Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation.

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: Figure 2. Metallo-aminopeptidase inhibitors induce synergistic inflammasome activation with DPP8/9 blockade (A and B) The indicated THP-1 cells were treated with VbP (10 mM), MeBs (10 mM), CHR-2797 (10 mM), or batimastat (10 mM) for 6 h before LDH release and immunoblot analyses. (C and D) OCI-AML2 or resting human CD3+ T cells were treated with VbP (10 mM) and/or MeBs (10 mM) for (C) 6 h or (D) 18 h before LDH release and immunoblot analyses. (E and F) THP-1 or MV4;11 cells were treated with VbP (10 mM), CQ31 (20 mM), and/or MeBs (10 mM) for 16 h before LDH release and immunoblot analyses. (G) WT or Casp1/ RAW 264.7 cells were treated with VbP (10 mM) and/or MeBs (10 mM) for 6 h before LDH release and immunoblot analyses. (H and I) WT or NLRP1/ human N/TERT-1 keratinocytes were treated with VbP (0.2 mM), MeBs (20 mM), or CQ31 (20 mM) for 24 h before IL-1b release and immunoblot analyses. Data are means ± SEM of three biological replicates. All data, including immunoblots, are representative of three or more independent experiments. ***p < 0.001, **p < 0.01, *p < 0.05 by two-sided Student’s t test. n.s., not significant. See also Figure S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Actinonin Enzo ALX-260-128 Apstatin SCBT sc-201309 Amastatin Millipore-Sigma A1276 SC57461A Tocris 3107 HFI142 R&D Systems 5627 Fumagillin ApexBio A4407 1,10-phenathroline Millipore-Sigma 131377 Captopril R&D Systems 4455 Sitagliptin Thermo Fisher Scientific 11-101-5083 Compound 5385 (5385) (Okondo et al.)2 N/A Z-prolyl prolinal Millipore-Sigma SML0205 S17092 Millipore-Sigma SML0181 Butabindide Oxalate Thermo Fisher Scientific 1323/10 Geldenamycin (GA) SCBT sc-200617A Brefeldin A (BFA) BioLegend 42060 lipopolyaccharide (LPS) Invivogen tlrl-3pelps Nigericin Cayman 11437 Cycloheximide (CHX) Millipore-Sigma C7698 Puromycin (Puro) Millipore-Sigma P7255 Ala-Pro-7-amino-4-methylcoumarin (AP-AMC) MP Biomedicals 03AMC04210 Ala-7-amino-4-methylcoumarin (A-AMC; Sigma) Millipore-Sigma A4302 Leu-7-amino-4-methylcoumarin (L-AMC) SCBT sc-218643 Doxycycline (DOX) Cayman 14422 FuGENE HD Promega E2311 Critical commercial assays Pierce LDH Cytotoxicity Assay Kit Life Technologies PI88953 DCA Protein Assay kit Bio-Rad 5000111 IL-1b ELISA Assay R&D systems CellTiter-Glo Cell Viability Assay Promega G7573 CytoTox-Fluor cytotoxicity Assay Promega G9262 MycoAlert Mycoplasma Detection Kit Lonza LT07-318 Deposited data TMT proteomics raw data This Study ProteomeXchange: PXD038233 RNA-seq raw data This Study GSE200454 Experimental models: Cell lines HEK 293T ATCC CRL-3216 HEK 293T + CASP1 + GSDMD (Johnson et al.)5 N/A HEK 293T PEPD / This Study N/A HEK 293T DPP8 / /DPP9 / This Study N/A

Techniques: Activation Assay, Western Blot

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation.

