Journal: Cancers

Article Title: PRMT5/WDR77 Enhances the Proliferation of Squamous Cell Carcinoma via the ΔNp63α-p21 Axis.

doi: 10.3390/cancers16223789

Figure Lengend Snippet: Figure 1. PRMT5 is inversely correlated with SCC survival. (A) Statistical analysis of the TCGA- HNSC database comparing the expression of PRMT5 (left) and WDR77 (right) in cancer (red) vs. normal (blue) groups, utilizing Wilcoxon Rank-Sum Tests. Patient counts: HNSCC (513 tumors and 44 normal). Black dots represent median expression levels. (B) Kaplan–Meier survival curve for PRMT5 and WDR77 expression in the TCGA-HNSC database (n = 513). Patients were categorized by median PRMT5 (upper panel) and WDR77 (lower panel) expression levels. Statistical significance was determined using the Log-Rank Tests, with visualization provided by the ggsurvplot function from the survminer R package [29]. (C) Statistical analysis of DepMap data and display of Chronos Dependency Scores for six subtypes of SCC. Chronos Dependency Scores, derived from cell depletion assays, indicate gene essentiality, where lower (more negative) scores reflect higher gene essentiality. (D) Immunohistochemical staining for PRMT5 (left) and ∆Np63α (right) in HNSCC patient specimens. The top panels show low-magnification views. Scale bars: 400 µm (top) and 100 µm (bottom).

Article Snippet: The list of antibodies used in this study includes: PRMT5 (Cell Signaling, Danvers, MA, USA, Cat. No. 79998), WDR77 (Cell Signaling, Danvers, MA, USA, Cat. No. 2823), ∆Np63α (Cell Signaling, Danvers, MA, USA, Cat. No. 39692), p21 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-71811), β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-47778), and HSC70 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-7298).

Techniques: Expressing, Derivative Assay, Immunohistochemical staining, Staining

Journal: Cancers

Article Title: PRMT5/WDR77 Enhances the Proliferation of Squamous Cell Carcinoma via the ΔNp63α-p21 Axis.

doi: 10.3390/cancers16223789

Figure Lengend Snippet: Figure 2. PRMT5 and WDR77 regulate the HNSCC-specific transcriptome. (A) GSEA plots identified genes affected in both PRMT5KO and WDR77KO, with notably enriched RICK- MAN_HEAD_AND_NECK_CANCER C and E gene sets. (B) Heatmaps of GSEA results for both PRMT5KO and WDR77KO, highlighting the top five positively and negatively enriched gene sets. (C) UMAP visualizations depict cell types for each cluster and expression patterns of PRMT5, WDR77, and TP63. (D) Comparison of PRMT5 (left) or WDR77 (right), along with TP63, CDKN1A, and MKI67 gene expression, in PRMT5High vs. PRMT5Low (left) and in WDR77High vs. WDR77Low (right) subgroups in single HNSCC cells. p-values were calculated using the Wilcoxon Rank-Sum Tests, and the median (50th percentile) for each dataset is denoted by a solid line.

Article Snippet: The list of antibodies used in this study includes: PRMT5 (Cell Signaling, Danvers, MA, USA, Cat. No. 79998), WDR77 (Cell Signaling, Danvers, MA, USA, Cat. No. 2823), ∆Np63α (Cell Signaling, Danvers, MA, USA, Cat. No. 39692), p21 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-71811), β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-47778), and HSC70 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-7298).

Techniques: Expressing, Comparison, Gene Expression

Journal: Cancers

Article Title: PRMT5/WDR77 Enhances the Proliferation of Squamous Cell Carcinoma via the ΔNp63α-p21 Axis.

