Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 2. Neuronal activity bidirectionally modulates the phosphorylation state of Shank3. (A) The experiment protocol for extraction of Shank3 from rat cultured neocortical neurons for further quantitative mass spectrometry (MS) or Western blot analyses. (B) Volcano plot of quantitative MS data showing Shank3 residues that were differentially phosphorylated in tetrodotoxin (TTX)-treated samples compared to untreated controls. The log2 values of fold changes, if below zero, indicated hypophosphorylation (paired t-test: S1586, adjusted p=0.034142, S1614/5, 0.014444). (C) Top: diagram showing the location of S1586 and S1615 within the rat Shank3 protein. Functional domains: ANK = ankyrin repeat; SH3 = SRC homology 3; PDZ = PSD-95/Disc Large/ZO-1; Pro-rich = proline rich; SAM = sterile alpha motif. Bottom: homology comparison of sequences flanking rat S1586 and S1615 (matching mouse S1539) across species (human Shank3: NP_001358973.1; rat Shank3: NP_067708.2; mouse Shank3: UniprotKB: Q4ACU6.3). Phosphosites of interest are labeled in red; the only residue not conserved is shown in blue. (D, E) Representative Western blot using an antibody specific for phosphorylated S1615, showing changes in Shank3 phosphorylation after 10 min (D) or 24 hr (E) treatment with TTX or picrotoxin (PTX). (F) Quantification of the fold change of Shank3 S1615 phosphorylation in (D). Dashed line indicates the baseline untreated control (one-sample t-test: TTX, ***p=0.0005, PTX, **p=0.0035, n = 5 and 10 biological replicates, respectively). (G) Quantification of the fold change of Shank3 S1615 phosphorylation in (E) (one-sample t-test: TTX, ****p<0.0001, PTX, p = 0.6336, n = 7 and 7 biological replicates, respectively). Solid colored horizontal

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Extraction, Cell Culture, Mass Spectrometry, Western Blot, Functional Assay, Sterility, Comparison, Labeling, Residue, Control

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 3. Phosphorylation state modulates activity-dependent changes in the synaptic enrichment of Shank3. (A) Representative images of synaptic puncta colocalized with surface GluA2 (sGluA2) and Shank3 in neuron dendrites ± tetrodotoxin (TTX) (scale bar = 5 µm). (B) Quantification of synaptic sGluA2 intensity changes induced by scaling up and down protocols (number of neurons: untreated, n = 77, TTX, n = 40, picrotoxin [PTX], n = 29; Kruskal–Wallis test with post-hoc Dunn’s multiple comparison tests: Un vs. TTX, **p=0.0034, Un vs. PTX, *p=0.0408, TTX vs. PTX, ****p<0.0001). (C) Quantification of synaptic Shank3 intensity during scaling up and down protocols (Kruskal–Wallis test with post-hoc Dunn’s tests: Un vs. TTX, *p=0.0155, Un vs. PTX, *p=0.0205, TTX vs. PTX, ****p<0.0001). (D) Representative images of synaptic localization of wild-type Shank3 and Shank3 phospho-mutants (scale bar = 5 µm). (E) Quantification of synaptic intensity of Shank3 phospho-mutants (number of neurons: WT, n = 33, AA, n = 30, DD, n = 24; Kruskal–Wallis test with post-hoc Dunn’s tests: WT vs. AA, p>0.9999, WT vs. DD, *p=0.0395, AA vs. DD, **p=0.0039). (F) Quantification of the density of synaptic puncta containing Shank3 phospho-mutants (number of neurons: WT, n = 32, AA, n = 30, DD, n = 24; Kruskal–Wallis test: p=0.2814). For imaging experiments here and below, each data point represents a single pyramidal neuron, and data were collected from at least four independent experiments. Also see Figure 3—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Phospho-proteomics, Activity Assay, Comparison, Imaging

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 4. Increased PP2A activity maintains tetrodotoxin (TTX)-induced Shank3 hypophosphorylation. (A) Diagram showing the potential roles of kinases and phosphatases in regulating activity-dependent Shank3 phosphorylation. (B) Representative Western blot showing the impacts of inhibiting CAMKII (KN62, KN93) or PKA (H89) on Shank3 phosphorylation at baseline and upon TTX treatment. (C) Quantification of S1615 phosphorylation in (B) (two-way ANOVA with post-hoc Tukey’s test: DMSO vs. KN62, p>0.9999, DMSO vs. KN93, p=0.8148, DMSO vs. H89, p=0.9112, DMSO vs. picrotoxin (PTX), *p=0.0406, PTX vs. PTX/KN62, **p=0.0040, PTX vs. PTX/KN93, ****p<0.0001, PTX vs. PTX/H89, ****p<0.0001, n = 5 biological replicates). Dashed line indicates the DMSO control. (D) Quantification of PP2A activity after 1 hr TTX treatment (Un, n = 5, TTX, n = 5; paired t-test: **p=0.0018). (E) Quantification of PP2A activity after 24 hr TTX treatment (Un, n = 7, TTX, n = 7; paired t-test: *p=0.0129). (F, G) Western blot analyses showing changes in S1615 phosphorylation after 1 hr (F) or 24 hr (G) TTX treatment, with inhibition of PP2A by okadaic acid (OKA, 50 nM) during the

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Inhibition

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 5. PP2A activity is required for tetrodotoxin (TTX)-induced synaptic enrichment of Shank3. (A) Representative images of synaptic enrichment of endogenous Shank3 upon treatment with TTX and PP2A inhibitor fostriecin (FST) (scale bar = 10 µm). (B) Quantification of synaptic Shank3 intensity in (A) (number of neurons: DMSO, n = 26, FST, n = 28, TTX, n = 28, TTX/FST, n = 29; Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. FST, p>0.9999, DMSO vs. TTX, ***p=0.0002, FST vs. TTX/FST, p=0.1259, TTX vs. TTX/FST, p=0.1292). (C) Quantification of density of synapses containing Shank3 in (A) (Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. FST, p=0.9458, DMSO vs. TTX, **p=0.0051, FST vs. TTX/FST, p=0.2446, TTX vs. TTX/FST, *p=0.0273). (D) Representative images of synaptic enrichment of endogenous Shank3 upon treatment with TTX and PP1 inhibitor tautomycetin (TAUT) (scale bar = 10 µm). (E) Quantification of synaptic Shank3 intensity in (D) (number of neurons: DMSO, n = 26, TAUT, n = 21, TTX, n = 28, TTX/ TAUT, n = 32; Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. TAUT, *p=0.0315, DMSO vs. TTX, ***p=0.0006, TAUT vs. TTX/TAUT, ***p=0.0002, TTX vs. TTX/TAUT, *p=0.0392). (F) Quantification of density of synapses containing Shank3 in (D) (Kruskal–Wallis test with post-hoc Dunn’s tests: DMSO vs. TAUT, p=0.2450, DMSO vs. TTX, *p=0.0116, TAUT vs. TTX/TAUT, p=0.6552, TTX vs. TTX/TAUT, ***p=0.0007). Also see Figure 5—figure supplement 1 and Figure 5—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 6. Changes in the phosphorylation state of Shank3 are crucial for bidirectional synaptic scaling. (A, B) Representative miniature excitatory postsynaptic current (mEPSC) recordings from neurons overexpressing Shank3 WT (A) or DD mutant (B) during scaling up. (C) Quantification of average mEPSC amplitude in (A) (WT, n = 8, WT + tetrodotoxin [TTX], n = 9; unpaired two-tailed t-test: **p=0.0074). (D) Quantification of average mEPSC amplitude in (B) (number of neurons: DD, n = 12, DD + TTX, n = 14; unpaired two-tailed t-test: p=0.5708). (E, F) Representative traces of mEPSCs recorded from neurons overexpressing Shank3 WT (E) or AA mutant (F) during scaling down. (G) Quantification of average mEPSC amplitude in (E) (number of neurons: WT, n = 8, WT + bicuculline [BIC], n = 8; Mann–Whitney test: *p=0.0148). (H) Quantification of average mEPSC amplitude in (F) (AA, n = 9, AA + BIC, n = 14; unpaired two-tailed t-test: p=0.8612). Also see Figure 6—figure supplement 1, Figure 6—figure supplement 2, and Figure 6—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Phospho-proteomics, Mutagenesis, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 7. Brief PP2A inactivation reverses scaling up. (A) Representative images showing the effects of 1 hr fostriecin (FST) treatment on synaptic sGluA2 intensity in neurons expressing Shank3 WT or AA, after 24 hr of tetrodotoxin (TTX) to scale up synaptic strengths (scale bar = 10 µm). (B) Quantification of synaptic sGluA2 intensity in (A) (number of cells: WT/TTX, n = 22, WT/TTX/FST, n = 23, AA/TTX, n = 26, AA/TTX/FST, n = 25; Mann–Whitney test: WT/TTX vs. WT/TTX/FST, ***p=0.0007, AA/TTX vs. AA/TTX/FST, p=0.3739). (C) Quantification of synaptic Shank3 intensity in (A) (Mann–Whitney test: WT/TTX vs. WT/TTX/FST, **p=0.0090, AA/TTX vs. AA/TTX/FST, p=0.7296). (D) Quantification of the density of puncta containing sGluA2 and Shank3 (Mann–Whitney test: WT/TTX vs. WT/TTX/FST, **p=0.0016, AA/TTX vs. AA/TTX/FST, p=0.7017). Each data point indicates a cell, and the total number (n) was pooled from five independent experiments. Also see Figure 7—source data 1.