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: Figure 4. Amino acid depletion does not activate NLRP1 or CARD8 (A) Schematic of amino acid recycling from proteins. (B) HEK 293T cells were cultured in media supplemented with [U-13C]glutamine and [U-13C]leucine for 3 weeks prior to replacement with unlabeled media and simultaneous treatment with VbP (10 mM), MeBs (10 mM) or bortezomib (Bort., 10 mM). Following 6 h of treatment, cell extracts were profiled for levels of glutamine-derived 13C-labeled amino acids. Data are means ± SEM of four biological replicates. (C) Heatmap of amino acid levels in WT, DPP8/9/, and PEPD/ HEK 293T cells treated with VbP (10 mM) and/or MeBs (10 mM) for 3 h. (D) CASP1/ MV4;11 cells were deprived of amino acids or treated with MeBs (10 mM), VbP (10 mM) or Torin1 (10 mM) for 6 h before immunoblot analysis of amino acid deprivation markers. (E) MV4;11 cells were cultured in amino acid-free RPMI supplemented with or without MEM amino acids and treated with MeBs (10 mM) and/or VbP (10 mM) for 6 h, followed by LDH release and immunoblot analysis. Data are means ± SEM of three biological replicates unless otherwise stated. ***p < 0.001, **p < 0.01 by two- sided Student’s t test. n.s., not significant. See also Figure S3 and Table S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Actinonin Enzo ALX-260-128 Apstatin SCBT sc-201309 Amastatin Millipore-Sigma A1276 SC57461A Tocris 3107 HFI142 R&D Systems 5627 Fumagillin ApexBio A4407 1,10-phenathroline Millipore-Sigma 131377 Captopril R&D Systems 4455 Sitagliptin Thermo Fisher Scientific 11-101-5083 Compound 5385 (5385) (Okondo et al.)2 N/A Z-prolyl prolinal Millipore-Sigma SML0205 S17092 Millipore-Sigma SML0181 Butabindide Oxalate Thermo Fisher Scientific 1323/10 Geldenamycin (GA) SCBT sc-200617A Brefeldin A (BFA) BioLegend 42060 lipopolyaccharide (LPS) Invivogen tlrl-3pelps Nigericin Cayman 11437 Cycloheximide (CHX) Millipore-Sigma C7698 Puromycin (Puro) Millipore-Sigma P7255 Ala-Pro-7-amino-4-methylcoumarin (AP-AMC) MP Biomedicals 03AMC04210 Ala-7-amino-4-methylcoumarin (A-AMC; Sigma) Millipore-Sigma A4302 Leu-7-amino-4-methylcoumarin (L-AMC) SCBT sc-218643 Doxycycline (DOX) Cayman 14422 FuGENE HD Promega E2311 Critical commercial assays Pierce LDH Cytotoxicity Assay Kit Life Technologies PI88953 DCA Protein Assay kit Bio-Rad 5000111 IL-1b ELISA Assay R&D systems CellTiter-Glo Cell Viability Assay Promega G7573 CytoTox-Fluor cytotoxicity Assay Promega G9262 MycoAlert Mycoplasma Detection Kit Lonza LT07-318 Deposited data TMT proteomics raw data This Study ProteomeXchange: PXD038233 RNA-seq raw data This Study GSE200454 Experimental models: Cell lines HEK 293T ATCC CRL-3216 HEK 293T + CASP1 + GSDMD (Johnson et al.)5 N/A HEK 293T PEPD / This Study N/A HEK 293T DPP8 / /DPP9 / This Study N/A

Techniques: Cell Culture, Derivative Assay, Labeling, Western Blot

Journal: Cell reports

Article Title: Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation.