doi: 10.3390/cancers16223789

Figure Lengend Snippet: Figure 3. Depletion of PRMT5 or WDR77 inhibits cell proliferation. (A) CRISPR-mediated depletion of PRMT5 or WDR77 in SCC cells using four sgRNAs (two for each gene). (B) Cellular competition-based GFP dropout assays in HSC5, FaDu, and Cal33 cells treated with sgRosa26, sgCDK1, sgPRMT5-1, sgPRMT5-2, sgWDR77-1, and sgWDR77-2. Normalized to P0. (C) MTT-based proliferation assays in the indicated SCC cell lines. Cells were treated with DMSO or the PRMT5 inhibitor PF-06939999 (4 µmol/L) for 48 h. Data were normalized to DMSO controls (n = 3 biologically independent replicates). The p-values were calculated using two-tailed unpaired Student’s t-tests. (D) Flow cytometry of HSC5 cells treated with sgNeg, sgPRMT5-1, and sgWDR77-2 (n = 3 biologically independent samples). The p-values were calculated using Two-Way ANOVA.

Article Snippet: The list of antibodies used in this study includes: PRMT5 (Cell Signaling, Danvers, MA, USA, Cat. No. 79998), WDR77 (Cell Signaling, Danvers, MA, USA, Cat. No. 2823), ∆Np63α (Cell Signaling, Danvers, MA, USA, Cat. No. 39692), p21 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-71811), β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-47778), and HSC70 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-7298).

Techniques: CRISPR, Two Tailed Test, Flow Cytometry

Journal: Cancers

Article Title: PRMT5/WDR77 Enhances the Proliferation of Squamous Cell Carcinoma via the ΔNp63α-p21 Axis.

doi: 10.3390/cancers16223789

Figure Lengend Snippet: Figure 4. PRMT5 and WDR77 modulate the ∆Np63α-p21 pathway. (A) qRT-PCR for PRMT5, WDR77 and TP63 transcript levels in FaDu cells transduced with sgPRMT5-1 and sgWDR77-2, normalized to GAPDH. The p-values were calculated using One-Way ANOVA. (B) Western blot of PRMT5, WDR77, ∆Np63α, and p21 expression in FaDu cells transduced with sgNeg (empty vector control), sgPRMT5-1, sgPRMT5-2, sgWDR77-1, and sgWDR77-2. (C) FaDu cells were infected with sgNeg, sgPRMT5-1, and sgWDR77-2, with or without additional treatment of 1 µmol/L lactacystin for 24 h, as described [36]. (D) MTT-based proliferation assays in FaDu scramble cells (control) and FaDu cells overexpressing CRISPR-resistant ∆Np63α cDNAs (∆Np63α CR). Cells were infected with sgNeg, sgPRMT5-1, and sgWDR77-2. Data are presented as means ± S.D., normalized to sgNeg (n = 3 biologically independent samples). The p-values were calculated using One-Way ANOVA. (E) Confirmation of PRMT5KO and WDR77KO via Western blot in FaDu scramble cells (control) and FaDu cells overexpressing ∆Np63α CR. Cells were treated with sgNeg, sgPRMT5-1, and sgWDR77-2. (F) Assessment of PRMT5, WDR77, ∆Np63α, and p21 expression via Western blot in FaDu scramble cells (control) and FaDu cells overexpressing ∆Np63α CR. Cells were treated with sgNeg (empty vector control), sgp63-1, and sgp63-2. The uncropped bolts were shown in Supplementary Figure S7.

Article Snippet: The list of antibodies used in this study includes: PRMT5 (Cell Signaling, Danvers, MA, USA, Cat. No. 79998), WDR77 (Cell Signaling, Danvers, MA, USA, Cat. No. 2823), ∆Np63α (Cell Signaling, Danvers, MA, USA, Cat. No. 39692), p21 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-71811), β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-47778), and HSC70 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-7298).

