Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 enhances host m6A modification and promotes MAT2A expression. ( A ), ( B ) Caco2 cells were infected with SARS-CoV-2 at a MOI of 2. ( A ) Total RNA was extracted at 24, 48, and 72 h post-infection (hpi) and analyzed by dot blot to examine m6A modification. Methylene blue staining was used as a loading control.( B ) Expression levels of m6A machinery proteins in Caco-2 cells infected with SARS-CoV-2. ( C ) MeRIP-seq analysis identified m6A peaks in the SARS-CoV-2 genome isolated from Caco-2 cells infected with SARS-CoV-2 at a MOI of 4. ( D ) Relative abundance of SARS-CoV-2 RNA in 1 µg of total RNA from MeRIP-seq samples. ( E ) Schematic representation illustrating the relationship between MAT2A and m6A modification. ( F ) Protein levels of MAT2A and MAT2B in Caco-2 cells infected with SARS-CoV-2. The α2 and α2’ are two isoforms of the MAT2A protein. ( G ) MAT2A was knocked down in 293 T cells using short hairpin RNA (shRNA), with a non-targeted shRNA (shNC) serving as the control. ( H ), ( I ) The shNC or shMAT2A#1 293 T cell lines were cultured in DMEM containing 2% FBS for 24 h. ( H ) Levels of m6A modification in the indicated 293 T cells. ( I ) Concentrations of methionine and S-adenosylmethionine (SAM) were quantified by UPLC-MS/MS in the indicated cell lines. Data are presented as mean ± SEM of three independent experiments. Statistical significance was determined, with * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Modification, Expressing, Infection, Dot Blot, Staining, Control, Isolation, shRNA, Cell Culture, Tandem Mass Spectroscopy

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 nsp14 increases host m6A modification by inducing MAT2A expression. ( A ) Expression of MAT2A in 293 T cells transfected with varying doses of HA-nsp14 plasmid. ( B ) Immunoblots of 293 T cells transfected with plasmids encoding either wild-type nsp14, the H268A mutant, or the D331/G333A mutant. ( C ) Dot blot analysis of m6A modification in 293 T cells transfected with increasing doses of HA-nsp14 plasmid. ( D ) MeRIP-RT-qPCR analysis showing the levels of m6A in HA-nsp14 mRNA. Fold enrichment of m6A was normalized to the IgG control. ( E ), ( F ) Quantification of m6A modification in 293 T cells transfected with plasmids encoding either wild-type nsp14 or the D331/G333A mutant. ( G ) Comparative DNA sequence analysis of plasmids encoding wild-type HA-nsp14 and the D331/G333A mutant. ( H ) RT-qPCR analysis of mRNA abundance in the experiment described in panel f. The grayscale intensity was measured using Image J software. Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with * P < 0.05, ** P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Modification, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Dot Blot, Quantitative RT-PCR, Control, Sequencing, Software

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 nsp14 upregulates MAT2A expression by activating the mTORC1 signalling pathway. (A) Immunoblots of Caco2 cells infected with SARS-CoV-2 at a MOI of 2, analyzed at 24, 48, and 72 hpi. (B) Immunoblots of 293 T transfected with HA-nsp14 plasmid in a dose-dependent manner. (C) Transcript levels of MAT2A in 293 T cells transfected with indicated plasmids. (D) Immunoblots of 293 T cells grown overnight in the absence of serum, then treated with either vehicle (H2O) or insulin (500 nM, 30 min), or 293 T cells transfected with HA-nsp14 and treated with either vehicle (DMSO) or rapamycin (20 nM). (E – F) RAPTOR was knocked down in 293 T cells using a shRNA. (E) Immunoblots of the indicated 293 T cells transfected with HA-nsp14 in a dosedependent manner. (F) Quantification of m6A modification in the indicated 293 T cells by dot blot analysis. Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with *P < 0.05, **P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Expressing, Western Blot, Infection, Transfection, Plasmid Preparation, shRNA, Modification, Dot Blot