doi: 10.1016/j.celrep.2022.111965

Figure Lengend Snippet: Figure 6. AP inhibition induces cIAP1 and NT degradation independently (A) WT or SMAC//HTRA2/ THP-1 cells were treated with MeBs (10 mM), Batimastat (10 mM), or BV6 (5 mM) for 24h prior to immunoblot analysis. (B) Chemical structures of MeBs (top) and CQ83 (bottom). (C) THP-1 cells were treated with MeBs (1 mM), CQ83 (1 mM), and/or VbP (10 mM), and cell viability was assessed by CellTiter-Glo (CTG) after 6 h. Data are means ± SEM of four biological replicates. (D and E) Scatterplot (D) and immunoblots (E) depict the proteins enriched by CQ83 and competed by MeBs as determined by TMT-MS (D) or immunoblotting (E). (F) CETSA analyses of bestatin (10 mM), Batimastat (10 mM), BV6 (5 mM), and GDC-0152 (5 mM) in HEK 293T cell lysates. (G) MV4;11 cells were treated with the indicated aminopeptidase inhibitors or IAP agonists (BV6, GDC-0152) for 24 h prior to immunoblotting analysis. All compounds were treated at 10 mM except the following: fumagillin (3 mM); compound 5385 (DPP7 inhibitor; 20 mM), compound 8j (selective DPP8/9 inhibitor; 20 mM), and sitagliptin (DPP4 inhibitor; 20 mM), VbP (2 mM), BV6 (5 mM), and GDC-0152 (5 mM). (H) MV4;11 cells were treated with VbP (10 mM), MeBs (10 mM), and/or GDC-0152 (5 mM) for 6 h prior to LDH and immunoblot analyses.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Actinonin Enzo ALX-260-128 Apstatin SCBT sc-201309 Amastatin Millipore-Sigma A1276 SC57461A Tocris 3107 HFI142 R&D Systems 5627 Fumagillin ApexBio A4407 1,10-phenathroline Millipore-Sigma 131377 Captopril R&D Systems 4455 Sitagliptin Thermo Fisher Scientific 11-101-5083 Compound 5385 (5385) (Okondo et al.)2 N/A Z-prolyl prolinal Millipore-Sigma SML0205 S17092 Millipore-Sigma SML0181 Butabindide Oxalate Thermo Fisher Scientific 1323/10 Geldenamycin (GA) SCBT sc-200617A Brefeldin A (BFA) BioLegend 42060 lipopolyaccharide (LPS) Invivogen tlrl-3pelps Nigericin Cayman 11437 Cycloheximide (CHX) Millipore-Sigma C7698 Puromycin (Puro) Millipore-Sigma P7255 Ala-Pro-7-amino-4-methylcoumarin (AP-AMC) MP Biomedicals 03AMC04210 Ala-7-amino-4-methylcoumarin (A-AMC; Sigma) Millipore-Sigma A4302 Leu-7-amino-4-methylcoumarin (L-AMC) SCBT sc-218643 Doxycycline (DOX) Cayman 14422 FuGENE HD Promega E2311 Critical commercial assays Pierce LDH Cytotoxicity Assay Kit Life Technologies PI88953 DCA Protein Assay kit Bio-Rad 5000111 IL-1b ELISA Assay R&D systems CellTiter-Glo Cell Viability Assay Promega G7573 CytoTox-Fluor cytotoxicity Assay Promega G9262 MycoAlert Mycoplasma Detection Kit Lonza LT07-318 Deposited data TMT proteomics raw data This Study ProteomeXchange: PXD038233 RNA-seq raw data This Study GSE200454 Experimental models: Cell lines HEK 293T ATCC CRL-3216 HEK 293T + CASP1 + GSDMD (Johnson et al.)5 N/A HEK 293T PEPD / This Study N/A HEK 293T DPP8 / /DPP9 / This Study N/A

Techniques: Inhibition, Western Blot

Journal: Cell reports

Article Title: Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects.

