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: Communications biology

Article Title: PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth.

doi: 10.1038/s42003-024-06290-7

Figure Lengend Snippet: Fig. 1 | PRMT5 expression levels are increased in lung cancer cells. a, b PRMT5 expression is elevated in a lung adenocarcinoma and b lung squamous cell carci- noma. RNA-seq data from The Cancer Genome Atlas (TCGA) were analysed on the UCSC Xena browser. c Correlation between STAT3 and PRMT5 protein levels in 12 NSCLC cell lines (NCI-H23, LU65, CORL105, CHAGOK1, NCI-H3255, NCI-

Article Snippet: STAT3, PRMT5, and MEP50 protein levels were determined by western blotting using an antibody for the indicated tag [STAT3 (Myc-tag): GeneTex (GTX115046), PRMT5 (Flag-tag): SIGMA (SAB4301135), MEP50 (His-tag): Proteintech (10001-0-AP)]. h Quantification of PRMT5/MEP50 complex-mediated STAT3 methylation.

Techniques: Expressing, RNA Sequencing

Journal: Communications Biology

Article Title: PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth

doi: 10.1038/s42003-024-06290-7

Figure Lengend Snippet: a, b Expression of a PRMT5 and b MEP50 was stably knocked down in HCC827 and A549 cells. Short hairpin RNAs (shRNAs) targeting PRMT5 and MEP50 were expressed by recombinant lentivirus. Protein levels were detected by western blotting using the antibody for PRMT5 (abcam; ab10941) or MEP50 (abcam; ab154190). PRMT5 or MEP50 protein levels were quantified with normalization to the β-actin expression intensity. c , d Knockdown of c PRMT5 and d MEP50 attenuated cell proliferation. First, 50,000 cells were seeded on a 12-well plate in triplicate. Viable cells were counted by trypan blue exclusion. Results are shown as the mean ± standard deviation (SD) from three experiments. Tukey’s honestly significant difference test was applied for statistical comparisons. The relative cell number indicates the total cell number/Total cell number at time 0 of each cell . e , f PRMT5 is involved in CSC maintenance. First, 1000 cells were seeded on a six-well ultra-low-attachment plate in triplicate, and then spheres were counted after 7 days of growth. Results are shown as the mean ± SD from three experiments. e Sphere number from HCC827 cells. f Sphere number from A549 cells. In e and f , Tukey’s honestly significant difference test was applied for statistical comparisons. g , h Knockdown of PRMT5 attenuates the CSC population in HCC827 cells. The population of CSCs was assessed by examining aldehyde dehydrogenase (ALDH) activity using flow cytometry. g Representative flow cytometry plots indicate side scatter (SSC) versus intensity of FITC fluorescence. ALDH activity was based on FITC intensity. Cells having high ALDH activity were plotted in the gated area. A group treated with an ALDH inhibitor, 4-diethylaminobenzaldehyde (DEAB), was used as a negative control. The gating strategy is shown in Supplementary Fig. . h The CSC population is shown as the mean ± SD from three independent experiments. Unpaired two-tailed Student’s t-test was applied for statistical comparisons. i , j Knockdown of PRMT5 attenuates tumour growth. A549 cells with shPRMT5 or shMEP50 expression induced by the Tet-ON system were established. These cells also stably expressed luciferase by recombinant lentivirus. shRNAs for PRMT5 or MEP50 inducible cells were transplanted into 7-week-old nude mice at 1 × 10 6 cells ( n = 4). When tumour volume reached approximately 150 mm 3 , doxycycline (1 mg/mL) administration was started. Tumour volume was measured by In Vivo Imaging System (IVIS). The individual datapoints are in Supplementary Fig. . Representative IVIS images are shown in j (4 weeks). Results are shown as the mean ± standard error of the mean (SEM). Tukey’s honestly significant difference test was applied for statistical comparisons. The individual datapoints are in Supplementary Fig. . Source data are provided in Supplementary Data . Full immunoblot images are shown in Supplementary Fig. .

Article Snippet: PRMT5-bound MEP50 or STAT3 was detected by western blotting using an anti-MEP50 antibody (Abcam; ab154190) or an anti-STAT3 antibody (CST; 9139).