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: β-Coronaviruses stimulate SAM synthesis through the mTORC1 pathway to facilitate viral replication. ( A ) Caco2 cells were infected with HCoV-OC43, and total RNA was measured for m6A modification by dot blot analysis. ( B ) Immunoblot of Caco2 cells infected with HCoV-OC43. ( C ) Immunoblot of 293 T cells transfected with plasmids encoding SARS-CoV-1, SARS-CoV-2, and HCoV-OC43 nsp14. ZIKV-NS5 (an N7-MTase) was used as a negative control. ( D ) Immunoblot of MAT2A knockdown Caco2 cells infected with HCoV-OC43. ( E ) – ( H ) Caco2 and HRT-18 cells were cultured in the absence of methionine (Met-free). ( E ) Immunoblot of Caco2 cells infected with HCoV-OC43 with L-methionine (L-Met) supplemented in a dose-dependent manner. ( F ) Immunoblot of HRT-18 cells treated as in ( E ). ( G ) Immunoblot of Caco2 cells infected with HCoV-OC43 with SAM supplemented in a dose-dependent manner. ( H ) Immunoblot of HRT-18 cells treated as in ( G ). Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with * P < 0.05, ** P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Infection, Modification, Dot Blot, Western Blot, Transfection, Negative Control, Knockdown, Cell Culture

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: Primer sequences used for real-time PCR.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Sequencing

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A expression was upregulated in inflamed gingival tissues.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Expressing

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A inhibition decreased P. gingivalis -induced inflammatory responses in hGfs.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Inhibition

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A-knockdown reduced inflammation in P. gingivalis -infected hGFs.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Knockdown, Infection

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: NF-κB/MAPK pathway was involved in the enhanced MAT2A expression.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Expressing

Journal: Nature Communications

Article Title: Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

doi: 10.1038/s41467-024-50908-7

Figure Lengend Snippet: A Gene expression level of MAT2A in prostate cancer patients indicated subgroups (Primary, n = 714; CRPC, n = 316; NEPC, n = 19). p -values are indicated. B Heatmap of ERG and MAT2A expression values in the cohort of CRPC patients described in A . (Padj value 0.0014). C Gene expression level of MAT2A in CRPC described in A and divided in ERG positive ( n = 113) and negative ( n = 203). Adjusted p -value 0.0014. D Immunoblot of VCaP control (Ctrl) and MAT2Akd VCaP cells (Sh1, Sh2) with indicated Ab ( n = 3 independent experiments). E Sphere formation assay (SFA) in indicated cell lines ( n = 3 independent experiments with n = 4 (for sh1) and n = 3 (for ctrl and sh2) biological replicates. Bottom, representative images of tumor spheres. Scale bars, 100 µm. F Growth curve of VCaP xenograft from VCaP control (Ctrl) and VCaP with stable MAT2A knockdown (Sh1 and Sh2) were engrafted and growth was monitored by caliper every 2 days ( n = 5 mice /group). G Tumor weight of indicated tumor xenografts ( n = 5 mice/group). H Representative sections from the indicated tumor xenografts. Scale bars, 200 µm. Right, Immunoscore of Ki67 by Aperio tool ( n = 3 mice Ctrl, n = 5 mice Sh1, n = 5 mice Sh2). I Immunoblot of RWPE-1 cells with stable expression of ERG, MAT2A and ERG + MAT2A with indicated Ab ( n = 3 independent experiments). J SFA in indicated cell lines ( n = 3 independent experiments with 3 biological replicates). K Fold change of mRNA levels of NANOG and SOX2 in RWPE-1 cells with stable expression of ERG, MAT2A and ERG + MAT2A. ( n = 5-6 biological replicates). L Growth curve of RWPE-1 xenografts. Indicated cell lines were engrafted and growth was monitored by caliper every 2 days ( n = 6 mice/group). M Tumor weight of indicated tumor xenografts ( n = 6). N Representative sections from the indicated tumor xenografts. Scale bars are 200 µm. O Immunoscore of Ki67 using the Aperio tool ( n = 6 mice/group). Molecular weights are indicated in kilodaltons (kDa). All error bars, mean ± SD. For box-and-whisker plots in A and C , the line inside the box shows the median value. The bounds of the box represent the 25th–75th percentiles, with whiskers at minimum and maximum values. One-way-ANOVA was used to test significant differences between groups in all panels, except for ( K ) where 2-way-ANOVA was used. Data presented in F and L are independent replicates derived from individual mice. * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Article Snippet: For MST experiments, Histidine-tagged MAT2A protein (BPS Bioscience, cat 71401) was labelled with fluorescent dye using Monolith His-Tag Labeling Kit (cat MO-L008) (NanoTemper Technologies GmbH).