doi: 10.1016/j.celrep.2021.109337

Figure Lengend Snippet: Figure 1. Activation of the p53 pathway in Shashi-XLID iPSCs and U2OS cells (A) Whole-cell extracts from control (CTRL) and RBMX-DRGG (clones DRGG1, DRGG2) iPSCs were immunoblotted with anti-RBMX and anti-b-actin antibodies. Molecular mass markers are shown on the left in kDa. (B) GSEA analysis of p53 consensus targets (Fischer, 2017) using fragments per kilobase of transcript per million mapped reads (FPKM) (Table S1). (C) qRT-PCR assays were performed for p53, CDKN1A, and BBC3 in CTRL, DRGG1, and DRGG2 iPSCs. The data were normalized to GAPDH. n = 2 independent experiments. *p < 0.05, **p < 0.01; ns, not significant; by unpaired t test. Mean ± SEM. (D) Immunofluorescence staining of hiPSC colonies with anti-p53 and anti-cleaved caspase-3 (CC3) antibodies in CTRL and DRGG1 cells counterstained with DAPI. Scale bars, 100 mm. Inset scale bars, 50 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GST-RBMX-166-391 This study N/A pMDM4 This study N/A Chemicals, peptides, and recombinant proteins EPZ015666 Caymanchem Cat# 17285 PRMT5/MEP50 Active human Sigma-Aldrich Cat# SRP0145 ANTI-FLAG M2 Affinity Gel Sigma-Aldrich Cat# A2220 Recombinant Human BDNF Protein R&D Cat# 248-BD-010 Recombinant Human GDNF Protein R&D Cat# 212-GD-010 N2 Thermo Scientific Cat# 17502048 B27 Thermo Scientific Cat# 17504044 Critical commercial assays Caspase-3/7 Green Dye for Apoptosis Incucyte Cat# 4440 Deposited data Raw and analyzed data This study GSE156923 Experimental models: Cell lines U2OS ATCC Cat# HTB-96 HEK293 ATCC Cat# CRL-1573 RBMX-DRGG1 This study N/A RBMX-DRGG2 This study N/A Oligonucleotides Primers and probes Table S5 N/A Software and algorithms ImageJ PMID: 22930834 RRID: SCR_003070; https://imagej.net/ ATtRACT PMID: 27055826 https://attract.cnic.es/ Integrative Genomics Viewer PMID: 21221095 RRID: SCR_011793; https://www. broadinstitute.org/igv/ STAR v2.7.1a PMID: 23104886 RRID: SCR_015899; https://github.com/ alexdobin/STAR ComplexHeatmap R package PMID: 27207943 RRID: SCR_017270; https://bioconductor. org/packages/release/bioc/html/ ComplexHeatmap.html rMATS v4.0.2 PMID: 25480548 RRID: SCR_013049; http://rnaseq-mats. sourceforge.net/ HOMER v4.11.1 PMID: 20513432 RRID: SCR_010881; http://homer.ucsd. edu/homer/ STRING v 11.0 PMID: 30476243 RRID: SCR_005223; https://string-db.org/ cgi/input?sessionId=rm6l8BxmjY3N&input_ page_show_search=on Gene Set Enrichment Analysis PMID: 16199517 RRID: SCR_003199; https://www. gsea-msigdb.org/gsea/index.jspPMID: 12808457 Adobe Illustrator N/A RRID: SCR_010279; https://www.adobe. com/products/illustrator.html GraphPad Prism N/A RRID: SCR_002798; https://www. graphpad.com:443/

Techniques: Activation Assay, Control, Clone Assay, Quantitative RT-PCR, Staining

Journal: Cell reports

Article Title: Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects.

