Journal: Blood Cancer Journal

Article Title: Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations

doi: 10.1038/s41408-023-00799-6

Figure Lengend Snippet: MCL cell lines were treated with a two-fold serial dilution of a PARP inhibitor AZD2281, b ATR inhibitor AZD6738, c p53 reactivator APR-246, or d CDK4/6 inhibitor abemaciclib for 3 days and the viability were evaluated using CellTiter-Glo. Similarly, the viability of MCL cells treated with a two-fold serial dilution of e PRMT5 inhibitors GSK3326595 or f LLY-283 for 6 days were evaluated due to longer responding time required for PRMT5 inhibitors. g Viability of primary MCL cells from R/R patients treated with a two-fold serial dilution of PRMT5 inhibitors GSK3326595 for only 3 days. The primary cells stop growing after a few days in culture, therefore we shortened the treatment period.

Article Snippet: To validate the target specificity of the PRMT5 inhibitors, we knocked out PRMT5 in three MCL cell lines (Maver-1, Granta-519, and Z-138) with different TP53 and ATM status (Fig. and Supplementary Table S ) using CRISPR-Cas9 gene editing and evaluated the cytotoxic effect of the inhibitors.

Techniques: Serial Dilution

Journal: Blood Cancer Journal

Article Title: Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations

doi: 10.1038/s41408-023-00799-6

Figure Lengend Snippet: a , b PRMT5 ( a ) and WDR77 ( b ) expression analyzed by RNA-seq in clinical specimens from unpaired ibrutinib-sensitive or -resistant patients with MCL. c PRMT5 protein expression was determined by western blotting in clinical specimens from MCL patients sensitive or resistant to ibrutinib treatment. The PRMT5 bands were quantitated using ImageJ and then normalized with a corresponding loading control. d Left: Immunohistochemistry analysis of MCL tissue microarray (TMA) for the expression of PRMT5, H4R3me2s, and proliferation marker Ki67. Representative images of tissue staining are shown. Right: Quantification of PRMT5, H4R3me2s, and Ki67 staining signal by ImageJ was shown. P value was determined by two-tailed independent Student’s t test. Correlation between the indicated protein levels was determined by Pearson chi-square test. r correlation coefficient. e Kaplan survival analysis of MCL patients with high or low PRMT5 expression. For all panels: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns (not statistically significant), P ≥ 0.05.

Article Snippet: To validate the target specificity of the PRMT5 inhibitors, we knocked out PRMT5 in three MCL cell lines (Maver-1, Granta-519, and Z-138) with different TP53 and ATM status (Fig. and Supplementary Table S ) using CRISPR-Cas9 gene editing and evaluated the cytotoxic effect of the inhibitors.

Techniques: Expressing, RNA Sequencing, Western Blot, Control, Immunohistochemistry, Microarray, Marker, Staining, Two Tailed Test

Journal: Blood Cancer Journal

Article Title: Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations

doi: 10.1038/s41408-023-00799-6

Figure Lengend Snippet: a Indicated MCL cell lines were treated with PRMT5 inhibitor GSK3326595 and subjected to IF staining with anti-γH2AX, a marker for DNA damage. Scale bars = 20 μm. b γH2AX foci in ( a ) were quantified using ImageJ. c Expression of genes (AR, DNAPK, NHEJ1 and RAD51) in DDR pathways analyzed by real-time qPCR in Granta-519 and Z-138 cell lines treated with PRMT5 inhibitor GSK3326595. d Indicated cell lines were treated with PRMT5 inhibitors GSK3326595 (1 µM) and LLY-283 (1 µM) and subjected to RT-PCR analysis of MDM4 mRNA splicing. e Expression of PRMT5 and p53 pathway genes determined by immunoblotting in Granta-519 with or without PRMT5 KO. f Granta-519 cells were stained with anti-γH2AX for 2 h after exposure to 10 Gy γ-irradiation. Scale bars = 20 μm. γH2AX foci were quantitated using ImageJ. g Indicated cell lines were treated as in ( d ) before they were subjected to western blotting analysis of MDM4 and p53. h Indicated MCL cell lines were treated with PRMT5 inhibitor GSK3326595 or LLY-283 and subjected to apoptosis analysis. i Western blotting analysis of the expression of PRMT5 and p53 in the cell lines treated as in ( h ). j Expression of p53 target genes (MDM2, p21, and PUMA) analyzed by real-time qPCR in in Z-138, Granta-519 and JVM-2 cell lines treated with LLY-283. k A schematic illustration of the alternative splicing of MDM4 upon PRMT5 depletion stabilizing p53. For all panels: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns (not statistically significant), P ≥ 0.05.