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Expressing, MANN-WHITNEY

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet: Effects of LRRC37B overexpression on functional and synaptic properties of mouse CPNs, related to Figure 2 (A) Immunodetection of LRRC37B and ankyrin-G in the mouse cerebral (barrel) cortex after transfection of EGFP only or bicistronic expression of EGFP and human(h)LRRC37B-HA or chimpanzee(c)LRRC37B-HA cDNAs (arrows at the AIS). (B) Human (n = 62 from 9 animals from 3 litters) and chimpanzee (n = 18 neurons from 6 animals from 1 litter) LRRC37B colocalizes with ankyrin-G in mouse neurons transfected for LRRC37B and EGFP; note that in next panels, LRRC37B is for human LRRC37B (mean + SEM). (C) Corresponding quantification of the AIS length and position of mouse neurons transfected for LRRC37B (n = 62 from 9 animals from 3 litters) compared with control neurons (n = 59 from 9 animals from 3 litters) (B, mean + SEM; C, lines at median; Mann-Whitney tests). (D) Immunodetection of LRRC37A2-HIS in the mouse cerebral cortex after transfection of LRRC37A2-HIS and EGFP cDNAs. (E) Intrinsic properties of LRRC37B neurons (36 neurons from 12 animals from 4 litters) compared with control neurons (18 neurons from 7 animals from 4 litters) complementary to Figures 2 B–2F (lines at median; Mann-Whitney tests). (F) Phase plot analysis of single evoked AP from control versus LRRC37B transfected neurons complementary to Figure 2 F (see in ). (G) Phase plot analysis of single evoked AP of LRRC37B neurons (n = 10 neurons from 2 animals from 1 litter) and control neurons (n = 9 neurons from 2 animals from 1 litter) (see in ) (lines at median; Mann-Whitney tests). (H) Phase plot analysis of single evoked AP of LRRC37B neurons (n = 48 neurons from 16 animals from 8 litters) and control neurons (n = 25 neurons from 10 animals from 8 litters) (see in ) (lines at median; Mann-Whitney tests). (I) IV-curves (left, ionic currents) and maximum currents (right) of LRRC37B transfected neurons (n = 43 neurons) compared with of control neurons (n = 26 neurons) (left, mean + SEM; right, lines at median; Mann-Whitney tests). (J and K) Quantification of VGAT puncta, gephyrin-tdTomato puncta, and VGAT/gephyrin-tdTomato puncta in mouse neurons in utero electroporated for plasmids leading to a bicistronic expression of LRRC37B and EGFP (40 neurons for VGAT, 33 neurons for gephyrin quantifications, from 11 animals from 4 litters) or EGFP only (30 neurons for VGAT, 26 for gephyrin quantifications, from 8 animals from 3 litters) as well as gephyrin-tdTomato expression (lines at median; Mann-Whitney tests). (L) Excitatory and inhibitory postsynaptic potentials (E/I PSP) frequency and amplitude in LRRC37B transfected mouse neurons (10 neurons from 4 animals from 2 litters) versus control neurons (8 neurons from 4 animals from 2 litters) (lines at median; Mann-Whitney tests). ns, non-significant; ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: All constructs were verified by DNA sequencing. p-SCN1B-FLAG plasmid originates from Origene (RC209565); pSCN8A plasmid originates from Addgene (#162280) and previously described in DeKeyser et al.. pCAG-cre is a gift from Franck Polleux laboratory (United States) previously described in Hand et al.. pCAG-GEPH.FingR-tdTomato-IL2RGTC is a gift from Juan Burrone (United Kingdom), derived from pCAG_GPHN.FingR-mKate2-IL2RGTC (Addgene #46297) previously described Gross et al.. pdisplay-GPR158_ECTO, pdiplsay-SLIRTK2_ECTO and pdisplay-LRRTM1_ECTO were previously described., pCD4_ECTO is a gift from Luís Ribeiro (Joris de Wit’s laboratory), with the cDNA of the predicted extracellular domain of CD4, originating from pdisplay-CD4 (Addgene #51604) previously described, inserted into the pDisplay™ Mammalian Expression Vector (ThermoFisher V66020).

Techniques: Over Expression, Functional Assay, Immunodetection, Transfection, Expressing, MANN-WHITNEY, In Utero

Journal: Cell

Article Title: LRRC37B is a human modifier of voltage-gated sodium channels and axon excitability in cortical neurons

doi: 10.1016/j.cell.2023.11.028

Figure Lengend Snippet:

Article Snippet: All constructs were verified by DNA sequencing. p-SCN1B-FLAG plasmid originates from Origene (RC209565); pSCN8A plasmid originates from Addgene (#162280) and previously described in DeKeyser et al.. pCAG-cre is a gift from Franck Polleux laboratory (United States) previously described in Hand et al.. pCAG-GEPH.FingR-tdTomato-IL2RGTC is a gift from Juan Burrone (United Kingdom), derived from pCAG_GPHN.FingR-mKate2-IL2RGTC (Addgene #46297) previously described Gross et al.. pdisplay-GPR158_ECTO, pdiplsay-SLIRTK2_ECTO and pdisplay-LRRTM1_ECTO were previously described., pCD4_ECTO is a gift from Luís Ribeiro (Joris de Wit’s laboratory), with the cDNA of the predicted extracellular domain of CD4, originating from pdisplay-CD4 (Addgene #51604) previously described, inserted into the pDisplay™ Mammalian Expression Vector (ThermoFisher V66020).

Techniques: Recombinant, Plasmid Preparation, Magnetic Beads, Expressing, Comparison, Software

Journal: bioRxiv

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator, SUGP1

doi: 10.1101/2022.11.14.516533

Figure Lengend Snippet: (A) Diagram of split-APEX (sAPEX) proximity labeling experiments with AP-tagged DHX15 and EX-tagged SUGP1. BP, Biotin-Phenol. APEX, ascorbate peroxidase. Upon short incubation of H2O2, DHX15-SUGP1 interaction-dependent reconstitution of APEX activity oxidizes BP into biotin-phenoxyl radicals, which then biotinylated proteins within several nanometers of radius. (B) Protein blots (left) and quantification (right) of biotinylated proteins labeled by DHX15-SUGP1 interaction-reconstituted sAPEX activity. Ponceau S protein stain, loading control for total protein. Streptavidin IRDye, detection of biotinylated proteins. FRB-nls, FRB control protein fused with an SV40 nuclear localization signal. (C) Diagram of DHX15’s primary domain structure and sites of mutations tested in (E, F, H). (D) Predicted protein complex structure of DHX15 interaction with SUGP1 G-patch domain by ColabFold . Arrows, sites of three mutations, and the corresponding functional centers. Colors of domains and mutations match (C). (E) Protein blots (left) and quantification (right) of biotinylated proteins labeled by wild-type versus mutant DHX15-SUGP1 interaction reconstituted split-APEX activity. (F) Protein blots of biotinylated proteins labeled in (E), enriched by streptavidin-coated bead pull-down experiments. Cell lysates were collected after 24 hours of AP-DHX15 and EX-SUGP1 co-transfection. Wild-type and mutant AP-DHX15 expression induced by doxycycline addition for 24 hours. (G) Quantification of pull-downs in (F). (H) Similar to (F), except that wild-type and mutant AP-DHX15 expressions were induced for 4 hours. (I) Quantification of pull-downs in (H). (J) Model. Top row, hDHX15/ctPrp43 at ATP-bound open (PDB ID: 5ltk), G-patch domain-bound semi-open (PDB ID: 6sh6), and ADP-bound closed (PDB ID: 5dou) states. Middle row, side view of the open, semi-open, and closed states. Bottom row, cartoon representations of the structures, the G-patch domain (pink) binds to DHX15 at a semi-open state.

Article Snippet: To enrich biotinylated protein from the total protein lysates, 60 μL streptavidin-coated magnetic beads (Pierce, Thermo Scientific 88817) were used for each 1 mg of total protein lysates collected after APEX labeling.

Techniques: Labeling, Incubation, Activity Assay, Staining, Control, Functional Assay, Mutagenesis, Cotransfection, Expressing

Journal: bioRxiv

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator, SUGP1

doi: 10.1101/2022.11.14.516533

Figure Lengend Snippet: (A) Diagram of SUGP1’s primary domain structure. (B) Protein blots and quantification of biotinylated proteins labeled by FL versus truncated SUGP1-DHX15 split-APEX activity. Ponceau S protein stain, loading control for total protein. Streptavidin IRDye, detection of biotinylated proteins. FRB-nls, FRB control protein fused with an SV40 nuclear localization signal. ΔGp, G-patch domain truncation. ΔULM, ULM truncation. ΔS1&2, SURP1, and SURP2 truncation. (C) Protein blots of biotinylated proteins labeled in (B), enriched by streptavidin pull-down experiments. (D) Quantification of pull-downs in (C). (E) Fluorescent microscopy images of biotinylation signals (Streptavidin, magenta), antibodies detecting HA-tagged SUGP1-EX, FL versus truncations (HA, red), FLAG-tagged AP-DHX15 (FLAG, green), nuclear DNA dye (DAPI, blue), and merged channels. (F) Fluorescent microscopy images of antibody detecting HA-tagged SUGP1.W387A-EX (HA, red), nuclear DNA dye (DAPI, blue), and merged channels. (G) Protein blots and quantification of biotinylated proteins labeled by full-length (FL), ΔULM, versus W387A mutant SUGP1-DHX15 interaction reconstituted split-APEX activity. Ponceau S protein stain, loading control for total protein. Streptavidin IRDye, detection of biotinylated proteins. Biotinylated-HA was detected by merging the streptavidin channel (green) with the HA channel (red).

Article Snippet: To enrich biotinylated protein from the total protein lysates, 60 μL streptavidin-coated magnetic beads (Pierce, Thermo Scientific 88817) were used for each 1 mg of total protein lysates collected after APEX labeling.

Techniques: Labeling, Activity Assay, Staining, Control, Microscopy, Mutagenesis

Journal: bioRxiv

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator, SUGP1

doi: 10.1101/2022.11.14.516533

Figure Lengend Snippet: (A) Protein blots of biotinylated proteins labeled by full-length (FL) versus truncated SUGP1-DHX15 interaction reconstituted split-APEX activity, enriched by streptavidin pull-down experiments. ΔGp, G-patch domain truncation. ΔC, C-terminus truncation. (B) Quantification of pull-downs in (A).

Article Snippet: To enrich biotinylated protein from the total protein lysates, 60 μL streptavidin-coated magnetic beads (Pierce, Thermo Scientific 88817) were used for each 1 mg of total protein lysates collected after APEX labeling.

Techniques: Labeling, Activity Assay

Journal: bioRxiv

Article Title: Splicing quality control mediated by DHX15 and its G-patch activator, SUGP1

doi: 10.1101/2022.11.14.516533

Figure Lengend Snippet: (A) Protein blots and quantification of biotinylated proteins labeled by full-length (FL) versus truncated SUGP1-DHX15 interaction reconstituted split-APEX activity. Ponceau S protein stain, loading control for total protein. Streptavidin IRDye, detection of biotinylated proteins. FRB-nls, FRB control protein fused with an SV40 nuclear localization signal. ΔGp, G-patch domain truncation. Gp-nls, G-patch domain alone fused with an SV40 nuclear localization signal. (B) Fluorescent microscopy images of biotinylation signals (Streptavidin, magenta), antibodies detecting HA-tagged SUGP1-EX, FL versus Gp-nls (HA, red), FLAG-tagged AP-DHX15 (FLAG, green), nuclear DNA dye (DAPI, blue), and merged channels. (C) Model. A sampling-and-recruitment model of DHX15-SUGP1 interaction during early splicing QC.

Article Snippet: To enrich biotinylated protein from the total protein lysates, 60 μL streptavidin-coated magnetic beads (Pierce, Thermo Scientific 88817) were used for each 1 mg of total protein lysates collected after APEX labeling.

Techniques: Labeling, Activity Assay, Staining, Control, Microscopy, Sampling

Journal: Nucleic Acids Research

Article Title: An autism spectrum disorder mutation in Topoisomerase 3β causes accumulation of covalent mRNA intermediates by disrupting metal binding within the zinc finger domain

doi: 10.1093/nar/gkaf1138

Figure Lengend Snippet: The autism-linked C666R mutation in human TOP3B causes an accumulation of TOP3B•mRNA covalent intermediates. ( A ) AlphaFold predicted structure of full-length human TOP3B (UniProtKB: O95985 ) with disease-linked mutation residues (P378, R472, and C666) and the catalytic active site tyrosine (Y336) labeled with black arrows (top). Diagram of TOP3B functional domains with disease-linked mutations labeled with red lines (bottom). ( B ) Schematic of TOP3B topoisomerase cycle on mRNA. The synthetic “self-trapping” R338W mutation blocks the mRNA rejoining step, leading to the accumulation of TOP3B•mRNA covalent intermediates. Addition of LDS denatures noncovalently linked TOP3B from substrate mRNAs. ( C ) Schematic of Neuro2A cell-based TOP3B activity assay. Oligo-dT magnetic beads are used to isolate mRNA under strong protein denaturing lysis and wash conditions [i.e. 0.5% (w/v) LDS and 500 mM LiCl] allowing for selective isolation of TOP3B•mRNA covalent intermediates. ( D ) Anti-FLAG western blot of WT and mutant TOP3B-3xFLAG in Neuro2A cells. mEGFP was used as a transfection control and tubulin was used as a loading control. NTC = no template control. ( E ) Anti-FLAG slot blot of WT and mutant TOP3B•mRNA covalent intermediates isolated from Neuro2A cells (nitrocellulose membrane). Free mRNA was stained with methylene blue (positively charged nylon membrane) and served as a loading control. ( F ) TOP3B activity levels were assessed by quantifying TOP3B•mRNA covalent intermediate levels [signal in panel (E)] normalized by steady state protein levels [signal in panel (D)]. TOP3B-FLAG protein levels were first normalized by the mEGFP transfection control. Data were then set relative to the R338W mutant. n = 3 biological replicates. Comparisons were made using a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons.* = P <.05, *** = P ≤.001, **** = P ≤.0001. ns = not significant. Exact P -values are reported in .