Techniques: Quantitative RT-PCR, Transduction, Western Blot, Expressing, Plasmid Preparation, Control, Infection, CRISPR

Journal: Cancers

Article Title: PRMT5/WDR77 Enhances the Proliferation of Squamous Cell Carcinoma via the ΔNp63α-p21 Axis.

doi: 10.3390/cancers16223789

Figure Lengend Snippet: Figure 5. PRMT5 or WDR77 depletion inhibits tumor growth in vivo. (A,B) Tumor volume caliper measurements and tumor weight measurements are means ± S.D. (n = 8). FaDu cells transduced with sgNeg, sgPRMT5-1, and sgWDR77-2 were injected subcutaneously into both rear flanks of nude mice (n = 4), with 5 × 104 cells per injection. Tumors were measured, weighed, and collected after 22 days. Mice were initially labeled as groups 1, 2, and 3, with the treatment groups blinded to ensure unbiased measurements until all data collection was completed. The p-values were calculated using One-Way ANOVA. (C) Representative images of tumors from each group. (D) Western blot assessment of PRMT5, WDR77, ∆Np63α, and p21 expression in the tumor samples from mice. The uncropped bolts were shown in Supplementary Figure S7.

Article Snippet: The list of antibodies used in this study includes: PRMT5 (Cell Signaling, Danvers, MA, USA, Cat. No. 79998), WDR77 (Cell Signaling, Danvers, MA, USA, Cat. No. 2823), ∆Np63α (Cell Signaling, Danvers, MA, USA, Cat. No. 39692), p21 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-71811), β-Actin (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-47778), and HSC70 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat. No. sc-7298).

Techniques: In Vivo, Transduction, Injection, Labeling, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Methylation of H4R3 by PRMT1 and PRMT5.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Effects of acetylation on Arg-3 methylation catalyzed by PRMT5. Reaction buffer contained 50 mm Hepes, pH 8.0, 10 mm NaCl, and 1 mm DTT. The concentrations of PRMT5, [14C]AdoMet, and H4 peptide were 0.1, 30, and 200 μm respectively. The reaction time was 1 h.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Kinetic parameters of PRMT5 catalysis The catalytic activity of PRMT5 on each peptide was tested with the radioactive filter binding assay. Varied concentrations of peptide (0–10 μ m ) and 30 μ m of [ 14 C]AdoMet were incubated at 30 °C for 5 min in the reaction buffer (50 m m HEPES (pH 8.0), 50 m m NaCl, 1 m m EDTA and 0.5 m m DTT) prior to the addition of PRMT5. The methylated products were loaded onto P81 filter paper and quantified by liquid scintillation. Calculated methylation rate was plotted as a function of peptide concentration, and the data were fitted with Michaelis-Menten equation.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Activity Assay, Filter-binding Assay, Incubation, Methylation, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Acetylation on H4 protein inhibits its methylation by PRMT1, although it promotes its methylation by PRMT5. A, p300 acetylation inhibits H4 methylation by PRMT1. H4 protein was incubated with acetyl-CoA in the absence or presence of p300 before submission to PRMT1 methylation with [14C]AdoMet. Methylated H4 bands were separated by 15% SDS-PAGE and visualized by storage phosphor scan, which is the same method as for B–D. B, p300 acetylation promotes H4 methylation by PRMT5. H4 protein was incubated with acetyl-CoA in the absence or presence of p300 before submission to PRMT5 methylation. C, MOF acetylation inhibits H4 methylation by PRMT1. H4 protein was incubated with acetyl-CoA in the absence or presence of MOF before submission to PRMT1 methylation. D, MOF acetylation promotes H4 methylation by PRMT5. H4 protein was incubated with acetyl-CoA in the absence or presence of MOF before submission to PRMT5 methylation.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Methylation, Incubation, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: Histone H4 Acetylation Differentially Modulates Arginine Methylation by an in Cis Mechanism *

doi: 10.1074/jbc.M110.207258

Figure Lengend Snippet: Summary of the effects of lysine acetylation on Arg-3 methylation in H4. Acetylation of the N-terminal H4 tail reciprocally affects PRMT1-mediated Arg-3 methylation and PRMT5-mediated Arg-3 methylation. A solid line means that the effect of acetylation is appreciably strong, and a dotted line means that the effect of acetylation is relatively weak.

Article Snippet: Active FLAG-tagged human recombinant PRMT5/MEP50 was purchased from BPS Bioscience, Inc. Radioactive Methylation Assays The methylation assays of different H4 peptide substrates were performed using 14 C-isotope labeled AdoMet at 30 °C.

Techniques: Methylation

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