Techniques: Expressing, Stable Transfection, Recombinant, Western Blot, Standard Deviation, Activity Assay, Flow Cytometry, Fluorescence, Negative Control, Two Tailed Test, Luciferase, In Vivo Imaging

Journal: Communications Biology

Article Title: PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth

doi: 10.1038/s42003-024-06290-7

Figure Lengend Snippet: a , b Sphere numbers with IL-6 treatment in shControl- and shPRMT5-expressing a HCC827 and b A549 cells. Cells were treated with recombinant IL-6 (50 ng/mL) and recombinant sIL-6 (50 ng/mL) for 7 days, after which the numbers of spheres were determined. The results are shown as the mean ± standard deviation (SD) from three experiments. Tukey’s honestly significant difference test was applied for statistical comparisons. c Western blot analysis of STAT3 activation after 50 ng/ml recombinant IL-6 and 50 ng/ml recombinant sIL-6 treatment for 7 days in control and PRMT5-knockdown cell-derived spheres. PRMT5 and STAT3 protein levels were detected by western blotting using an antibody for PRMT5 (abcam; ab10941) or STAT3 (CST; 12640). STAT3 activation was monitored by western blotting using an anti-phosphorylated STAT3 (Y705) antibody (CST; 9138). PRMT5 expression levels were quantified with normalization to β-actin expression intensity and phosphorylated STAT3 intensity was normalized to STAT3 expression intensity. d, e STAT3 transcriptional activity in PRMT5-knockdown d) HCC827 and e) A549 cells. STAT3 activity was measured by luciferase assays using a STAT3 reporter plasmid. Results are expressed as firefly/ Renilla ratios and presented as the mean ± SD from three experiments. Tukey’s honestly significant difference test was applied for statistical comparisons. f Schematic diagram of arginine residues methylated by PRMT5 in human STAT3. TAD: Transcriptional Activation Domain. g In vitro methylation of STAT3. Myc-tagged wild-type STAT3 (STAT3 WT) or its mutants were transiently expressed in HEK293T cells and purified by immunoprecipitation using an antibody for Myc-tag. Methylation of STAT3 by recombinant human Flag-tagged PRMT5/His-tagged MEP50 complex was detected by western blotting using an anti-symmetric dimethyl arginine antibody (Merck Millipore; 07-413). STAT3, PRMT5, and MEP50 protein levels were determined by western blotting using an antibody for the indicated tag [STAT3 (Myc-tag): GeneTex (GTX115046), PRMT5 (Flag-tag): SIGMA (SAB4301135), MEP50 (His-tag): Proteintech (10001-0-AP)]. h Quantification of PRMT5/MEP50 complex-mediated STAT3 methylation. Methylated STAT3 was quantified using MTase-Glo TM Methyltransferase assay kit (Promega). The results are shown as the mean ± standard deviation (SD) from three experiments. Unpaired two-tailed Student’s t-test was applied for statistical comparisons. i Methylation of arginine residue 609 (R609) in STAT3 by PRMT5 in STAT3-KO BEAS2B cells. STAT3 was knocked out using the CRISPR–Cas9 system. Myc-tagged STAT3 wild type (WT) and its mutants were transiently expressed in STAT3-knockout BEAS2B cells. Methylated STAT3 was detected using an anti-symmetric dimethyl arginine antibody (Merck Millipore; 07-413). Expression of HA-tagged PRMT5 was detected using an anti-HA antibody (abcam; ab130275), and Myc-tagged STAT3 was detected using a STAT3 antibody (CST; 9139). j Methylation of STAT3 R609 is necessary for its activation. STAT3 activity was measured by luciferase assays. STAT3 reporter plasmid and Myc-tagged STAT3 WT and its mutants were transiently expressed in STAT3-KO BEAS2B cells. Results are expressed as firefly/ Renilla ratios and presented as the mean ± standard deviation (SD) from three experiments. k Interaction of PRMT5 and STAT3 via MEP50. The PRMT5–MEP50–STAT3 complex was detected by immunoprecipitation using an anti-PRMT5 antibody (abcam; ab10941). PRMT5-bound MEP50 or STAT3 was detected by western blotting using an anti-MEP50 antibody (Abcam; ab154190) or an anti-STAT3 antibody (CST; 9139). Representative results from two independent experiments are shown in a–e and g–j, while those from three independent experiments are shown in k. Source data are provided in Supplementary Data . Full immunoblot images are shown in Supplementary Fig. .

Article Snippet: PRMT5-bound MEP50 or STAT3 was detected by western blotting using an anti-MEP50 antibody (Abcam; ab154190) or an anti-STAT3 antibody (CST; 9139).

Techniques: Expressing, Recombinant, Standard Deviation, Western Blot, Activation Assay, Derivative Assay, Activity Assay, Luciferase, Plasmid Preparation, Methylation, In Vitro, Purification, Immunoprecipitation, FLAG-tag, Two Tailed Test, Residue, CRISPR, Knock-Out