Article Snippet: To validate the target specificity of the PRMT5 inhibitors, we knocked out PRMT5 in three MCL cell lines (Maver-1, Granta-519, and Z-138) with different TP53 and ATM status (Fig. and Supplementary Table S ) using CRISPR-Cas9 gene editing and evaluated the cytotoxic effect of the inhibitors.

Techniques: Staining, Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Irradiation, Alternative Splicing

Journal: Blood Cancer Journal

Article Title: Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations

doi: 10.1038/s41408-023-00799-6

Figure Lengend Snippet: NSG mice xenografts implanted with a Granta-519 or d Maver-1 were treated with either vehicle solvent or PRMT5 inhibitor GSK3326595 (100 mg/kg, daily). Tumor volumes were measured and plotted against the time of treatment ( n = 5). Statistical significance between vehicle and PRMT5 inhibitor treatment was determined by a two-way ANOVA test. Tumor weights of b Granta-519 and e Maver-1 xenografts from ( a ) and ( d ), respectively, were measured at the endpoint of treatment. Statistical significance between vehicle and PRMT5 inhibitor treatment was determined by a two-tailed independent Student’s t test. c , f Western blotting analysis of the expression of PRMT5, H4R3me2s, p53, and p21 in xenograft tumors from ( a ) and ( d ), respectively. g Granta-519 cells with or without PRMT5 KO were injected into NSG mice. Tumor volume was monitored and plotted. Statistical significance between vehicle and PRMT5 inhibitor treatment was determined by two-way ANOVA test. h Tumor weights at the endpoint are presented. Statistical significance between vehicle and PRMT5 inhibitor treatment was determined by two-tailed independent Student’s t test. i PRMT5 expression in tumors of mice was confirmed using western blotting. j PDX models were derived from a MCL patient who had TP53 mutations. The PDX mice were treated with vehicle or GSK3326595 (100 mg/kg, daily). Tumor sizes were measured at different days and plotted relative to the number of days post treatment ( n = 5). Statistical significance between vehicle and PRMT5 inhibitor treatment was determined by a two-way ANOVA test. k , l Tumor weight measurement, statistical analysis, and western blotting were performed as described in ( b ) and ( c ). m PDX models derived from a MCL patient who had relapsed after CD19 CAR T-cell therapy were treated with GSK3326595 (100 mg/kg, daily). Tumor sizes were measured at different days and plotted against the time of treatment ( n = 5). Statistical significance between vehicle and PRMT5 inhibitor treatment was determined by two-way ANOVA test. n , o Tumor weight measurement, statistical analysis, and western blotting were performed as described in ( b ) and ( c ). For all panels: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns (not statistically significant), P ≥ 0.05.

Article Snippet: To validate the target specificity of the PRMT5 inhibitors, we knocked out PRMT5 in three MCL cell lines (Maver-1, Granta-519, and Z-138) with different TP53 and ATM status (Fig. and Supplementary Table S ) using CRISPR-Cas9 gene editing and evaluated the cytotoxic effect of the inhibitors.