Article Snippet: pCMV6/TOP3B(WT)-FLAG plasmid that encodes isoform 1 of human TOP3B (RefSeq: NM_003 935) was obtained from Origene Technologies (catalog #RC223204).

Techniques: Mutagenesis, Labeling, Functional Assay, Activity Assay, Magnetic Beads, Lysis, Isolation, Western Blot, Transfection, Control, Dot Blot, Membrane, Staining

Journal: Nucleic Acids Research

Article Title: An autism spectrum disorder mutation in Topoisomerase 3β causes accumulation of covalent mRNA intermediates by disrupting metal binding within the zinc finger domain

doi: 10.1093/nar/gkaf1138

Figure Lengend Snippet: TOP3B-C666R•mRNA covalent intermediates are sensitive to inhibiting the ubiquitin-proteasome pathway, phenocopying the “self-trapping” R338W mutant. ( A ) The reported proteasomal degradation pathway of unresolved TOP3B•RNA covalent intermediates that are sensitive to TAK243 (ubiquitin E1 activating enzyme inhibitor) and MG132 (proteasome inhibitor). ( B ) Anti-FLAG slot blot of WT and mutant TOP3B•mRNA covalent intermediates isolated from Neuro2A cells treated with vehicle or 10 µM TAK243 for 3 h. NTC = no template control. ( C ) Quantification of panel (B). n = 3 biological replicates. ( D ) Anti-FLAG slot blot of WT and mutant TOP3B•mRNA covalent intermediates isolated from Neuro2A cells treated with vehicle or 10 µM MG132 for 3 h. ( E ) Quantification of panel (D). n = 3 biological replicates. ( F ) Anti-V5 slot blot of WT and mutant TOP3B•mRNA covalent intermediates isolated from Neuro2A cells co-transfected with either an empty plasmid control (Empty) or a plasmid encoding 3xV5-Ubiquitin (3xV5-Ub). ( G ) Quantification of panel (F). n = 3 biological replicates. Free mRNA was stained with methylene blue and served as a loading control for all experiments. Comparisons were made using a one-way ANOVA with Dunnett’s multiple comparisons. ** = P ≤.01, *** = P ≤.001, **** = P ≤.0001. ns = not significant. Exact P -values are reported in .

Article Snippet: pCMV6/TOP3B(WT)-FLAG plasmid that encodes isoform 1 of human TOP3B (RefSeq: NM_003 935) was obtained from Origene Technologies (catalog #RC223204).

Techniques: Ubiquitin Proteomics, Mutagenesis, Dot Blot, Isolation, Control, Transfection, Plasmid Preparation, Staining

Journal: Nucleic Acids Research

Article Title: An autism spectrum disorder mutation in Topoisomerase 3β causes accumulation of covalent mRNA intermediates by disrupting metal binding within the zinc finger domain

doi: 10.1093/nar/gkaf1138

Figure Lengend Snippet: C666R disrupts a D1C3-type metal binding motif within the ZnF domain. ( A ) AlphaFold 3 predicted structure of human TOP3B depicting putative zinc binding motifs and Zn 2+ coordination (UniProtKB: O95985 ). The C666 residue is predicted to coordinate Zn 2+ as part of a D1C3 motif. ( B ) Anti-FLAG western blot of WT and mutant TOP3B-3xFLAG in Neuro2A cells. mEGFP was used as a transfection control and tubulin was used as a loading control. NTC = no template control. ( C ) Anti-FLAG slot blot of WT and mutant TOP3B•mRNA covalent intermediates isolated from Neuro2A cells. Free mRNA was stained with methylene blue and served as a loading control. ( D ) TOP3B activity levels were assessed by quantifying TOP3B•mRNA covalent intermediate levels [signal in panel (C)] normalized by steady state protein levels [signal in panel (B)]. TOP3B-3xFLAG protein levels were first normalized by the mEGFP transfection control. Data were then set relative to the R338W mutant. n = 3 biological replicates. Comparisons were made using a one-way ANOVA with Dunnett’s multiple comparisons. * = P <.05, ** = P ≤.01, **** = P ≤.0001. ns = not significant. Exact P -values are reported in .

Article Snippet: pCMV6/TOP3B(WT)-FLAG plasmid that encodes isoform 1 of human TOP3B (RefSeq: NM_003 935) was obtained from Origene Technologies (catalog #RC223204).

Techniques: Binding Assay, Residue, Western Blot, Mutagenesis, Transfection, Control, Dot Blot, Isolation, Staining, Activity Assay

Journal: Nucleic Acids Research

Article Title: An autism spectrum disorder mutation in Topoisomerase 3β causes accumulation of covalent mRNA intermediates by disrupting metal binding within the zinc finger domain

doi: 10.1093/nar/gkaf1138

Figure Lengend Snippet: The C666R mutation leads to loss of metal coordination in the ZnF domain. ( A ) Schematic of recombinant FLAG-MBP-twinSTII, FLAG-MBP-ZnF(WT)-twinSTII, and mutant FLAG-MBP-ZnF-twinSTII proteins. ( B ) SDS–PAGE and Coomassie stain of recombinant proteins. ( C, D ) Stoichiometry of Zn ( C ) and Fe ( D ) and each indicated recombinant protein as determined by ICP-MS. n = 4 separate ICP-MS samples that were each analyzed in triplicate. ( E ) The published cryo-EM structure of TOP3B bound to TDRD3 (PDB: 9CAH) overlayed with the reported density for the metals in motif 2 with either a Zn (top) or Fe (bottom) being coordinated. Both metals fit into the reported density within the D1C3 motif that consists of C666. Comparisons were made using a one-way ANOVA with Dunnett’s multiple comparisons. **** = P ≤.0001. Exact P -values are reported in . ICP-MS data is also found in .

Article Snippet: pCMV6/TOP3B(WT)-FLAG plasmid that encodes isoform 1 of human TOP3B (RefSeq: NM_003 935) was obtained from Origene Technologies (catalog #RC223204).

Techniques: Mutagenesis, Recombinant, SDS Page, Staining, Cryo-EM Sample Prep

Journal: Nucleic Acids Research

Article Title: An autism spectrum disorder mutation in Topoisomerase 3β causes accumulation of covalent mRNA intermediates by disrupting metal binding within the zinc finger domain

doi: 10.1093/nar/gkaf1138

Figure Lengend Snippet: Unresolved TOP3B•mRNA covalent intermediates are toxic to primary neurons. ( A ) Schematic of single cell longitudinal fluorescence microscopy of primary rat cortical neurons. ( B ) Representative images of primary neurons co-transfected with mApple to mark cell bodies (green outline). Transfected primary neurons are imaged taken every 24 h; for simplicity, only days 1–5 are shown. Neuron 16 (top) survives the entire time course, whereas neuron 4 (bottom) death can be seen at day 4 (red). ( C ) Cumulative risk of death of primary cortical neurons expressing the mEGFP-NES or the indicated version of mEGFP-NES-TOP3B. Data were combined and stratified from six separate experiments imaged every 24 h for 10 days. n = total number of neurons imaged. The indicated comparisons were made using a Cox proportional hazard test. ** = P ≤.01. *** = P ≤.001. ns = not significant. Exact P -values and all possible comparisons are reported in .

Article Snippet: pCMV6/TOP3B(WT)-FLAG plasmid that encodes isoform 1 of human TOP3B (RefSeq: NM_003 935) was obtained from Origene Technologies (catalog #RC223204).

Techniques: Fluorescence, Microscopy, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: An autism spectrum disorder mutation in Topoisomerase 3β causes accumulation of covalent mRNA intermediates by disrupting metal binding within the zinc finger domain

doi: 10.1093/nar/gkaf1138

Figure Lengend Snippet: Unresolved TOP3B•mRNA covalent intermediates cause ribosome collisions. ( A ) Schematic of unresolved TOP3B•mRNA covalent intermediates leading to collided ribosomes. ( B ) Anti-FLAG western blot confirming inducible expression of the indicated Flp-In T-REx 293 cell lines after 24 h vehicle or doxycycline (Dox; 1 µg/ml final). Tubulin was used as a loading control. ( C ) Anti-FLAG slot blot of TOP3B•mRNA covalent intermediates isolated from the indicated Flp-In T-REx 293 cell lines after 24 h doxycycline (1 µg/ml final). Free mRNA was stained with methylene blue and served as a loading control. ( D, E ) Polysome analysis (10–50% (w/v) sucrose gradients) of the S7 micrococcal nuclease (RNase)-treated lysates of catalytically inactive Y336F mutant ( D ) or “self-trapping” R338W mutant ( E ) Flp-In T-REx 293 cell lines after 7-day vehicle or doxycycline (1 µg/ml final). ( F ) Quantification of each ribosomal species reported as the % fold change between + and − Dox induction in the indicated TOP3B mutant. n = 6 biological replicates. Comparisons were made using a two-way ANOVA with Sidak’s multiple comparisons. ( G ) Anti-FLAG, anti-RPS6 and anti-RPL7 western blots of nuclease-resistant disomes (fractions 10) and trisomes (fraction 12). Recombinant MBP-mEGFP was spiked in as a loading control. ( H ) Quantification of panel (G). 3xFLAG-TOP3B levels were first normalized by RPS6. Data were then set relative to Y336F + Dox in each fraction (disome or trisome). n = 3 biological replicates. Comparisons were made using a two-tailed unpaired t -test with Welch’s correction. * = P ≤.05. ns = not significant. Exact P -values for comparisons in panels (F) and (H) are reported in .

Article Snippet: pCMV6/TOP3B(WT)-FLAG plasmid that encodes isoform 1 of human TOP3B (RefSeq: NM_003 935) was obtained from Origene Technologies (catalog #RC223204).

Techniques: Western Blot, Expressing, Control, Dot Blot, Isolation, Staining, Mutagenesis, Recombinant, Two Tailed Test

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm. (B) Functional annotation of AMIGO2-interacting proteins detected by GFP pull-down followed by MS in SKmel147 cells stably expressing AMIGO2-GFP (see Table S4). (C) PTK7 and GFP immunoblots following GFP pull-down from 501MEL cells stably expressing AMIGO2-GFP. (D) Full-length PTK7 (FL-PTK7), C-terminal fragments CTF1- and CTF2-PTK7, and FOXM1 immunoblots of 501MEL cells 72 hr post-infection with shSCR or shPTK7 (shP7 #1 and #2). Actin was used as a loading control. (E) Relative growth curves of 501MEL (left) and SKmel147 (right) cells stably transduced with shSCR or shPTK7 (shP7 #1 and #2). Values are normalized to seeding control (n = 3). (F) Percent Annexin V-positive cells 6 days post-transduction for same cells as in (E). (G) FL-PTK7, CTF-PTK7, and FOXM1 immunoblots of 501MEL cells 48 hr post-transduction with shSCR or shAMIGO2 (shA2 #1 and #2). Actin was used as a loading control. (H) FL-PTK7, CTF-PTK7, FOXM1, and AMIGO2 immunoblots of 501MEL cells untreated or treated with JQ1 (JQ1[+]) for 72 hr. Tubulin was used as a loading control. (I) CTF2-PTK7 immunoblot of nuclear lysates from same cell as in (G) (left). Lamin B1 was used as loading control. Signal quantification (right), normalized to Lamin B1, relative to shSCR (n = 3). All values and error bars represent mean ± SD or ± SEM. See also Figures S3 and S4.