Techniques: Expressing, Western Blot, Control, Tube Formation Assay, Knockdown, Whisker Assay, Derivative Assay

Journal: Nature Communications

Article Title: Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

doi: 10.1038/s41467-024-50908-7

Figure Lengend Snippet: A Number of genes affected by knockdown of MAT2A (Sh1 and Sh2) in VCaP cells ( p -value ≤ 0.05). B Principal component analysis (PCA) plot of VCaP cell lines, colored according to the condition (Ctrl, Sh1, Sh2). C Heatmap of differentially expressed genes in MAT2Akd (VCaP Sh1 and VCaP Sh2) versus control VCaP cells (VCaP Ctrl). Replicates ( n = 3 biological replicates) are indicated. D Hallmark enrichment analysis of MAT2Akd VCaP cells (Sh1 and Sh2) compared to control cells, performed with cameraPR function. Down-regulated pathways are colored in blue, up-regulated pathways are colored in red. E Functional annotation of top-ranking features of MAT2Akd VCaP cells (Sh1 and Sh2) performed with enrichR (cell markers augmented). F Fold changes of selected oncogenic drivers in MAT2Akd cells (Sh1 and Sh2) versus VCaP control, evaluated by RNA-seq. G Cumulative gene expression level of down-regulated genes in MAT2Akd VCaP cells (Sh1 and Sh2) in the cohort of primary ( n = 714), CRPC ( n = 316) and NEPC ( n = 19) patients. H Fold changes of selected canonical androgen genes in MAT2Akd (Sh1 and Sh2) versus VCaP control cells evaluated by RNA-seq. I Cumulative gene expression level of up-regulated genes in MAT2Akd VCaP cells (Sh1 and Sh2) in the cohort of primary ( n = 714), CRPC ( n = 316) and NEPC ( n = 19) patients. J Venn diagram showing the overlap between down-regulated genes in VCaP MAT2Akd cells (Sh1 and Sh2) and gene set associated with NEPC. Test: Fisher exact test. Fold changes of selected NEPC ( K ) and cancer stem cell genes ( L ), repressed by MAT2Akd in VCaP cells (Sh1 and Sh2). M Hallmark enrichment analysis of ERG + MAT2A RWPE-1 cells compared to RWPE-1 MAT2A performed with cameraPR function. Down-regulated pathways are colored in blue, up-regulated pathways are colored in red. N Fold change of selected CSC- and EMT-related genes up-regulated in RWPE-1 ERG + MAT2A versus RWPE-1 MAT2A. For box-and-whisker plots in G and I , the line inside the box shows the median value. The bounds of the box represent the 25th–75th percentiles, with whiskers at minimum and maximum values. One-way-ANOVA was used to test significant differences between groups.

Article Snippet: For MST experiments, Histidine-tagged MAT2A protein (BPS Bioscience, cat 71401) was labelled with fluorescent dye using Monolith His-Tag Labeling Kit (cat MO-L008) (NanoTemper Technologies GmbH).

Techniques: Knockdown, Control, Functional Assay, RNA Sequencing Assay, Expressing, Whisker Assay