Techniques: Solvent, Two Tailed Test, Western Blot, Expressing, Injection, Derivative Assay

Journal: Blood Cancer Journal

Article Title: Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations

doi: 10.1038/s41408-023-00799-6

Figure Lengend Snippet: a Cell viability of Granta-519 and Maver-1 cells treated with LLY-283 (LLY, 0.25 µM), AZD2281 (2.5 µM), or in combination. b Cell viability of Granta-519 and Maver-1 cells treated with GSK3326595 (GSK, 0.6 µM), AZD6738 (0.6 µM), or their combination. c NSG mice implanted with Granta-519 and treated with vehicle, GSK3326595 (GSK, 100 mg/kg, daily), AZD6738 (25 mg/kg, daily), or the combination of both inhibitors. Tumor volumes were measured on specified days. Average tumor volumes were plotted against the number of days after treatment ( n = 5). Statistical significance values relative to inhibitors combination were determined by two-way ANOVA test. d Tumor weight measured at the endpoint of treatment. Statistical significance values relative to inhibitors combination was determined by two-tailed independent Student’s t test. e Western blotting analysis of the expression of PRMT5, H4R3me2s, and p53 in representative tumors from mice in ( d ). For all panels: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns (not statistically significant), P ≥ 0.05.

Article Snippet: To validate the target specificity of the PRMT5 inhibitors, we knocked out PRMT5 in three MCL cell lines (Maver-1, Granta-519, and Z-138) with different TP53 and ATM status (Fig. and Supplementary Table S ) using CRISPR-Cas9 gene editing and evaluated the cytotoxic effect of the inhibitors.

Techniques: Two Tailed Test, Western Blot, Expressing

Journal: Blood Cancer Journal

Article Title: Exploiting PRMT5 as a target for combination therapy in mantle cell lymphoma characterized by frequent ATM and TP53 mutations

doi: 10.1038/s41408-023-00799-6

Figure Lengend Snippet: a Relative CDK4 expression levels analyzed using the RNA-seq dataset in Fig. . b Indicated MCL cells treated with 1 μM GSK3326595 (GSK) and subjected to cell cycle analysis. c Cell viability of JVM-2 and Maver-1 cells treated with LLY-283 (LLY), abemaciclib (ABE), or the combination of both inhibitors. d Tumor volumes of Maver-1-derived xenografts treated with vehicle, GSK3326595 (GSK, 100 mg/kg, daily), abemaciclib (ABE, 10 mg/kg, daily), or the combination of both inhibitors were measured on specified days. Average tumor volumes were plotted against the number of days after treatment ( n = 5). Statistical significance values relative to inhibitors combination was determined by two-way ANOVA test. e Tumor mass weight measured at the endpoint of treatment. Statistical significance values relative to inhibitors combination were determined by two-tailed independent Student’s t test. f Western blotting analysis of the expression of PRMT5, H4R3me2s, CDK4, and p-Rb in the representative tumors from ( e ). For all panels: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns (not statistically significant), P ≥ 0.05.

Article Snippet: To validate the target specificity of the PRMT5 inhibitors, we knocked out PRMT5 in three MCL cell lines (Maver-1, Granta-519, and Z-138) with different TP53 and ATM status (Fig. and Supplementary Table S ) using CRISPR-Cas9 gene editing and evaluated the cytotoxic effect of the inhibitors.

Techniques: Expressing, RNA Sequencing, Cell Cycle Assay, Derivative Assay, Two Tailed Test, Western Blot

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Onametostat, a PfPRMT5 inhibitor, exhibits antimalarial activity to Plasmodium falciparum

doi: 10.1128/aac.00176-24

Figure Lengend Snippet: Screening 11 PRMT5 inhibitors in P. falciparum. ( A ) Eleven commercially available PRMT inhibitors were selected. Among them, eight compounds are potent PRMT5 inhibitor candidates. ( B ) All the compounds were screened for in vitro activity against asexual P. falciparum parasites at 0, 1, 10, and 100 µM, respectively.

Article Snippet: The PRMT5 inhibitor PF-06855800 was purchased from ChemieTek, while the rest of the PRMT inhibitors were from Selleck Chemicals.