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining, Functional Assay, Western Blot, Infection, Control, Transduction

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, DNA Library Preparation, Blocking Assay, Extraction, TA Cloning, Sequencing, RNA Sequencing, Western Blot, Mass Spectrometry, Expressing, Software

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) The cleavage motifs derived from PIAS1 (LTYD*G and NGVD*G) were used to virtually screen the entire human proteome for proteins sharing the same sequences. The human proteome dataset containing approximately 20,000 human protein-coding genes represented by the canonical protein sequence was downloaded from UniProtKB/Swiss-Prot. (B) 16 additional proteins were extracted from the screen. 8 proteins carry the LTYD*G motif (left) and 8 proteins carry the NGVD*G motif (right). 6 proteins (underlined) were selected for further validation. (C) Protein downregulation during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot showing the downregulation of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes the cleaved fragment for EHMT2. (D) Caspase inhibition blocks the degradation of YTHDF2, MAGEA10, SORT1 MTA1 and EHMT2. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot showing the protein levels of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes cleaved EHMT2 fragment.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Derivative Assay, Sequencing, Western Blot, Inhibition

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Schematic representation showing the relative positions of Cas9 target sites for small guide RNAs sg-1 to sg-3. (B) Akata (EBV+) cells were used to establish stable cell lines using 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated cross-linking of BCR. YTHDF2 and viral protein (ZTA and RTA) expression levels were monitored by Western Blot using antibodies as indicated. (C) RNAs from YTHDF2-depleted and control Akata cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (D) P3HR-1 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with TPA and sodium butyrate (NaBu) to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (E) RNAs from YTHDF2-depleted and control P3HR-1 cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (F) SUN-719 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with Gemcitabine to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (G) Akata (EBV+) cells were used to establish control and YTHDF2 overexpression cell line as indicated. The cells were untreated or lytically induced by anti-IgG treatment. The expression of YTHDF2 as monitored by anti-YTHDF2 and anti-Myc antibodies. Viral protein expression levels were monitored by Western Blot using antibodies as indicated. (H) Extracellular virion-associated DNA from cells treated in panel G was extracted and the relative EBV viral copy numbers were calculated by q-PCR analysis using primers specific to BALF5. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p<0.001. See also - .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Plasmid Preparation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2 or 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG treatment for 24 or 48 hrs as indicated. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (A) EIF4H depletion promotes the expression of EBV ZTA and RTA. (B) MAGEA10 depletion does not affect EBV protein expression. (C) SORT1 depletion does not significantly affect EBV protein expression. (D) EHMT2 depletion does not affect EBV protein expression. Arrowhead denotes cleaved fragments. (E) MTA1 depletion does not uniformly affect EBV protein expression but slightly enhances the expression of its homolog MTA2.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Expressing, Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) YTHDF2-depleted and control Akata (EBV+) cells were lytically induced with anti-IgG for 0 to 48 hrs. (B) YTHDF2-depleted and control P3HR1 cells were lytically induced with TPA and NaBu for 0 to 48 hrs. (C) YTHDF2-depleted and control SNU-719 cells were lytically induced with TPA and NaBu for 0 to 48 hrs. Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p< 0.01; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Western Blot showing YTHDF2 downregulation by IgG cross-linking induced BCR activation. Akata (EBV+) and Akata-4E3 (EBV-) cells were treated with anti-IgG antibody as indicated. YTHDF2 and viral protein expression levels were monitored by Western Blot. Arrowheads denote cleaved YTHDF2 in the longer exposure blot. (B) Caspase inhibition blocks YTHDF2 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Arrowheads denote cleaved YTHDF2. (C) Functional domains and putative cleavage sites in YTHDF2. CaspDB was used to predict the potential cleavage sites in YTHDF2. The locations of the putative cleavage sites D166 and D367 were labeled as indicated. CNOT1 binding domain: responsible for the degradation of associated RNA; P/Q/N rich region: aggregation-prone region; YTH domain: responsible for binding to m 6 A-modified RNA. (D) Schematic representation of V5-tagged YTHDF2 with two putative cleavage sites. Red oval, anti-YTHDF2 monoclonal antibody recognition site. (E-F). Wild-type V5-YTHDF2 was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using either anti-YTHDF2 (E) or anti-V5 (F) antibodies. The relative position of predicted cleavage fragments was labeled as indicated. (G-H) YTHDF2 (D166A/D367A) mutant protein was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. (I) Motif analysis showing the conservation of the two cleavage sites and the surrounding amino acids. Amino acid sequences were extracted from 97 (D166) and 80 (D367) vertebrate species and motif logos were generated using WebLogo. (J) Structure modeling of full-length YTHDF2 by I-TASSER. The two cleavage sites D166 and D367 are labeled as indicated. N and C denote N-terminus and C-terminus, respectively. (K) Triple depletion of caspase-3, -8 and -6 reduces YTHDF2 and PIAS1 degradation and blocks viral protein accumulation. The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. The expression of caspases, cleaved caspases, YTHDF2, PIAS1 and viral proteins (ZTA and RTA) was monitored by Western Blot using antibodies as indicated. Arrowheads denote cleaved fragments. See also - and Table S2.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot, Activation Assay, Expressing, Inhibition, Functional Assay, Labeling, Binding Assay, Modification, Incubation, Recombinant, Mutagenesis, Generated

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-C) Akata (EBV+) cells were lytically induced with anti-IgG for 0, 6, 12, 24 and 48 hrs (A). P3HR1 (B) and SNU-719 (C) cells were lytically induced with TPA and NaBu for 0 6, 12, 24 and 48 hrs. YTHDF2, EBV ZTA and RTA, cleaved caspase substrate (CASP sub.), cleaved PARP, cleaved CASP3 and cleaved CASP8 were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls. (D-F) Apoptotic induction by an intrinsic trigger promotes EBV reactivation. Akata (EBV+) (D), P3HR1 (E) and SNU-719 (F) cells were untreated or treated with increasing amount of Taxol for 48 hrs. YTHDF2, EBV ZTA and RTA, and cleaved caspase substrate (CASP sub.) were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata EBV(+) cells were transduced with lenti-vector control or Myc-CASP8 to establish stable cell lines. The cells were treated with anti-IgG for 0, 24 and 48 hrs. (A) CASP8, EBV ZTA and RTA were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls. (B) Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of vector control at 0 hr was set as 1. (C) Total RNA was extracted and then EBV lytic (ZTA and RTA) and latent (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1 and LMP2) genes were analyzed by RT-qPCR. The value of vector control at 0 hr was set as 1 Results from three biological replicates are presented. Error bars indicate ±SD. *, p< 0.05; **, p< 0.01; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. (A-B) Total RNA was extracted and then EBV ZTA and RTA mRNA levels were analyzed by RT-qPCR. (C) Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-B) Immunofluorescence assay showing YTHDF2 downregulation and EBV EAD upregulation in apoptotic Akata (EBV+) cells upon lytic induction. Akata (EBV+) cells were either untreated (control) or treated with anti-IgG antibody for 24 and 48 hrs as indicated. (A) The cells were stained with Propidium Iodide (PI) and then permeabilized for immnunostaining with anti-YTHDF2 antibody. (B) Cells were permeabilized and co-immunostained with anti-YTHDF2 and anti-EBV EAD antibodies as indicated. (C-D) Immunofluorescence assay showing YTHDF2 downregulation and EBV EAD upregulation in apoptotic SNU-719 cells upon lytic induction. SNU-719 cells were either untreated (control) or treated with TPA and NaBu for 24 and 48 hrs as indicated. (C) Cells were stained with Propidium Iodide (PI) and then permeabilized for immunostaining with anti-YTHDF2 antibody. (D) Cells were permeabilized and co-immunostained with anti-YTHDF2 and anti-EBV EAD antibodies as indicated. Scale bar, 20 μm

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Immunofluorescence, Staining, Immunostaining

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: The Akata (EBV+) (A), P3HR-1 (B) and SNU-719 (C) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then lytically induced with anti-IgG antibody or TPA/NaBu as indicated for 48 hrs. Western Blot showing the protein levels of RIP, phospho-RIP (p-RIP) and phospho-RIP3 (p-RIP3) using antibodies as indicated. β-actin blots were included as controls.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) The design of CRISPR/Cas9-resistant YTHDF2 variant was based on the sg-2 protospacer adjacent motif (PAM). D166A/D367A mutations were introduced into the PAM-mutated YTHDF2. Both constructs were cloned into a lentiviral vector with a C-terminal Myc-tag. (B-C) WT and cleavage-resistant YTHDF2 suppresses EBV replication. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing YTHDF2 and EBV protein expression levels in these cell lines upon IgG cross-linking as indicated (B). Arrowheads denote cleaved fragments. Extracellular and intracellular viral DNA was measured by qPCR using primers specific to BALF5 (C). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001. (D) Schematic representation of 5 YTHDF2 cleavage-mimicking fragments. These fragments were cloned into a lentiviral vector with a C-terminal Myc-tag. (E) SNU-719 cells were transduced with lentiviruses carrying vector control or individual fragment to establish stable cell lines. Western Blot analysis showing YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by adding TPA (20 ng/ml) for 24 hrs. (F) Akata (EBV+) cells were transduced with lentiviruses carrying vector control or individual fragment to establish stable cell lines. Western Blot analysis showing YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. Shorter and longer exposures were included to show the differences in protein levels. (G) Caspase-mediated cleavage impairs YTHDF2 binding to CNOT1. Halo-V5-tagged WT YTHDF2 and the individual fragments were co-transfected with HA-tagged CNOT1 SH domain into 293T cells as indicated. Co-immunoprecipitation (Co-IP) experiments were performed using anti-V5 antibody-conjugated magnetic beads. The immunoprecipitated samples and total cell lysates (Input) were analyzed by Western Blot with antibodies as indicated. (H) Model showing the functional consequences of YTHDF2 cleavage in CNOT1 binding and the targeting of m 6 A-modified RNA. See also and .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: CRISPR, Variant Assay, Construct, Clone Assay, Plasmid Preparation, Western Blot, Expressing, Transduction, Stable Transfection, Binding Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Functional Assay, Modification