doi: 10.1016/j.celrep.2021.109337

Figure Lengend Snippet: Figure 3. RBMX regulates MDM4 alternative splicing through its RGG/RG motif (A) Schematic representation of MDM4 exon-intron boundaries and the MDM4-FL or MDM4-S isoforms by regulation of exon 6 inclusion or exclusion. Primers used to detect MDM4 alternative splicing are indicated with black arrows. The premature termination codon generated by exon 6 exclusion is indicated with a red arrow. (B) Sashimi plots of MDM4 alternative splicing events between CTRL and DRGG1 iPSCs. (C) Semiquantitative RT-PCR of MDM4 transcripts in CTRL, DRGG1, and DRGG2. Four repeats were performed (n = 4), and the exon 6 exclusion ratio is shown in percentage expressed as the mean ± SEM. *p < 0.05, **p < 0.01; by unpaired t test comparing each sample to the CTRL. (D) RT-PCR for examining MDM4 transcripts in siCTRL, siRBMX, and siPRMT5 cells. Three repeats were performed (n = 3), and the exon 6 exclusion ratio is shown in percentage expressed as the mean ± SEM. **p < 0.01, ***p < 0.001; by unpaired t test comparing each sample to siCTRL. (E) Immunoblotting of U2OS transfected with siCTRL, siRBMX, and siPRMT5 with indicated antibodies.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GST-RBMX-166-391 This study N/A pMDM4 This study N/A Chemicals, peptides, and recombinant proteins EPZ015666 Caymanchem Cat# 17285 PRMT5/MEP50 Active human Sigma-Aldrich Cat# SRP0145 ANTI-FLAG M2 Affinity Gel Sigma-Aldrich Cat# A2220 Recombinant Human BDNF Protein R&D Cat# 248-BD-010 Recombinant Human GDNF Protein R&D Cat# 212-GD-010 N2 Thermo Scientific Cat# 17502048 B27 Thermo Scientific Cat# 17504044 Critical commercial assays Caspase-3/7 Green Dye for Apoptosis Incucyte Cat# 4440 Deposited data Raw and analyzed data This study GSE156923 Experimental models: Cell lines U2OS ATCC Cat# HTB-96 HEK293 ATCC Cat# CRL-1573 RBMX-DRGG1 This study N/A RBMX-DRGG2 This study N/A Oligonucleotides Primers and probes Table S5 N/A Software and algorithms ImageJ PMID: 22930834 RRID: SCR_003070; https://imagej.net/ ATtRACT PMID: 27055826 https://attract.cnic.es/ Integrative Genomics Viewer PMID: 21221095 RRID: SCR_011793; https://www. broadinstitute.org/igv/ STAR v2.7.1a PMID: 23104886 RRID: SCR_015899; https://github.com/ alexdobin/STAR ComplexHeatmap R package PMID: 27207943 RRID: SCR_017270; https://bioconductor. org/packages/release/bioc/html/ ComplexHeatmap.html rMATS v4.0.2 PMID: 25480548 RRID: SCR_013049; http://rnaseq-mats. sourceforge.net/ HOMER v4.11.1 PMID: 20513432 RRID: SCR_010881; http://homer.ucsd. edu/homer/ STRING v 11.0 PMID: 30476243 RRID: SCR_005223; https://string-db.org/ cgi/input?sessionId=rm6l8BxmjY3N&input_ page_show_search=on Gene Set Enrichment Analysis PMID: 16199517 RRID: SCR_003199; https://www. gsea-msigdb.org/gsea/index.jspPMID: 12808457 Adobe Illustrator N/A RRID: SCR_010279; https://www.adobe. com/products/illustrator.html GraphPad Prism N/A RRID: SCR_002798; https://www. graphpad.com:443/

Techniques: Alternative Splicing, Generated, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

Journal: Cell reports

Article Title: Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects.