Techniques: In Vitro, Activity Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Onametostat, a PfPRMT5 inhibitor, exhibits antimalarial activity to Plasmodium falciparum

doi: 10.1128/aac.00176-24

Figure Lengend Snippet: The effects of onametostat on P. falciparum . ( A ) The activity of the purified PfPRMT5 was significantly inhibited by onametostat at a molar ratio of 1:1 and 1:2 of PRMT5 to onametostat. The activities were measured using in vitro methyltransferase activity assays and shown as relative light units (RLU). ( B ) Comparison of the in vitro susceptibility of ΔPfPRMT5 parasite line with 3D7 exposed to onametostat. ( C ) The level of H3R2me2s was detected by Western blot after parasites at the schizont stage were treated with either dimethyl sulfoxide (DMSO) or onametostat at IC 50 for 3 h. H3 was used as a loading control. The numbers show the relative levels of H3R2me2s normalized with H3. ( D ) Onametostat at IC 50 and IC 90 did not significantly disturb schizont egress. The egress rate was defined as the ratio of naturally ruptured mature schizonts from three replicates. ( E ) Onametostat at IC 50 and IC 90 concentrations substantially disturbed merozoite invasion. Invasion rates of parasites were estimated using purified schizonts. Error bars in all panels indicate the mean ± SD from three replicates. *< I>P < 0.05, **< I>P < 0.01.

Article Snippet: The PRMT5 inhibitor PF-06855800 was purchased from ChemieTek, while the rest of the PRMT inhibitors were from Selleck Chemicals.

Techniques: Activity Assay, Purification, In Vitro, Comparison, Western Blot, Control

Journal: Nature Communications

Article Title: PRMT5 activates AKT via methylation to promote tumor metastasis

doi: 10.1038/s41467-022-31645-1

Figure Lengend Snippet: a The RNA expression of PRMT5 in increasing grades of neuroblastoma from the GSE49711 RNA-seq data series (Stage 1, n = 122, Stage 2, n = 91, Stage 3, n = 64, Stage 4, n = 184). Kruskal–Wallis with Dunn’s multiple comparisons test was used to determine p values, which were corrected with the Benjamini–Hochberg method.). b – d PRMT5 expression levels (left) and patient survival probability (right) in stage 3 and stage 4 neuroblastoma patients in Kocak ( b , Stage 3, n = 91, Stage 4, n = 214), SEQC ( c , Stage 3, n = 63, Stage 4, n = 183), and Versteeg ( d , Stage 3, n = 13, Stage 4, n = 40) databases. P values were calculated with a two-sided Wilcoxon rank-sum test for boxplots (left) and p values were calculated with a log-rank test for survival curves (right). a – d Boxplot center represents mean, the box represents SD, and whiskers represent minimum and maximum. e The efficacy of PRMT5 small molecule inhibitor GSK3203591 (GSK591) was determined by MTS assay in CHLA20, NGP, and SK-N-BE (2) cells ( n = 3). The IC50 value was determined by nonlinear regression (curve fit) using log 10 (inhibitor) versus response (three parameters) model in GraphPad Prism. f Cell viability was measured by MTS assay in a scramble or shRNA targeting PRMT5 in NGP (left) and SK-N-BE (2) cells (right) ( n = 4). g Cleaved caspase-3 levels in neuroblastoma cell lines treated with increasing doses of GSK591. h Hoechst 33342 staining in cells treated with DMSO or 100 nM GSK591. Scale bars, 100 μm. i Apoptosis was measured by caspase-3/7 staining in CHLA20 cells treated with DMSO or GSK591. j Quantification of caspase-3/7 positive cells ( n = 6). f , j p values were indicated by a two-tailed unpaired Student’s t -test using Microsoft Excel; error bars represent SD ( e – j ). e – i Representative results from three independent experiments. Uncropped immunoblots are provided in the Source Data file.

Article Snippet: PRMT5 methyltransferase activity inhibitor GSK3203591 was purchased from Cayman Chemical (cat#1616391-87-7).