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) SNU-719 cells were transduced with lentiviruses carrying vector control or individual YTHDF2 fragment to establish stable cell lines (see ). RT-qPCR analysis showing EBV ZTA and RTA mRNA levels in these cell lines upon lytic induction by adding TPA (20 ng/ml) for 24 hrs. The value of vector control was set as 1. (B) Akata (EBV+) cells were transduced with lentiviruses carrying vector control or individual YTHDF2 fragment to establish stable cell lines . RT-qPCR analysis showing EBV ZTA and RTA mRNA levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. The value of vector control at 24 hrs was set as 1. (C) Akata (EBV+) cells were transduced with lentiviruses carrying vector control, Halo-tag, WT YTHDF2 or individual YTHDF2 fragment to establish stable cell lines. Western Blot analysis showing Halo, YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. Shorter and longer exposures were included to show the differences in protein levels. (D) Total mRNA was extracted from cells treated in panel (C). EBV ZTA and RTA mRNA levels were analyzed by RT-qPCR. The value of vector control at 24 hrs was set as 1 Results from three biological replicates are presented. Error bars indicate ±SD. N.S., not significant; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata (EBV+) cells were lytically induced by IgG-cross linking for 24 hrs. (A) Total RNA was subjected to m 6 A RIP, followed by RT-qPCR using indicated primers. Values are displayed as fold change over 10% input. GAPDH and Dicer are cellular negative and positive controls, respectively. (B) Cell lysate was collected to detect YTHDF2 binding of viral RNAs by RIP-qPCR. Values are displayed as fold change over 10% input. MALAT1 and SON are cellular negative and positive controls, respectively. Results from three biological replicates are presented. Error bars indicate ±SD. **, p< 0.01.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Quantitative RT-PCR, Binding Assay

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) A group of genes in the category of “ activation of cysteine-type endopeptidase activity involved in apoptotic process ” (also called “ caspase activation ”) were extracted from YTHDF2 target genes derived from YTHDF2 RIP-seq and PAR-CLIP-seq datasets ( , , ) (B-C) YTHDF2 reconstitution suppresses caspase-8 expression and subsequent caspase activation. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing the levels for caspase-8 (CASP8), cleaved caspase-8, and cleaved caspase substrates (CASP sub.) in these cell lines upon IgG cross-linking as indicated (B). CASP8 mRNA levels were analyzed by RT-qPCR using CASP8 primers (C). The value of vector control at 0 hr was set as 1. (D-E) Caspase-8 inhibition suppress EBV replication in YTHDF2-depleted cells. Control and YTHDF2-depleted Akata (EBV+) cells were either untreated or pretreated with caspase-8 inhibitor (Z-IETD-FMK, 50 μM) for 1 hr and then anti-IgG antibody was added for 0 to 48 hrs as indicated. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (D). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (E). The value of vector control at 0 hr was set as 1. (F-G) Caspase-8 depletion suppresses EBV replication in YTHDF2-depleted cells. YTHDF2-depleted Akata (EBV+) cells were transduced with lentivirus carrying control sgRNA or CASP8-sg1 to establish cell lines and then anti-IgG antibody was added for 0 to 48 hrs as indicated. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (F). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (G). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Activation Assay, Activity Assay, Derivative Assay, Expressing, Construct, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Inhibition, Transduction

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-B) YTHDF2 depletion promotes CASP8 mRNA expression. Akata (EBV+) cells and P3HR-1 cells carrying different sgRNA targeting YTHDF2 or control (sg-NC) were used to extract total RNA and qPCR analyses were performed a group of YTHDF2-targeted cellular genes involved in caspase activation. The values were normalized with a non YTHDF2 target HPRT1 . The values of sg-NC were set as 1. (C-D) CASP8 is modified by m 6 A and YTHDF2 binding to CASP8 . Akata (EBV+) cells were used to perform m6A RIP-qPCR (C) and YTHDF2 RIP-qPCR (D), respectively. Values are displayed as fold change over 10% input. (E-G) YTHDF2 depletion promotes caspase-8 protein expression and PIAS1 cleavage upon lytic induction. Akata (EBV+) cells (E), P3HR-1 cells (F) and SNU-719 cells (G) carrying different sgRNA targeting YTHDF2 or control (sg-NC) were lytically induced by anti-IgG, TPA and sodium butyrate (NaBu) and gemcitabine treatment for 24 hrs. Protein expression was monitored by Western Blot using antibodies as indicated. (H) CASP8 m 6 A peaks were extracted from MeT-DB V2.0 database. YTHDF2-PAR-CLIP data were retrieved from Wang et al.. The Exon-7 of CASP8 with highest m 6 A peaks were analyzed for conservation among sequences derived from 100 vertebrate species. 15 potential m 6 A motifs (M1-M15) were extracted based on m 6 A motif DRACH. (I) Motif logos were generated for 15 individual sites. Red cycles denote highly conserved motifs (M2, M3, M5, M8 and M12) across 100 vertebrate species. (J-K) WT and mutant CASP8 -Exon-7 were cloned into the m 6 A-null Renilla luciferase (RLuc) reporter (3’UTR region) that also express Firefly luciferase (FLuc) from a separate promoter (J). These three reporter plasmids were transfected into parental or YTHDF2-depleted (YTHDF2 KD) SNU719 cells. Relative Renilla to Filefly luciferase activity (RLuc/FLuc) was calculated (K). The value of WT in parental cells was set as 1. (L) Model illustrating YTHDF2 regulation of CASP8 mRNA and caspase-8 regulation of YTHDF2 and PIAS1 in EBV reactivation. Results from three biological replicates are presented. Error bars indicate ±SD. *, p< 0.05; **, p< 0.01; ***, p< 0.001. See also , and Table S3.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Expressing, Activation Assay, Modification, Binding Assay, Western Blot, Derivative Assay, Generated, Mutagenesis, Clone Assay, Luciferase, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Diagram summarizing the major writers, readers and erasers involved in the m 6 A RNA modification pathway. (B) The downregulation of m 6 A RNA modification pathway proteins during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot was performed using antibodies as indicated. N6AMT1 and β-actin blots were included as controls. (C) Caspase inhibition blocks the degradation of m 6 A RNA modification pathway proteins. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot was performed using antibodies as indicated. (D and E) V5-METTL14 (D) and V5-WTAP (E) were incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL14, anti-V5 and anti-WTAP antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. Arrowheads denote cleaved fragments. Star denotes non-specific bands. See also - .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Modification, Western Blot, Inhibition, Incubation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) V5-METTL3 was incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL3 and anti-V5 antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. The positions of weakly cleaved fragments were labelled by arrowhead. Star denotes non-specific bands. (B) V5-tagged WTAP D301A/D302A and D301A mutants were incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. Arrowheads denote cleaved fragments. (C) Sequence alignment of WTAP sequences from 10 representative species using the Constraint-based Multiple Alignment Tool (COBALT). The cleavage motifs were highlighted by yellow color. (D) Motif analysis showing the conservation of the WTAP D302 and the surrounding amino acids. Amino acid sequences were extracted from 97 vertebrate species and motif logos were generated using WebLogo. (E-F) Akata (EBV+) cells were used to establish stable cell lines using 2 different guide RNA constructs targeting YTHDF1 (D) and ALKBH5 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Incubation, Western Blot, Recombinant, Sequencing, Generated, Stable Transfection, Construct, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A), METTL14 (B), WTAP (C), VIRMA (D) and YTHDF3 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. See also and .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A and B), METTL14 (C and D), WTAP (E and F), VIRMA (G and H) and YTHDF3 (I and J) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation for 24 or 48 hrs. EBV ZTA and RTA mRNA expression levels were monitored by RT-qPCR (A, C, E, G and I). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (B, D, F, H and J). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Activation Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization of GCB‐resistant bladder cancer cells . (A) Schematic illustration of the establishment of GCB‐resistant bladder cancer cell lines (T24GCB and 5637GCB). (B) Cell viability of parental (T24, J82 and 5637) and GCB‐resistant (T24GCB and 5637GCB) bladder cancer cells was assessed using the MTT assay following 48 h treatment with a range of GCB concentrations (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 3 µM; n = 6 per group). (C, F) Colony formation assays were used to evaluate the clonogenic survival of T24, J82, 5637, T24GCB and 5637GCB cells treated with GCB (0, 0.0001, 0.001 and 0.01 µM) for 14 days ( n = 3 per group). (D, G) Migration assay was used to evaluate the migratory ability of the indicated bladder cancer cells treated with GCB (0 and 0.1 µM) for 24 h ( n = 3 per group). (E, H) Invasion assay was used to evaluate the invasive potential of bladder cancer cells treated with GCB (0 and 0.1 µM) for 24 h ( n = 3 per group). (I) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, CMPK1, NME23, TK1, TK2, NT5C2, CNT3, ABCB1 and ABCG2), epithelial‐mesenchymal transition markers (E‐Cadherin and Vimentin) and the anti‐apoptotic protein BCL‐2 in parental and resistant bladder cancer cells following treatment with 0.01 µM GCB for 24 h. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: MTT Assay, Migration, Invasion Assay, Western Blot, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein Rab27A in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: Functional Assay, MTT Assay, Cell Culture, Transmission Assay, Electron Microscopy, Derivative Assay, Concentration Assay, Imaging, Flow Cytometry, Staining, Fluorescence, Microscopy, Incubation, Western Blot, Colony Assay, Co-Culture Assay, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Identification of GCB resistance‐associated genes in GCB‐resistant bladder cancer cells and their EVs by RNA sequencing . (A) Schematic overview of total RNA sequencing comparing GCB‐sensitive bladder cancer cells (T24 and 5637) with their GCB‐resistant counterparts (T24GCB and 5637GCB), as well as EVs derived from GCB‐sensitive (T24‐EVs and 5637‐EVs) and GCB‐resistant cells (T24GCB‐EVs and 5637GCB‐EVs). (B) The heatmap illustrates DEGs between GCB‐sensitive and GCB‐resistant cells and EVs. DEGs were identified using a fold change > 2 (Log 2 FC > 1.5 or <–1.5) and adjusted p < 0.01. The colour scale represents relative gene expression levels (blue = low, red = high). (C) RT‐qPCR validation of selected DEGs in GCB‐sensitive and GCB‐resistant bladder cancer cells, as well as their corresponding EVs. (D) Pathway enrichment analysis of DEGs identified in GCB‐resistant EVs identified several significantly enriched pathways, including the ROS pathway, peroxisome, Myc targets V2 and angiogenesis. (E) Comparative heatmap of proteomic profiling illustrates differential protein expression in GCB‐resistant versus GCB‐sensitive EVs. The analysis identified overlapping candidates with RNA‐seq data from both cell (T24GCB vs. T24) and EV (T24GCB‐EVs vs. T24‐EVs) datasets. (F and G) Canonical pathway analysis using IPA identified overlapping enriched pathways between GCB‐resistant and GCB‐sensitive EVs in (F) 5637GCB‐EVs versus 5637‐EVs and (G) T24GCB‐EVs versus T24‐EVs. GCB, gemcitabine; EVs, extracellular vesicles; DEGs, differentially expressed genes; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; ROS, reactive oxygen species; IPA, Ingenuity Pathway Analysis.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: RNA Sequencing, Derivative Assay, Gene Expression, Quantitative RT-PCR, Biomarker Discovery, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Functional characterization of H3C14 in GCB resistance in bladder cancer cells . (A) Western blot analysis of histone H3.2 (H3C14) protein expression in GCB‐sensitive bladder cancer cells (T24 and 5637) versus their GCB‐resistant counterparts (T24GCB and 5637GCB) and J82 cells. (B) Western blot analysis of histone H3.2 (H3C14) protein levels in EVs isolated from 5637, 5637GCB, T24, T24GCB and J82 cells. (C) Western blot analysis of histone H3.2 (H3C14) in T24 and 5637 cells after 24 h treatment with conditioned media (ConMed) or EVs derived from T24, T24GCB and 5637, 5637GCB cells. (D) RT‐qPCR analysis was used to evaluate the knockdown efficiency of H3C14 RNA in T24 and 5637 cells transfected with H3C14 siRNA (siH3C14#1 and siH3C14#2). (E) MTT assay was used to evaluate cell viability in T24 and 5637 cells transfected with scramble siRNA (Vector) or siH3C14 following treatment with a series of GCB concentrations (0–3 µM) for 48 h ( n = 6 per group). (F and I) Colony formation assays were used to evaluate measuring clonogenic survival clonogenicity of T24 and 5637 cells transfected with Vector or siH3C14 over 14 days ( n = 3 per group). (G and J) Migration assays were used to evaluate the migratory capacity of T24 and 5637 cells transfected with Vector or siH3C14 ( n = 3 per group). (H and K) Invasion assays were used to evaluate the invasive ability of T24 and 5637 cells transfected with Vector or siH3C14 ( n = 3 per group). (M and N) Flow cytometry analysis of apoptosis in T24 and 5637 cells transfected with Vector or siH3C14 following treatment with 0.01 µM gemcitabine. Apoptosis was assessed using PI and Annexin V staining ( n = 3 per group). (L) Western blot analysis of histone H3.2 (H3C14) and GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) in T24 and 5637 cells transfected with Vector, siH3C14#1, or siH3C14#2. For all panels, data are presented as mean ± SEM. An unpaired two‐tailed Student's t ‐test was used for comparisons between groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. All experiments were independently repeated at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; PI, propidium iodide.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: Functional Assay, Western Blot, Expressing, Isolation, Derivative Assay, Quantitative RT-PCR, Knockdown, Transfection, MTT Assay, Plasmid Preparation, Migration, Flow Cytometry, Staining, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Functional characterization of H3C14 gain‐of‐function in GCB‐resistant bladder cancer cells and its role in GCB sensitivity . (A) RT‐qPCR analysis was conducted to confirm H3C14 overexpression in T24GCB, 5637GCB and J82 cells transfected with H3C14‐GFP plasmid. (B) Representative fluorescence microscopy images show subcellular localization of H3C14‐GFP in T24GCB, 5637GCB, and J82 cells. Scale bar: 20 µm. (C) MTT assays were used to evaluate cell viability in T24GCB‐H3C14‐GFP, 5637GCB‐H3C14‐GFP and J82‐H3C14‐GFP cells after 48 h treatment with GCB concentrations ranging from 0 to 3 µM ( n = 6 per group). (D) Western blot analysis of histone H3.2 (H3C14) and GCB‐metabolizing enzymes and transporters (CNT1, ENT1, NT5C2 and CNT3) in T24GCB, 5637GCB and J82 cells transfected with either vector or H3C14‐GFP, with or without 0.01 µM GCB treatment. (E and F) Western blot analysis of histone H3.2 (H3C14) protein stability in T24 and T24GCB cells treated with cycloheximide (CHX, 50 µg/mL) for 0, 2, 4 or 6 h, and with MG132 (10 µM) for 6 h. (G) Schematic diagram of xenograft tumour model in nude mice—T24GCB‐Vector or T24GCB‐H3C14 cells were subcutaneously injected, followed by GCB treatment (1 mg/kg every 2 days for 28 days). (H) Representative tumour images from the four treatment groups: Vector, Vector + GCB, H3C14 and H3C14 + GCB ( n = 3 mice per group). (I) Tumour weight comparison among the four groups ( n = 3 mice per group). (J) Tumour growth curves present changes in tumour volume over time across treatment groups ( n = 3 mice per group). (K) Histogram of final tumour weights from each treatment group ( n = 3 mice per group). (L–O) IHC staining of H3C14, CNT3 and TK1 in xenograft tumours from the four groups. H‐scores were calculated to quantify expression levels ( n = 3 mice per group). (P–R) Kaplan–Meier survival analysis of patients with bladder cancer from TCGA datasets based on high versus low expression of H3C14, CNT3 and TK1. (S) TCGA survival analysis based on high H3C14 and low CNT3 expression revealed the highest survival probability in this subgroup. Data are presented as mean ± SEM. Statistical comparisons were made using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were independently repeated at least three times. GCB, gemcitabine; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; IHC, Immunohistochemical; TCGA, The Cancer Genome Atlas.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: Functional Assay, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Injection, Comparison, Immunohistochemistry, Expressing, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemical staining

