients due to amplification of PRMT5 and EZH2. (g-h) Survival analysis of PRMT5 and EZH2 gene by Kaplan Meier curve: we clearly observe that high PRMT5 & EZH2 expression is associated with poor patient survival.

Article Snippet: The antibodies Anti-PRMT5 antibody (#2252) Histone (H4) antibody (#2935), H3 antibody (#4499), EZH2 antibody (#5246), H3K27 (#9733), anti-beta actin antibody (#4967), Ki67 antibody (#D3B5) was obtained from Cell Signaling Technology (CST) USA.

Techniques: Amplification, Expressing

Journal: bioRxiv

Article Title: Epigallocatechin-3-gallate inhibit the protein arginine methyltransferase 5 and Enhancer of Zeste homolog 2 in breast cancer both in vitro and in vivo

doi: 10.1101/2024.02.18.580855

Figure Lengend Snippet: (a-c) Interaction profile of EGCG with PRMT5(4X60, 4X61, 4X63). The hydrogen bonds are shown in green dotted lines, the interacting amino acid residues, as well as the ligand Sfg, are shown in blue/black color ball and stick model, the non-bonded interactions are depicted with residues with starbursts. (d-e) interaction profile of EGCG with EZH2 (5HYN & 4mi5). The ligands are shown in the middle, and the hydrogen bonds are shown with purple arrows, and the arrowheads display the H-bond donor-acceptor relationship. The π-cation interaction of both EGCG and Sfg with the lysine side chain is depicted with a red arrow. The hydrogen bonds are shown in green dotted lines, the interacting amino acid residues, as well as the ligand SFG, are shown in blue/black color ball and stick model, the non-bonded interactions are depicted with residues with starbursts

Article Snippet: The antibodies Anti-PRMT5 antibody (#2252) Histone (H4) antibody (#2935), H3 antibody (#4499), EZH2 antibody (#5246), H3K27 (#9733), anti-beta actin antibody (#4967), Ki67 antibody (#D3B5) was obtained from Cell Signaling Technology (CST) USA.

Techniques:

Journal: bioRxiv

Article Title: Epigallocatechin-3-gallate inhibit the protein arginine methyltransferase 5 and Enhancer of Zeste homolog 2 in breast cancer both in vitro and in vivo

doi: 10.1101/2024.02.18.580855

Figure Lengend Snippet: SPR Stenographs of immobilized PRMT5 & EZH2 on CM5 chip. (a &c): PRMT5 & PRMT5 immobilization on CM5 sensor chip (b & d): EZH2 & PRMT5 blanks

Article Snippet: The antibodies Anti-PRMT5 antibody (#2252) Histone (H4) antibody (#2935), H3 antibody (#4499), EZH2 antibody (#5246), H3K27 (#9733), anti-beta actin antibody (#4967), Ki67 antibody (#D3B5) was obtained from Cell Signaling Technology (CST) USA.

Techniques:

Journal: bioRxiv

Article Title: Epigallocatechin-3-gallate inhibit the protein arginine methyltransferase 5 and Enhancer of Zeste homolog 2 in breast cancer both in vitro and in vivo

doi: 10.1101/2024.02.18.580855

Figure Lengend Snippet: Surface Plasmon resonance Studies: The sensorgram of EGCG on PRMT5 & EZH2 complexes on cm5 sensor chip, (a) dose–response sensorgram of EGCG with immobilized PRMT5-MEP50. (b) Fitting of response and concentration using BIAcore T200 Evaluation software version 2.0. (c) dose–response sensorgram of EGCG with immobilized EZH2. (d) Fitting of response and concentration using BIAcore T200 Evaluation software version 2.0. The response units RU versus protein concentration plot linear. The obtained Ka, kd & KD values are mentioned: for PRMT5 Ka: 2.07E+02, kd 3.61 E-03, & KD-1.74E-05, EZH2: Ka-82.78, kd:3.63 E-03 & KD:4.39E-05 respectively

Article Snippet: The antibodies Anti-PRMT5 antibody (#2252) Histone (H4) antibody (#2935), H3 antibody (#4499), EZH2 antibody (#5246), H3K27 (#9733), anti-beta actin antibody (#4967), Ki67 antibody (#D3B5) was obtained from Cell Signaling Technology (CST) USA.

Techniques: SPR Assay, Concentration Assay, Software, Protein Concentration

Journal: bioRxiv

Article Title: Epigallocatechin-3-gallate inhibit the protein arginine methyltransferase 5 and Enhancer of Zeste homolog 2 in breast cancer both in vitro and in vivo

doi: 10.1101/2024.02.18.580855

Figure Lengend Snippet: The MDA-MB231 cells were exposed to increasing concentration of either EGCG (0.1, 1, 10 μM) for 24 hours. Protein lysate was prepared as explained in methods and analyzed for H4R3me2s & H3K27me3 using immunoblotting. The β-actin was used as a loading control. (a) Western blots (b) protein intensity graphs (c) ELISA of In-vitro methylation which was carried using EZH2 and PRMT5-MEP50 enzyme complex and histones as a substrate.

Article Snippet: The antibodies Anti-PRMT5 antibody (#2252) Histone (H4) antibody (#2935), H3 antibody (#4499), EZH2 antibody (#5246), H3K27 (#9733), anti-beta actin antibody (#4967), Ki67 antibody (#D3B5) was obtained from Cell Signaling Technology (CST) USA.

Techniques: Concentration Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, In Vitro, Methylation

Journal: bioRxiv

Article Title: Epigallocatechin-3-gallate inhibit the protein arginine methyltransferase 5 and Enhancer of Zeste homolog 2 in breast cancer both in vitro and in vivo

doi: 10.1101/2024.02.18.580855

Figure Lengend Snippet: The mice were divided into three groups at randomly with 7 mice per group: (1) control group-1 (2) control group-2 (3) treatment group. The mice were injected MDA-MB-231 cells subcutaneously on both flaks. Tumor volume was measured every three days. The tumor volume once reached 100mm 3 started the EGCG (100mg/kg) administration orally to treatment group for a period of 21 days. (a) comparison of EGCG treated vs control from left to right 1 st is control mice, remaining are from 2 nd to 7 th bearing tumors at day 1 st of treatment (b) tumor growth measurement top panel in control animals down panel is of treatment (c) Tumor volume comparison graphs (control vs treated mice). (d) Body weight measurement graphs (control vs treated). (e-f) The western blot analysis of tumor lysates from treatment group, levels of PRMT5 & EZH2 and H3R3mes and H3K27me3, β-actin was used as loading control.. (g) tumor weight comparison graphs (control vs treated)

Article Snippet: The antibodies Anti-PRMT5 antibody (#2252) Histone (H4) antibody (#2935), H3 antibody (#4499), EZH2 antibody (#5246), H3K27 (#9733), anti-beta actin antibody (#4967), Ki67 antibody (#D3B5) was obtained from Cell Signaling Technology (CST) USA.

Techniques: Control, Injection, Comparison, Western Blot