Journal: Nature Communications

Article Title: Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

doi: 10.1038/s41467-024-50908-7

Figure Lengend Snippet: A Schematic representation of ex-vivo SFA assay, starting from LuCaP 35. B Ex-vivo SFA from dissociated LuCaP 35 under treatment with the indicated concentration of PF-9366 Right, images of spheres at the indicated concentration ( n = 6 biological replicates). Scale bars are 100 µm. C Ex-vivo SFA from dissociated LuCaP 35 under treatment with the indicated concentration of AG-270. Scale bars are 100 µm. Right, images of spheres at the indicated concentration ( n = 3 biological replicates). D Schematic representation of systemic in vivo treatment of LuCaP 35 tumors with vehicle or AG-270. E Growth curve of LuCaP 35 xenografts. Mice ( n = 7/group) were treated by oral gavage with either vehicle or AG-270. Tumor growth was monitored every 2 days with caliper. F Ex-vivo SFA assay from explanted LuCaP 35 tumors ( n = 3 tumors/group) after treatment with either vehicle or AG-270. Right, images of spheres of indicated treatment groups. Scale bars are 100 µm. G Representative sections from the indicated LuCaP tumors. Scale bars are 200 µm. H Immunoscore of Ki67, ERG, EZH2, AR and CHGA of tumor xenografts shown in ( E ) (n = 7 mice/group/; n = 7 biological replicates) using the Aperio tool. I Representative immunohistochemical evaluation of MAT2A (left) and quantification (right) in prostate tissue from WT (Pb-Cre;ERGflox/flox; n = 4 mice, n = 8 technical replicates) and ERG + /PTEN-(Pb-Cre4;Ptenflox/flox;Rosa26ERG/ERG; n = 6 mice, n = 40 technical replicates). Scale bars, 200 µm. J Schematic representation of 3D organoids assay derived from ERG/PTEN prostates to evaluate the reversion of the organoid phenotype. K Organoids treated with the indicated doses of PF-9366 ( n = 8 biological replicates). Right, representative images are shown. Scale bars, 100 µm. L Organoids treated with the indicated doses of AG-270 ( n = 8 biological replicates). Right, representative images are shown. Scale bars are 100 µm. All error bars, mean ± s.d. Ordinary one-way-ANOVA was used to test significant differences between groups in all panels, except for ( K , L ) where was used two-way-ANOVA. P -value was determined using unpaired t -test in F – H and I . * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001. Panels A , B and J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: For MST experiments, Histidine-tagged MAT2A protein (BPS Bioscience, cat 71401) was labelled with fluorescent dye using Monolith His-Tag Labeling Kit (cat MO-L008) (NanoTemper Technologies GmbH).

Techniques: Ex Vivo, Concentration Assay, In Vivo, Immunohistochemical staining, Derivative Assay

Journal: Nature Communications

Article Title: Epigenome-wide impact of MAT2A sustains the androgen-indifferent state and confers synthetic vulnerability in ERG fusion-positive prostate cancer

doi: 10.1038/s41467-024-50908-7

Figure Lengend Snippet: A Immunoblot of MAT2A in ERG positive and negative cell lines ( n = 2 independent experiments). B SFA from LNCaPabl cells under treatment with the indicated concentration of AG-270 (Left) ( n = 6 biological replicates for each drug concentration) or PF-9366 (Right) ( n = 3 biological replicates for each drug concentration). Bottom, images of spheres at the indicated concentration. C SFA from LNCaP cells under treatment with the indicated concentration of AG-270 (Left) ( n = 6 biological replicates for each drug concentration) or PF-9366 (Right) ( n = 3 biological replicates for each drug concentration). Bottom, images of spheres at the indicated concentration. D SFA from 22Rv1 cells under treatment with the indicated concentration of AG-270 (Left) ( n = 3 biological replicates for each drug concentration) or PF-9366 (Right) ( n = 3 biological replicates for each drug concentration). Bottom, images of spheres at the indicated concentration. E Immunoblot of H3K4me2 in ERG positive and negative cell lines ( n = 2 independent experiments). F Immunoblot with indicated antibodies in LNCaPabl cells after treatment with the indicated dose of AG-270. Right, densitometry analysis of the indicated markers ( n = 2 independent experiments). G Schematic representation of ex-vivo SFA from dissociated patient-derived xenograft LuCaP 145.2. H Immunoblot of MAT2A in patient-derived xenograft LuCaP 145.2 ( n = 2 independent experiments). I Ex-vivo SFA from dissociated LuCaP 145.2 under treatment with the indicated concentration of AG-270 (Left) ( n = 3 biological replicates for each drug concentration) or PF-9366 (Right) ( n = 6 biological replicates for each drug concentration). Bottom, images of spheres at the indicated concentration. J Schematic representation of organoid establishment from LuCaP 145.2 treatment with AG-270, and evaluation of NE markers. K Number and images of organoids at the indicated concentrations ( n = 7 biological replicates). L Immunoblot of NE markers in LuCaP 145.2 organoids treated with AG-270. Molecular weights are expressed in kDa ( n = 2 biological experiments). Scale bars indicated in all images are 50 µm except for B and I which are 100 µm. All error bars, mean ± s.d. * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001. ns = no significant. One-way-ANOVA was used to test significant differences between groups in all panels, except for K where two-sided t -test was used. Panels G and J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: For MST experiments, Histidine-tagged MAT2A protein (BPS Bioscience, cat 71401) was labelled with fluorescent dye using Monolith His-Tag Labeling Kit (cat MO-L008) (NanoTemper Technologies GmbH).

Techniques: Western Blot, Concentration Assay, Ex Vivo, Derivative Assay