Techniques: RNA Expression, RNA Sequencing, Expressing, MTS Assay, shRNA, Staining, Two Tailed Test, Western Blot

Journal: Nature Communications

Article Title: PRMT5 activates AKT via methylation to promote tumor metastasis

doi: 10.1038/s41467-022-31645-1

Figure Lengend Snippet: a Schematic diagram of PRMT5 inhibitor GSK3326595 (GSK595) in vivo study in the kidney renal capsule implantation xenograft model. b Tumor mass of CHLA20 iRFP720-Luc xenograft tumors from mice treated with vehicle ( n = 9) or GSK595 ( n = 10) (top) and representative in vivo bioluminescent images and quantification of tumor (bottom). Tumor mass was calculated by subtracting the weight of the normal kidney from the weight of the kidney that was implanted with tumor cells. Representative ex vivo images of the tumor by light microscope ( c ) or bioluminescence ( d ). e Tumor mass of NGP iRFP720-Luc xenograft tumors from mice treated with vehicle ( n = 7) or GSK595 ( n = 9) (top) and representative in vivo bioluminescent images and quantification of tumor (bottom). Representative ex vivo images of the tumor by light microscope ( f ) or bioluminescence ( g ). h Representative ex vivo bioluminescent images of liver from mice bearing CHLA 20 or NGP xenograft tumors treated with vehicle or GSK595. i Representative charts of FACS analysis of iRFP720 positive human neuroblastoma cells in hepatocytes isolated from the whole liver from CHLA20 xenografted mice, NC negative control. j Quantification of the percentage of iRFP720 positive tumor cells determined by flow cytometry in hepatocytes isolated from the whole liver from CHLA20 xenografted mice. Vehicle, n = 9, GSK595, n = 7, NC, n = 4. p values were indicated by a two-tailed unpaired Student’s t -test using Microsoft Excel; error bars represent SD ( b , e , j ).

Article Snippet: PRMT5 methyltransferase activity inhibitor GSK3203591 was purchased from Cayman Chemical (cat#1616391-87-7).

Techniques: In Vivo, Ex Vivo, Light Microscopy, Isolation, Negative Control, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: PRMT5 activates AKT via methylation to promote tumor metastasis

doi: 10.1038/s41467-022-31645-1

Figure Lengend Snippet: a Proteomics-based pathway screening by IPAD platform (immuno-paired-antibody detection assay) for the expression or modification of key proteins involved in more than 20 signaling pathways. Signals were normalized to internal levels of GAPDH and beta Tubulin. Heatmap showed differences between DMSO and GSK591 groups based on the average value from two independent experiments ( n = 2 biologically independent samples). The color scale represented log2 fold changes over DMSO treatment. b Immunoblots showing phosphorylation of AKT, and its downstream targets phospho-GSK3α and phospho-GSK3β in CHLA20 and NGP cells treated with DMSO or increasing doses of GSK591. c Western blots of AKT phosphorylation and AKT downstream targets phospho-GSK3α and phospho-GSK3β in the presence and absence of PRMT5 in NGP or BE2 cells harboring scramble or shPRMT5. d Levels of phosphorylated AKT, GSK3α, and GSK3β with or without EGF stimulation in DMSO and GSK591-treated CHLA20 and NGP cells. e Activated AKT and its targets phospho-GSK3α and phospho-GSK3β were detected by Western blotting in xenograft tumors from vehicle or GSK595-treated mice. b – d Representative results from three independent experiments. Uncropped immunoblots are provided in the Source Data file.

Article Snippet: PRMT5 methyltransferase activity inhibitor GSK3203591 was purchased from Cayman Chemical (cat#1616391-87-7).