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization of H3.2 (H3C14)‐carrying EVs in GCB Resistance . (A) Schematic illustration of the isolation of EV subpopulations from T24GCB cells using MicroBeads Pan, targeting CD9, CD63 and CD81. (B) MTT assay was used to assess the viability of T24 and 5637 cells treated with PBS, ultracentrifugation‐derived T24GCB‐EVs (or 5637GCB‐EVs), or MicroBeads Pan‐isolated EVs (+MicroBeads Pan), or EVs not captured by MicroBeads Pan (–MicroBeads Pan). Cells were subsequently treated with a series of GCB concentrations (0–3 µM) for 48 h ( n = 6 per group). (C) Mass spectrometry‐based comparative proteomic profiling of Transport‐EVs and Excretion‐EVs. The heatmap illustrates differentially expressed proteins, with red indicating higher expression and blue indicating lower expression levels. (D) Western blot analysis of EV‐associated markers (CD9 and CD81), histone H3.2 (H3C14), LAMB1 and CD147 in EVs derived from ultracentrifugation, MicroBeads Pan‐enriched (+MicroBeads Pan), or non‐captured (–MicroBeads Pan) fractions from T24GCB‐EVs and 5637GCB‐EVs. EVs may be broadly classified into Transport‐EVs (either CD9+, CD63+, or CD81+) and Excretion‐EVs (lacking CD9, CD63 and CD81). (E) Western blot analysis of histone H3.2 (H3C14), Alix, LAMB1 and CD147 in EVs isolated via ultracentrifugation or immunoprecipitation using magnetic beads conjugated with IgG, anti‐CD147, or anti‐LAMB1 antibodies from T24GCB‐EVs. (F–I) Imaging flow cytometry analysis of EVs derived from T24‐EVs, T24GCB‐EVs and T24GCB‐H3C14‐EVs (TG‐H3C14 EVs). EVs were stained with a lipid bilayer dye and labelled with CD9‐APC, CD63‐FITC, CD81‐PE, LAMB1‐AF568 and CD147‐Cy7 antibodies. Percentage gated values represent the proportion of EVs co‐expressing the indicated surface markers: (F) CD63⁺CD9⁺, CD63⁺CD81⁺, CD63⁺LAMB1⁺ and CD63⁺CD147⁺ (G) CD9⁺CD81⁺, CD9⁺LAMB1⁺ and CD9⁺CD147⁺ (H) CD81⁺LAMB1⁺ and CD81⁺CD147⁺ (I) CD147⁺LAMB1⁺ For all panels, data are presented as mean ± SEM. Statistical significance was determined using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were repeated independently at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; PBS, paraformaldehyde.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: Isolation, MTT Assay, Derivative Assay, Mass Spectrometry, Expressing, Western Blot, Immunoprecipitation, Magnetic Beads, Imaging, Flow Cytometry, Staining, Two Tailed Test, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 1. The FoxP3 Reporter LV Shows Expression Selective for the Treg Cell Lineage (A) Endogenous human FOXP3 gene shows location of regulatory regions (promoter, CNS1, CNS2, CNS3, and 30 UTR) included in vector. Vector maps show design of CNS123p-mStrawberry and CNS123p-FoxP3-mStrawberry constructs within the pCCL vector backbone. (B) Analysis of human hematopoietic cell lines transduced with CNS123p-mStrawberry. Histograms show endogenous FoxP3 status of each cell line (left) and mStrawberry expression (right) in each cell line transduced with CNS123p-mStrawberry. Plots show mStrawberry expression in human hematopoietic cell lines over a range of vector copy numbers (n = 12 per cell line). (C) Activated human CD4 cells transduced with different doses of CNS123p-mStrawberry. Histograms show mStrawberry expression in viable CD4+ cells analyzed 4 days after activation. (see also Figure S2B). (D) Experimental design to evaluate in vivo lineage-specific expression of CNS123p-mStrawberry. Lin HSPCs were isolated from CD45.2 FoxP3-prom-GFP mice and transduced with CNS123p-mStrawberry. Transduced lin HSPCs were transplanted into lethally irradiated congenic CD45.1 recipients. CD45.2 donor cells within each hematopoietic lineage were analyzed at 20 weeks post-transplant for mStrawberry reporter LV expression (see also Figure S2A). (E) Histograms depict mStrawberry reporter expression in each hematopoietic lineage in bone marrow, thymus, and spleen of engrafted mice. Each individual histogram line represents mStrawberry expression for an individual mouse (n = 9 mice; see also Figure S2C). (F) y axis represents the percentage of mStrawberry+ cells within each hematopoietic lineage in the BM, spleen, and thymus (n = 9 mice). Data in (E) represent mean ± SD.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Expressing, Plasmid Preparation, Construct, Transduction, Activation Assay, In Vivo, Isolation, Irradiation

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 2. Lineage-Specific FoxP3 Expression Restores Treg Cell Development from Scurfy (FoxP3 Mutant) HSCs (A) Transplant setup to evaluate Treg cell development. Scurfy (FoxP3mut) mice were rescued with WT CD45.1 splenocytes at birth to allow survival into adulthood to serve as bone marrow donors. Lin HSPCs were isolated from rescued scurfy (FoxP3mut) or wild-type (FoxP3-prom-GFP) donor mice and transduced with CNS123p-mStrawberry or CNS123p-FoxP3-mStrawberry. Transduced lin HSPCs were transplanted into lethally irradiated WT CD45.1 congenic recipients. After 12 weeks, donor cells from each transplant cohort were evaluated for thymic and splenic reconstitution of Treg cells. Treg cell populations from each group were identified as CD4+mStrawberry+ cells (uncorrected scurfy Treg cells [Sf-Treg cells]; corrected scurfy Treg cells [cSf-Treg cells]) or CD4+GFP+ cells (wild- type Treg cells [WT-Treg cells]). (B) Lineage distribution of total donor thymocytes in mice reconstituted with Sf, cSf, or WT BM lin cells (n = 3–5 mice/arm). (C) Thymic Treg cell reconstitution. FACS plots show donor CD45.2+CD4SP cells in the thymus of transplant recipients. Gates delineate thymic Sf-Treg cells, cSf-Treg cells, and WT-Treg cells. Bottom panel shows expression of the Treg cell surface markers CD25, GITR, and CTLA4 within each putative Treg cell population (surface marker expression for Sf-Treg cells and cSf-Treg cells is shown for mStrawberryhigh cells or cells expressing the top 50% of mStrawberry expression). Histograms depict one representative experiment of 3 (see also Figure S2D). (D) FACS sort for splenic Treg and Tconv cells. Splenic Treg cell populations (mStrawberryhigh or GFP+ gates) were FACS sorted for a Treg cell suppression assay. Tconv cell populations (mStrawberry or GFP gates) were sorted for an iTreg cell induction assay. (E) In vitro Treg cell suppression assay. Sorted Treg cells (shown in D) were co-cultured with responder T cells (Tresp cells) (congenic WT CD4+ cells labeled with a fluorescent proliferation dye) at a 1:1 ratio in the presence of bead-bound CD3 and CD28 antibodies. Histograms depict Tresp cell proliferation in one of three representative experiments. Bar graph shows proliferation index for each Treg cell culture condition (n = 6–9 Tresp cell cultures per arm from 3 different Treg cell sources per arm; data normalized to internal ‘‘no Treg cell’’ control for each experiment). (F) Sorted splenic Tconv cells (shown in D) were activated with CD3 and CD28 antibodies in the presence of 20 ng/mL interleukin-2 (IL-2) and 20 ng/mL TGF-b for 4 days to induce iTreg cells. FACS plots show mStrawberry or GFP expression from each group (n = 3 mice/group). Data in (B), (C), (E), and (F) are presented as mean ± SD. Data in (B) were analyzed by Kruskal-Wallis test for each lineage. Data in (E) are analyzed by Kruskal- Wallis test for overall comparison for all groups and Mann-Whitney U test for pairwise comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Expressing, Mutagenesis, Isolation, Transduction, Irradiation, Marker, Suppression Assay, In Vitro, Cell Culture, Labeling, Control, Comparison, MANN-WHITNEY