doi: 10.1016/j.celrep.2021.109337

Figure Lengend Snippet: Figure 4. RBMX RGG/RG motif mediates interaction with Sam68 and SRSF1 for RNA binding (A) Schematic representation of SILAC-based RBMX-interactome analysis. (B) Correlation analysis of two biological repeats of SILAC-based RBMX-interactome. U2OS cultured in light (Lys 0; Arg 0) or heavy (Lys 4, Arg 6) medium were transfected with pcDNA3.1 or FLAG-RBMX. Immunoprecipitation was performed with an equal amount of FLAG-M2 beads and mixed at a 1:1 ratio for tandem mass spectrometry (MS/MS) analysis. (C) GO analysis of potential RBMX-binding proteins by STRING. Bar plot presents the top five enriched biological processes, and the significance is presented with log10(false discovery rate [FDR]). (D) HEK293 were transiently transfected with pcDNA3.1, FLAG-RBMX, and FLAG-RBMX-DRGG. Extracts were subjected to co-immunoprecipitation analysis using anti-FLAG antibody, and the bound proteins immunoblotted after SDS-PAGE with anti-Sam68, anti-SRSF1, or anti-FLAG antibodies are indicated. (E) Same as in (D) except the lysates were either incubated or not with RNase I before the anti-FLAG immunoprecipitation step. (F) U2OS cells were transfected with siRNAs and analyzed by immunoblotting with the indicated antibodies. (G) RT-PCR was performed to examine MDM4 transcripts in siCTRL, siRBMX, siSam68, and siSRSF1 U2OS cells. Histogram denotes % exclusion. n = 3 in- dependent experiments. *p < 0.05, **p < 0.01, ***p < 0.001; by unpaired t test comparing each sample to the siCTRL. Mean ± SEM. (H) U2OS cells transfected with GFP-SRSF1, FLAG-RBMX, and the minigene pMDM4 were cross-linked with 1% formaldehyde and RIP was performed using IgG, anti-GFP, and anti-FLAG antibodies, respectively. qRT-PCR was used to assess the bound MDM4 RNA expressed as fold enrichment over IgG. n = 2 independent experiments. *p < 0.05, **p < 0.01; by unpaired t test. Mean ± SEM. (I) U2OS cells transfected with the indicated siRNA were further transfected with GFP-SRSF1 and pMDM4. RIP was performed and qRT-PCR was used to assess MDM4 recovery in each RIP sample, expressed as fold enrichment over pull-down with IgG. n = 3 independent experiments. *p < 0.05, **p < 0.01; by unpaired t test comparing each sample to the siCTRL. Mean ± SEM.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GST-RBMX-166-391 This study N/A pMDM4 This study N/A Chemicals, peptides, and recombinant proteins EPZ015666 Caymanchem Cat# 17285 PRMT5/MEP50 Active human Sigma-Aldrich Cat# SRP0145 ANTI-FLAG M2 Affinity Gel Sigma-Aldrich Cat# A2220 Recombinant Human BDNF Protein R&D Cat# 248-BD-010 Recombinant Human GDNF Protein R&D Cat# 212-GD-010 N2 Thermo Scientific Cat# 17502048 B27 Thermo Scientific Cat# 17504044 Critical commercial assays Caspase-3/7 Green Dye for Apoptosis Incucyte Cat# 4440 Deposited data Raw and analyzed data This study GSE156923 Experimental models: Cell lines U2OS ATCC Cat# HTB-96 HEK293 ATCC Cat# CRL-1573 RBMX-DRGG1 This study N/A RBMX-DRGG2 This study N/A Oligonucleotides Primers and probes Table S5 N/A Software and algorithms ImageJ PMID: 22930834 RRID: SCR_003070; https://imagej.net/ ATtRACT PMID: 27055826 https://attract.cnic.es/ Integrative Genomics Viewer PMID: 21221095 RRID: SCR_011793; https://www. broadinstitute.org/igv/ STAR v2.7.1a PMID: 23104886 RRID: SCR_015899; https://github.com/ alexdobin/STAR ComplexHeatmap R package PMID: 27207943 RRID: SCR_017270; https://bioconductor. org/packages/release/bioc/html/ ComplexHeatmap.html rMATS v4.0.2 PMID: 25480548 RRID: SCR_013049; http://rnaseq-mats. sourceforge.net/ HOMER v4.11.1 PMID: 20513432 RRID: SCR_010881; http://homer.ucsd. edu/homer/ STRING v 11.0 PMID: 30476243 RRID: SCR_005223; https://string-db.org/ cgi/input?sessionId=rm6l8BxmjY3N&input_ page_show_search=on Gene Set Enrichment Analysis PMID: 16199517 RRID: SCR_003199; https://www. gsea-msigdb.org/gsea/index.jspPMID: 12808457 Adobe Illustrator N/A RRID: SCR_010279; https://www.adobe. com/products/illustrator.html GraphPad Prism N/A RRID: SCR_002798; https://www. graphpad.com:443/