Techniques: Detection Assay, Expressing, Modification, Protein-Protein interactions, Western Blot, Phospho-proteomics

Journal: Nature Communications

Article Title: PRMT5 activates AKT via methylation to promote tumor metastasis

doi: 10.1038/s41467-022-31645-1

Figure Lengend Snippet: a PRMT5/AKT interaction captured by co-immunoprecipitation (co-IP). The lysate was immunoprecipitated with anti-PRMT5 antibody followed by immunoblotting with anti-AKT antibody in CHLA20 and NGP cells (top), and the reciprocal co-immunoprecipitation was shown in the middle. BRG1 and bait protein PRMT5 were shown as positive controls for the PRMT5 IP, whereas GSK3β and bait protein AKT served as positive controls for AKT IP. The input was used as internal controls (bottom). b Immunoprecipitation of AKT1 followed by a Western blotting analysis of symmetric dimethylarginine (SDMA) of AKT1, phosphorylation of AKT1 on Thr308 and Ser473 in DMSO or GSK591-treated CHLA20 and NGP cells. c AKT1 phosphorylation was detected by immunoblotting in a scramble or PRMT5 knockdown cells when forced expressing a wild-type PRMT5 or an enzymatic deficient form of PRMT5. d Analysis of AKT1 phosphorylation in DMSO or GSK591-treated CHLA20 and NGP cells expressing PRMT5 wild type or enzyme dead mutant. e In vitro methylation assay showing the methylation of AKT1 wild type and R15K mutant by recombinant PRMT5/MEP50 (top), Ponceau S staining of the membrane showing equal loading of each sample (middle), and Western blotting analysis showing an equal amount of HA-tagged proteins pulled down by anti-HA beads (bottom). f Analysis of SDMA and phosphorylation of AKT1 wild type or R15K mutant in CHLA20 and NGP cells with (right) or without (left) EGF stimulation. Cells were transfected with AKT1 wild type or R15K mutant. In the case of EGF stimulation, 24 h post-transfection, cells were serum-starved overnight and then treated with 10 ng/mL EGF for 15 min before harvest. a – d , f Representative results from three independent experiments. e Representative results from two independent biological samples. Uncropped immunoblots are provided in the Source Data file.

Article Snippet: PRMT5 methyltransferase activity inhibitor GSK3203591 was purchased from Cayman Chemical (cat#1616391-87-7).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Phospho-proteomics, Knockdown, Expressing, Mutagenesis, In Vitro, Methylation, Recombinant, Staining, Membrane, Transfection

Journal: Nature Communications

Article Title: PRMT5 activates AKT via methylation to promote tumor metastasis

doi: 10.1038/s41467-022-31645-1

Figure Lengend Snippet: a Western blotting analysis of the distribution of activated AKT1 in cytosolic or membrane fractions from DMSO or GSK591-treated CHLA20 cells in the absence or presence of EGF. b The presence of activated AKT1 in cytosolic and membrane fractions from scramble or PRMT5 knockdown cells with or without EGF stimulation. c Colocalization of AKT1 wild type or R15K mutant with plasma membrane by immunofluorescence visualized under confocal microscopy in CHLA 20 cells in the absence or presence of EGF. Scale bars, 100 μm. d The binding to phosphatidylserine (PS) of AKT1 wild type or R15K mutant was examined by the presence of HA-tagged protein in the eluate or flowthrough fraction after incubation with phosphatidylserine coated agarose beads by Western blotting. Top, elution from PS beads; middle, flowthrough after incubation; bottom, HA-tagged protein eluted from anti-HA beads. e The association of AKT1 wild type or R15K mutant with PDK1 or mTORC2 was analyzed by Western blotting in CHLA20 with or without EGF stimulation. Exogenous AKT1 wild type or R15K mutant was pulldown by HA beads, and the precipitants were analyzed by Western blotting for PDK1 and SIN1. f The interaction of AKT1 with PDK1 and mTORC2 was measured by immunoprecipitation of endogenous AKT1 followed by Western blotting against PDK1 and SIN1 in SK-N-BE(2) and NGP cells with or without PRMT5 knockdown. g The recruitment of PDK1 and mTORC2 was examined in DMSO or GSK591-treated CHLA20 and NGP cells. a – g Representative results from three independent experiments. Uncropped immunoblots are provided in the Source Data file.