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 3. The Lineage-Specific FoxP3 cDNA Vector Generates Functional Treg Cells Capable of Rescuing the Scurfy Mouse (A) Assay for in vivo Treg cell function: three groups of CD4 cells containing putative Treg cells were generated by congenic bone marrow transplants: uncorrected scurfy CD4 (Sf-CD4; no. 1); corrected scurfy CD4 (cSf-CD4; no. 2); and wild-type CD4 (WT-CD4; no. 3). To obtain Sf-CD4 and cSf-CD4, CD45.2 Lin HSPCs were isolated from scurfy donors (rescued at birth by CD45.1 splenocytes) and transduced with either CNS123p-mStrawberry (Sf-CD4; no. 1) or CNS123p-FoxP3- mStrawberry (cSf-CD4; no. 2). To obtain WT-CD4, CD45.2 Lin HSPCs were isolated from FoxP3-prom-GFP donors and transduced with CNS123p- mStrawberry (no. 3). Transduced cells were transplanted into lethally irradiated congenic (CD45.1) recipients. 8 weeks post-transplant, donor CD45.2+CD4+ cells were purified with magnetic beads from the spleens of transplant recipients and injected intraperitoneally into scurfy neonates. Scurfy neonates and age-matched WT receiving PBS injection were also analyzed as control conditions no. 4 and no. 5. The autoimmune phenotype of all groups was evaluated at 21 days of life (see also Figure S3). (B) Photographs of scurfy mice or WT controls at 21 days. White arrows highlight ear skin phenotype in mice from each group. Ear skin inflammation (char- acterized by small, thickened, scaly ears) is seen in untreated scurfy mice (Sf + PBS) and scurfy mice receiving uncorrected scurfy CD4 cells (‘‘Sf + Sf-CD4’’). Normal ear skin without inflammation is seen in WT controls (‘‘WT + PBS’’) and scurfy mice receiving wild-type or corrected scurfy CD4 cells (‘‘Sf + WT-CD4’’ and ‘‘Sf + cSf-CD4’’). (C) Spleen-to-body-weight ratio for rescued scurfy mice or WT littermate controls. (D) Activated (CD44+CD62L) CD4 T cells (expressed as a percentage of total CD4 splenocytes) in the spleens of rescued scurfy mice or WT littermate controls. (E) Serum cytokine levels in rescued scurfy mice or WT littermate controls. Data in (C)–(E) are presented as mean ± SD. Data on (C)–(E) represent n = 3 independent experiments pooled for analysis with a total of 3–6 mice/arm. Data in (C)–(E) were analyzed by Kruskal-Wallis test for overall comparison for all groups, and Mann-Whitney U test was performed for pairwise comparisons. *p < 0.05; **p < 0.01.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Plasmid Preparation, Functional Assay, In Vivo, Cell Function Assay, Generated, Isolation, Transduction, Irradiation, Magnetic Beads, Injection, Control, Comparison, MANN-WHITNEY

Journal: Cell stem cell

Article Title: Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.

doi: 10.1016/j.stem.2018.12.003

Figure Lengend Snippet: Figure 4. The FoxP3 Reporter Vector CNS123p-mStrawberry Shows Treg Cell Lineage-Selective Expression in a Humanized Mouse Model (A) Experimental setup for humanized mouse models. Cord blood CD34+ HSPCs were transduced with CNS123p-mStrawberry and transplanted into neonatal NSG mice (B, C, E, and F) or NSG-SGM3 mice (D). 12–16 weeks post-transplant, engrafted hCD45+ cells were analyzed for mStrawberry expression. (B) mStrawberry reporter expression in each hematopoietic lineage. Each overlaid histogram represents mStrawberry expression in an individual mouse (n = 10–14 mice humanized with 2 different cord blood CD34+ donors; see also Figures S4A–S4C). (C) Percentage of mStrawberry+ cells in each lineage shown in (B). (D) Co-expression of FoxP3 and mStrawberry in humanized mice. Splenic human CD4+ cells were FACS sorted into mStrawberry+ and mStrawberry pop- ulations followed by intracellular staining for FoxP3 expression. Left panel shows sorting of human CD4+ cells by mStrawberry expression, and right panel shows FoxP3 expression in sorted populations. Results are representative of 2 independent experiments. (E) CNS2 methylation analysis of T cell populations from humanized mice. Figure shows the locations of CNS2 within the endogenous FOXP3 gene and CNS2 within the viral genome. Red arrows indicate differential primer binding sites for amplification of endogenous or viral CNS2. FACS plot shows sorting gates used to define Treg cell (CD4+CD25+) and Tconv cell (CD4+CD25) populations in CD4-enriched cells isolated from the pooled spleens of 3–5 humanized mice. Heatmap represents the percentage of methylated reads detected at each of the 9 CpG sites within endogenous and viral CNS2. Results are representative of 2 independent experiments using pooled NSG cohorts humanized from 2 different CB CD34+ donors (see also Figures S4D–S4F). (F) Experimental setup for NSG competitive repopulation assay. ‘‘Test’’ CB CD34+ cells were transduced with either CNS123p-mStrawberry or CNS123p-FoxP3- mStrawberry, and ‘‘competitor’’ CD34+ cells were transduced with a UBC-mCitrine vector. Test and competitor cells were co-transplanted at a 1:1 ratio into NSG neonates, and the percentage of competitor (mCitrine+) CD45+ cells engrafted in the BM at 12 weeks was determined for each group (n = 6 mice per group; humanized from 2 different CB CD34+ donors). Data in (F) represent mean ± SD. Data in (F) were analyzed by Mann-Whitney U test.

Article Snippet: CpG dinucleotide methylation analysis of CNS2 of the human FoxP3 gene was performed by EpigenDx and determined by bisulfite treatment of RNase-treated genomic DNA, followed by PCR amplification and pyrosequencing (EpigenDx assay ADS783-FS2).

Techniques: Plasmid Preparation, Expressing, Transduction, Staining, Methylation, Binding Assay, Isolation, MANN-WHITNEY

Journal: Nature

Article Title: Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.

doi: 10.1038/s41586-024-08533-3

Figure Lengend Snippet: Fig. 3 | Structural mechanisms and amino acid preferences of functional KBTBD4 mutations. a, Cryo-EM map of LHC-bound KBTBD4 mutants with the two KBTBD4 protomers (slate and green), HDAC1 (pink), CoREST (orange) and InsP6 (red). Left, KBTBD4-PR; right, KBTBD4-TTYML. b, Ribbon diagram of the KBTBD4-PR–HDAC1–CoREST–InsP6 complex. Subunits of the complex are coloured the same way as in a. The hotspot arginine residue is shown in space filling model mode. InsP6 is shown in cyan and red sticks. c, Close-up view of the 4b-4c loops of KBTBD4-PR-A (slate) and KBTBD4-PR-B (green) after the β-propeller domain of the latter is superimposed onto that of the former. Side chains of two phenylalanine residues in the 4b-4c loop of KBTBD4-PR-A are shown in sticks. d, Close-up view of the 2b-2c loops of KBTBD4-A (slate) and KBTBD4-B (green) after the β-propeller domain of the former is superimposed

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used LSD1 (Bethyl Laboratories, A300-215A, Lot no. 2) RCOR1 (Cell Signaling Technology, #14567, Lot no. 1) GAPDH (Santa Cruz Biotechnology, sc-477724, Lot no. G2920; RRID: AB_627678) HA (Cell Signaling Technology, #3724, Lot no. 10) FLAG (Sigma-Aldrich, F1804, Lot no. #SLCN3722) KBTBD4 (Novus Biologicals, NBP1-88587, Lot no. A116815 ) HDAC1 (Cell Signaling Technology, #34589, Lot no. 4) HDAC2 (Cell Signaling Technology, #57156, Lot no. 1) Anti-Rabbit IgG HRP conjugate (Promega, Cat#W4011, RRID:AB_430833) Anti-Mouse IgG HRP conjugate (Promega, Cat#W4021, RRID:AB_430834) PierceTM Anti-HA Magnetic Beads (Thermo Scientific, #88837) Validation All antibodies used were commercial and validated for the appropriate application.

Techniques: Functional Assay, Cryo-EM Sample Prep, Residue

Journal: Nature

Article Title: Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.

doi: 10.1038/s41586-024-08533-3

Figure Lengend Snippet: Fig. 4 | Converging mechanism between KBTBD4 cancer mutations and UM171. a, Simplified ligand plot of UM171–KBTBD4–HDAC1 interactions. HDAC1 and KBTBD4 residues are denoted by pink and green circles, respectively. b, Superposition analysis of the β-propellers in protomer-B of the KBTBD4-WT and KBTBD4-PR dimers. The structural differences at several top surface loops are indicated by arrows. The 2b-2c loop is labelled. c, A comparison of UM171 (yellow and blue sticks), the side chain of Tyr312 of KBTBD4-TTYML-B (cyan and red sticks) and the side chain of Arg312 of KBTBD4-PR-B (green and blue sticks) at the active site pocket of HDAC1 (pink). The three complex structures are superimposed through HDAC1. Two phenylalanine residues outlining the entrance of the HDAC1 active site tunnel are shown in sticks. d, A comparison between UM171 (yellow and blue sticks) and the 2b-2c loop of KBTBD4-PR-B with the KBTBD4–UM171–HDAC1 structure superimposed with the KBTBD4- PR–HDAC1 structure through HDAC1. The side chains of key residues at the interface are shown in sticks. e, A comparison between UM171 (yellow and blue sticks) and the 2b-2c loop of KBTBD4-TTYML-B with the KBTBD4–UM171– HDAC1 structure superimposed with the KBTBD4-TTYML–HDAC1 structure through HDAC1. The side chains of key residues at the interface are shown in sticks. f, A close-up view of the inter-molecular interfaces among KBTBD4- TTYML (green), HDAC1 (pink, surface representation), CoREST (orange, surface representation) and InsP6 (cyan, orange and red sticks). The side chains of key KBTBD4-TTYML residues involved in InsP6 interaction and at the nearby 2b-2c loop are shown in sticks. Zn, zinc.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used LSD1 (Bethyl Laboratories, A300-215A, Lot no. 2) RCOR1 (Cell Signaling Technology, #14567, Lot no. 1) GAPDH (Santa Cruz Biotechnology, sc-477724, Lot no. G2920; RRID: AB_627678) HA (Cell Signaling Technology, #3724, Lot no. 10) FLAG (Sigma-Aldrich, F1804, Lot no. #SLCN3722) KBTBD4 (Novus Biologicals, NBP1-88587, Lot no. A116815 ) HDAC1 (Cell Signaling Technology, #34589, Lot no. 4) HDAC2 (Cell Signaling Technology, #57156, Lot no. 1) Anti-Rabbit IgG HRP conjugate (Promega, Cat#W4011, RRID:AB_430833) Anti-Mouse IgG HRP conjugate (Promega, Cat#W4021, RRID:AB_430834) PierceTM Anti-HA Magnetic Beads (Thermo Scientific, #88837) Validation All antibodies used were commercial and validated for the appropriate application.