Techniques: RNA Binding Assay, Multiplex sample analysis, Cell Culture, Transfection, Immunoprecipitation, Mass Spectrometry, Tandem Mass Spectroscopy, Binding Assay, SDS Page, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Cell reports

Article Title: Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects.

doi: 10.1016/j.celrep.2021.109337

Figure Lengend Snippet: Figure 5. RBMX colocalizes with SRSF1 in nuclear granules, and 1,6-HD treatment or PRMT5 depletion disrupts the colocalization (A) Immunostaining of RBMX and SRSF1 in U2OS cells fixed with 100% methanol. Scale bars, 10 mm. Inset scale bars, 4 mm. Histogram shows the colocalization of RBMX and SRSF1 within the ROI (region of interest) indicated by a dashed arrow. (B–D) Quantification of RBMX foci size (n = 142 foci; B), RBMX foci intensity (mean intensity per foci, n = 114 foci; C), and RBMX and SRSF1 Pearson correlation coefficient (n = 20 cells; D) were analyzed. ****p < 0.0001; by unpaired t test. Mean ± SEM. (E) U2OS cells were treated with 0.3 M 2,5-hexanediol (HD) or 1,6-HD for 10 min and analyzed by immunoblotting with the indicated antibodies.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GST-RBMX-166-391 This study N/A pMDM4 This study N/A Chemicals, peptides, and recombinant proteins EPZ015666 Caymanchem Cat# 17285 PRMT5/MEP50 Active human Sigma-Aldrich Cat# SRP0145 ANTI-FLAG M2 Affinity Gel Sigma-Aldrich Cat# A2220 Recombinant Human BDNF Protein R&D Cat# 248-BD-010 Recombinant Human GDNF Protein R&D Cat# 212-GD-010 N2 Thermo Scientific Cat# 17502048 B27 Thermo Scientific Cat# 17504044 Critical commercial assays Caspase-3/7 Green Dye for Apoptosis Incucyte Cat# 4440 Deposited data Raw and analyzed data This study GSE156923 Experimental models: Cell lines U2OS ATCC Cat# HTB-96 HEK293 ATCC Cat# CRL-1573 RBMX-DRGG1 This study N/A RBMX-DRGG2 This study N/A Oligonucleotides Primers and probes Table S5 N/A Software and algorithms ImageJ PMID: 22930834 RRID: SCR_003070; https://imagej.net/ ATtRACT PMID: 27055826 https://attract.cnic.es/ Integrative Genomics Viewer PMID: 21221095 RRID: SCR_011793; https://www. broadinstitute.org/igv/ STAR v2.7.1a PMID: 23104886 RRID: SCR_015899; https://github.com/ alexdobin/STAR ComplexHeatmap R package PMID: 27207943 RRID: SCR_017270; https://bioconductor. org/packages/release/bioc/html/ ComplexHeatmap.html rMATS v4.0.2 PMID: 25480548 RRID: SCR_013049; http://rnaseq-mats. sourceforge.net/ HOMER v4.11.1 PMID: 20513432 RRID: SCR_010881; http://homer.ucsd. edu/homer/ STRING v 11.0 PMID: 30476243 RRID: SCR_005223; https://string-db.org/ cgi/input?sessionId=rm6l8BxmjY3N&input_ page_show_search=on Gene Set Enrichment Analysis PMID: 16199517 RRID: SCR_003199; https://www. gsea-msigdb.org/gsea/index.jspPMID: 12808457 Adobe Illustrator N/A RRID: SCR_010279; https://www.adobe. com/products/illustrator.html GraphPad Prism N/A RRID: SCR_002798; https://www. graphpad.com:443/

Techniques: Immunostaining, Western Blot