Article Snippet: PRMT5 methyltransferase activity inhibitor GSK3203591 was purchased from Cayman Chemical (cat#1616391-87-7).

Techniques: Western Blot, Membrane, Knockdown, Mutagenesis, Clinical Proteomics, Immunofluorescence, Confocal Microscopy, Binding Assay, Incubation, Immunoprecipitation

Journal: Nature Communications

Article Title: PRMT5 activates AKT via methylation to promote tumor metastasis

doi: 10.1038/s41467-022-31645-1

Figure Lengend Snippet: a Immunoblots showing the expression of TWIST1, SNAIL, and ZEB1 in neuroblastoma cells treated with DMSO or increasing doses of GSK591 in CHLA20 and NGP cells. b Analysis of TWIST1, SNAIL, and ZEB1 protein levels in the scramble and shPRMT5 cells. c The expression of EMT transcription factor TWIST1, SNAIL, and ZEB1 was examined in xenograft tumors from mice treated with vehicle or GSK595 ( n = 4). d Representative images of DMSO or GSK591-treated cells migrated to the cleared space (wound) after 24 h. Scale bars, 100 μm. e Quantification of in vitro cell migration assay ( n = 6). The migration area was determined by measuring the total area of the wound using the ImageJ software. f Representative images of DMSO or GSK591-treated cells invaded to ECM coated membrane in transwell invasion assay. Scale bars, 100 μm. g Percentage of invasive cells normalized by cell numbers in the non-ECM coated 12-well plate using ImageJ ( n = 6). h Protein levels of TWIST1 and SNAIL were analyzed in DMSO or GSK591-treated cells transfected with a vector or constitutively activated AKT1. i The protein levels of TWIST1 and SNAIL in cells overexpressing PRMT5 wild type or enzyme activity deficient mutant by Western blotting. j Representative images of CHLA20 cells overexpressing vector, wild type PRMT5, or an enzymatic deficient form of PRMT5 invaded to ECM coated membrane in the transwell invasion assay (left). Scale bars, 100 μm. Percentage of invasive cells normalized by cell numbers in the non-ECM coated 12-well plate using ImageJ ( n = 6) (right). e , g p values were calculated by two-tailed unpaired Student’s t -test using Microsoft Excel. j p values were determined using one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent SD. a , b , d – j Representative results from three independent experiments and the results shown are from a representative experiment. Uncropped immunoblots are provided in the Source Data file.

Article Snippet: PRMT5 methyltransferase activity inhibitor GSK3203591 was purchased from Cayman Chemical (cat#1616391-87-7).

Techniques: Western Blot, Expressing, In Vitro, Cell Migration Assay, Migration, Software, Membrane, Transwell Invasion Assay, Transfection, Plasmid Preparation, Activity Assay, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: PRMT5 activates AKT via methylation to promote tumor metastasis

doi: 10.1038/s41467-022-31645-1

Figure Lengend Snippet: AKT is activated in a cascade of events. Upstream stimuli, such as growth factors and cytokines, induce the production of phosphatidylinositol (3,4,5) trisphosphates (PIP3) by phosphoinositide 3-kinase (PI3K). These phospholipids serve as plasma membrane docking sites for AKT and PDK1 at pleckstrin-homology (PH) domains. PDK1 phosphorylates AKT at Thr308 at the plasma membrane which partially activates AKT, whereas AKT is fully activated after phosphorylation at Ser473 by mTORC2. PRMT5 methylates AKT1 on R15 in the PH domain, by which it promotes AKT1 association with the plasma membrane and subsequent phosphorylation by PDK1 and mTORC2. Downstream of AKT signaling, PRMT5 increases the expression of EMT transcription factors, such as SNAIL, ZEB1, and TWIST1 augmenting the EMT program to promote tumor metastasis.

Article Snippet: PRMT5 methyltransferase activity inhibitor GSK3203591 was purchased from Cayman Chemical (cat#1616391-87-7).

Techniques: Clinical Proteomics, Membrane, Phospho-proteomics, Expressing