Techniques: Comparison

Journal: Nature

Article Title: Converging mechanism of UM171 and KBTBD4 neomorphic cancer mutations.

doi: 10.1038/s41586-024-08533-3

Figure Lengend Snippet: Fig. 5 | HDAC1/2 inhibitors block the neomorphic activity of KBTBD4 mutants. a, Steric clash between SAHA (yellow) and the central arginine residue at the 2b-2c loop of KBTBD4-PR-B. The KBTBD4-PR–HDAC1 complex structure is superimposed with the HDAC2–SAHA complex structure (PDB 4LXZ) through the HDAC subunits. b, Flow cytometry quantification of GFP+ cells for KBTBD4-null CoREST–GFP cells pre-treated with DMSO, CI-994 (10 µM), SAHA (10 µM) or RBC1HI (10 µM) for 1 h followed by dox-inducible overexpression of the indicated KBTBD4 variant. Data are mean ± s.d. of n = 3 biological replicates. c, Immunoblots of HA IP from 293T cells transfected with the indicated HA–KBTBD4 variant, pre-treated with MLN4924 (1 µM) for 3 h, and then treated with DMSO, UM171 (1 µM) or SAHA (10 µM) for 1 h. d, TR-FRET signal between fluorescein–LHC and anti-His CoraFluor-1-labelled antibody with indicated His–KBTBD4 mutant in the presence of DMSO, SAHA (10 µM), CI-994 (10 µM) or RBC1HI (10 µM) (n = 2 biological replicates). e, Ex vivo proliferation for ICB1572 (KBTBD4-PR), MED411FH (KBTBD4-WT) and RCMB28 (KBTBD4-WT) cells with RBC1HI treatment at indicated doses for 72 h. Data are mean ± s.d. across biological replicates from PDX cells derived from n = 5 (ICB1572), n = 3 (RCMB28) and n = 2 (MED411FH) implanted mice. f, Immunoblots showing LSD1, CoREST and GAPDH in ICB1572 after 24 h treatment with MLN4924 or RBC1HI at the indicated doses. Data in b and d and immunoblots in c and f are representative of two independent experiments. FACS-gating schemes and uncropped blots can be found in Supplementary Figs. 1a and 3, respectively.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used LSD1 (Bethyl Laboratories, A300-215A, Lot no. 2) RCOR1 (Cell Signaling Technology, #14567, Lot no. 1) GAPDH (Santa Cruz Biotechnology, sc-477724, Lot no. G2920; RRID: AB_627678) HA (Cell Signaling Technology, #3724, Lot no. 10) FLAG (Sigma-Aldrich, F1804, Lot no. #SLCN3722) KBTBD4 (Novus Biologicals, NBP1-88587, Lot no. A116815 ) HDAC1 (Cell Signaling Technology, #34589, Lot no. 4) HDAC2 (Cell Signaling Technology, #57156, Lot no. 1) Anti-Rabbit IgG HRP conjugate (Promega, Cat#W4011, RRID:AB_430833) Anti-Mouse IgG HRP conjugate (Promega, Cat#W4021, RRID:AB_430834) PierceTM Anti-HA Magnetic Beads (Thermo Scientific, #88837) Validation All antibodies used were commercial and validated for the appropriate application.

Techniques: Blocking Assay, Activity Assay, Residue, Flow Cytometry, Over Expression, Variant Assay, Western Blot, Transfection, Mutagenesis, Ex Vivo, Derivative Assay

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: PRSS55 deletion leads to impaired mitochondrial function in mouse testes and sperm. A in vitro sperm (marked by white cycle) migration in 10% MC4000 solution (n = 4). Capacitated sperm that migrate over 1 cm from the bottom of the capillary slide were counted under a microscope, 200 × magnification. Sperm were highlighted in white circle. B ATP levels in Prss55 −/− testes and sperm is shown as mean ± SE (n = 3). The liver in which PRSS55 is not expressed was used as unrelated control. C NAD+ , NADH levels and NAD+ /NADH ratio in wt and Prss55 −/− testicular cells were determined and presented as mean ± SE (n = 3). D The mitochondrial membrane potential (MMP) of spermatozoa from wt and Prss55 −/− mice was determined using JC-1 probes (n = 4). JC-1 polymer/JC-1 monomer fluorescence ratios were calculated and shown as mean ± SE (n = 4). (*, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, n.s., no significant difference.)

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: In Vitro, Migration, Microscopy, Control, Membrane, Polymer, Fluorescence

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: PRSS55 is localized in mitochondria and facilitates mitochondrial energy metabolism in cultured cell lines. A Immunofluorescence staining shows co-localization of PRSS55 and mitochondria in NIH-3T3 cells (PRSS55, green; MitoTracker, red; DAPI, blue). pixel profile analysis revealed colocalization of green and red signals. Empty vector plasmid (Ctrl) was utilized as a control. B Immunofluorescence staining shows co-localization of PRSS55 and mitochondria in matured sperm (PRSS55, green; COXIV, red; DAPI, blue). Mouse and rabbit IgG staining (Ctrl) was used as a negative control. C Mitochondrial fraction from Prss55 transfected HEK293T cells was subject to immunoblotting analysis. Tubulin was used as a marker for cytoplasm, histone H2A for nuclear, and COXIV for mitochondria (Total, total cell lysates; Mito, isolated mitochondria). D ATP levels in HEK293T cells transfected with PRSS55-Myc expression vector were detected and shown as mean ± SE (n = 4). E NAD+ , NADH levels and NAD+ /NADH ratio in PRSS55-overexpressed HEK293T cells are shown as mean ± SE (n = 3). (*, P ≤ 0.05, **, P ≤ 0.01.)

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Cell Culture, Immunofluorescence, Staining, Plasmid Preparation, Control, Negative Control, Transfection, Western Blot, Marker, Isolation, Expressing

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: Data-Independent Acquisition (DIA)-based quantitative proteomic analysis of wt and Prss55 −/− testicular layer 3 (TL3) cells and sperm. A Immunofluorescence staining of PRSS55 at the luminal side of the seminiferous tubules (PRSS55: red; PNA: green; DAPI: blue). B Work-flow displays the strategy of PRSS55-enrichment cells for both wild-type (wt) and Prss55 −/− mice. C Flow cytometry analysis of DNA ploid types of wt and Prss55 −/− mice T, L1, L2, and L3 testicular cells. D Immunoblotting analysis of PRSS55 levels in wild-type T, L1, L2, and L3 testicular cells. β-Actin was used as a loading control. E, F Volcano plot of TL3 ( E ) and sperm ( F ) proteomics showing significant (fold-change > 1.5, adjusted P ≤ 0.05, blue = down, red = up) differentially expressed proteins (DEPs). G , H Heatmap representation of TL3 DEPs ( G ) and sperm DEPs ( H )

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Data-independent acquisition, Immunofluorescence, Staining, Flow Cytometry, Western Blot, Control

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: DEPs between wt and Prss55 −/− TL3 cells and sperm are enriched in metabolic pathways. Top 5 significantly enriched GO and KEGG terms were selected to show potential functions of DEPs in TL3 cells A and sperm B and presented as bubble plots. Bubble size represents the number of DEPs. Bubble color represents the adjusted P. The x-axis shows the Z-score of proteins classified into each functional annotation. C Venn diagram exhibits the DEPs common in TL3 cells (red) and sperm (green). D The enriched canonical pathways identified in IPA by 153 DEPs common in testicular TL3 cells and sperm are indicated on the y-axis. On the x-axis, the enrichment score (- log 10 (P)) for each pathway is indicated by the bars. Color of each bar reflects its activation z-score upon IPA algorithm

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Functional Assay, Activation Assay

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: Branched chain amino acids were accumulated in Prss55 −/− testes and sperm. A, B Heatmap representation ( A ) and orthogonal partial least squares discrimination analysis (OPLS-DA) score plot B of untargeted testicular metabolomic profiles between wt and Prss55 − / − . (wt, n = 6; Prss55 − / − , n = 8) C Volcano plot of untargeted testicular metabolomics showing significant abundant metabolites (fold-change > 1.2, FDR ≤ 0.1, green = down, red = up). D The enrichment of the untargeted Prss55 −/− differential metabolites in the KEGG pathways sorted by -log 10 (P). Color of each bar reflects its enrichment ratio. E, F Relative contents of BCAAs in testes ( E , n = 4) and sperm ( F , n = 3) detected by targeted metabolomic analysis. G, H Determination of BCAAs in testes ( G , wt = 3, Prss55 − /− = 4) and sperm ( H , wt = 3, Prss55 − /− = 5) by ELISA. (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.)

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: Interaction of PRSS55 with BCKDK and BCKDHA. A The V5-PRSS55-Flag fusion protein was immunoprecipitated from the testis lysates of 3 Prss55 KI/KI mice with an anti-V5 mAb magnetic beads and detected by anti-V5 antibody. Wt mice were used as negative controls. B The volcano plot shows 56 proteins identified with LC/MS in the V5-PRSS55 precipitates. BCKDK and DBT were pointed. C The PRSS55-EGFP fusion protein was immunoprecipitated from the HEK293T cell lysates. Endogenous BCKDK and DBT were probed with their specific antibodies, respectively. GAPDH was shown as a loading control. D, E Co-IP of PRSS55 ( D ) or BCKDK ( E ) tagged as indicated with anti-tag antibodies shows the existence of BCKDK or PRSS55 in the precipitates from the cell lysates of co-transfected HEK293T cells by immunoblot. F Immunoblotting analysis of endogenous BCKDHA protein levels upon overexpression of PRSS55 and BCKDK (left), and quantitative analysis of relative intensities of BCKDHA protein levels (right). G The PRSS55-EGFP fusion protein was immunoprecipitated from the HEK293T cell lysates. Endogenous BCKDHA was probed with specific antibody. H, I Co-IP of PRSS55 ( H ) or BCKDHA ( I ) tagged as indicated with anti-tag antibodies shows the existence of BCKDHA or PRSS55 in the precipitates from the cell lysates of co-transfected HEK293T cells by immunoblot. J Immunoblotting analysis of isolated mitochondria between wt and Prss55 −/− testes (left), and quantitative analysis of relative intensities of BCKDK, p-BCKDHA, and BCKDHA protein levels (right, n = 3). Bubble size and color represent the relative protein level. Tubulin was used as a marker for cytoplasm, Histone H2A for nuclei, and COXIV for mitochondria. (* P ≤ 0.05, ** P ≤ 0.01)

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: Immunoprecipitation, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Control, Co-Immunoprecipitation Assay, Transfection, Western Blot, Over Expression, Isolation, Marker

Journal: Cell & Bioscience

Article Title: PRSS55 regulates BCAA metabolism and interacts with BCKDK and BCKDHA in mouse testes and sperm

doi: 10.1186/s13578-025-01511-w

Figure Lengend Snippet: Schematic illustration of PRSS55 participating in BCAA metabolism and energy homeostasis

Article Snippet: PRSS55 antibody (1:500, MA43587) was from Aviva Systems Biology; BCKDK (1:1000, ab151297) antibody and BCKDHA (1:1000, ab126173) antibody were from Abcam.

Techniques: