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: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Defining the contribution of skeletal muscle pyruvate dehydrogenase α1 to exercise performance and insulin action

doi: 10.1152/ajpendo.00241.2018

Figure Lengend Snippet: Quantitative PCR primer sequences

Article Snippet: The following antibodies were used: PDHα (cat. no. 3205, Cell Signaling), eukaryotic translation elongation factor 2 (cat. no. 2332, Cell signaling), ATP synthase subunit alpha, ubiquinol-cytochrome C reductase core protein 2, mitochondrially encoded cytochrome C oxidase I, succinate dehydrogenase subunit B, NADH/ubiquinone oxidoreductase subunit B8 (cat. no. MS-604, MitoSciences), acyl-Coenzyme A dehydrogenase, very long-chain; cat. no. ab-155138, Abcam), acyl-Coenzyme A dehydrogenase, long-chain (cat. no. ab-82853, Abcam), hexokinase 2 (HK2; cat. no. 2857, Cell Signaling), lactate dehydrogenase A (cat. no. ABN-896, MilliporeSigma), Akt (cat. no. 2920, Cell Signaling), pAkt S473 (cat. no. 4058, Cell Signaling), GSK3α/β (5676, Cell signaling), pGSK3α/β S21/S9 (9331, Cell signaling), PDK4 (cat. no. NBP1-07047SS, Novus Biologicals), phosphofructokinase 1 (cat. no. sc-377346, Santa Cruz Biotechnology).

Techniques: Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Defining the contribution of skeletal muscle pyruvate dehydrogenase α1 to exercise performance and insulin action

doi: 10.1152/ajpendo.00241.2018

Figure Lengend Snippet: Metabolic and mitochondrial proteins in HFD-fed PDHmKO and WT mice. WT and PDHmKO mice were fed either a chow or a HFD for 12 wk. Transcript abundance of Pdha1, Pdk2, and Pdk4 (A) and Cd36, Cact (Slc25a20), and Acadl (B) in skeletal muscle from chow- or HFD-fed PDHmKO and WT mice, normalized to Ppib (Chow, n = 5/6; HFD, n = 6/7). Representative blot of PDHα, HK2, LDHA, ACADVL (C), and ATP5A, UQCRC2, MTCO1, SDHB, and NDUFB8 (D) protein abundance in skeletal muscle from chow- or HFD-fed PDHmKO and WT mice. Bar graphs show quantification of protein abundance in skeletal muscle relative to ponceau (Chow, n = 6/6; HFD, n = 6/6) (E). Data reported as means ± SE two-way ANOVA, #P < 0.05, main effect of diet, *P < 0.05, main effect of genotype (A–E). HFD, high-fat diet; PDHmKO, tamoxifen-inducible Pdha1 knockout mice; WT, wild-type.

Article Snippet: The following antibodies were used: PDHα (cat. no. 3205, Cell Signaling), eukaryotic translation elongation factor 2 (cat. no. 2332, Cell signaling), ATP synthase subunit alpha, ubiquinol-cytochrome C reductase core protein 2, mitochondrially encoded cytochrome C oxidase I, succinate dehydrogenase subunit B, NADH/ubiquinone oxidoreductase subunit B8 (cat. no. MS-604, MitoSciences), acyl-Coenzyme A dehydrogenase, very long-chain; cat. no. ab-155138, Abcam), acyl-Coenzyme A dehydrogenase, long-chain (cat. no. ab-82853, Abcam), hexokinase 2 (HK2; cat. no. 2857, Cell Signaling), lactate dehydrogenase A (cat. no. ABN-896, MilliporeSigma), Akt (cat. no. 2920, Cell Signaling), pAkt S473 (cat. no. 4058, Cell Signaling), GSK3α/β (5676, Cell signaling), pGSK3α/β S21/S9 (9331, Cell signaling), PDK4 (cat. no. NBP1-07047SS, Novus Biologicals), phosphofructokinase 1 (cat. no. sc-377346, Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Knock-Out

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Defining the contribution of skeletal muscle pyruvate dehydrogenase α1 to exercise performance and insulin action

doi: 10.1152/ajpendo.00241.2018

Figure Lengend Snippet: PDHmKO mice have reduced running speed during voluntary wheel running (VWR). Weekly average speed (km/h) (A), distance run per 24-h period (B), and time/24 h spent running (C) over 21 days VWR (n = 7/6). Representative blots of PDHα, HK2, PFK1, LDH, PDK4, ACADVL, ACADL, and NDUFB8 protein abundance in skeletal muscle from sedentary (SED) or exercise-trained (EXT) PDHmKO and WT mice (D). Bar graphs show quantification of protein abundance in skeletal muscle relative to ponceau (SED, n = 5/5; EXT, n = 7/6) (E). Data reported as means ± SE 2-way ANOVA, Sidak’s post hoc test, *P < 0.05, PDHmKO compared with WT (A–C); 2-way ANOVA, Tukey’s post hoc test, #P < 0.05, EXT compared with SED, *P < 0.05, PDHmKO compared with WT (D–E). PDHmKO, tamoxifen-inducible Pdha1 knockout mice; WT, wild-type.

Article Snippet: The following antibodies were used: PDHα (cat. no. 3205, Cell Signaling), eukaryotic translation elongation factor 2 (cat. no. 2332, Cell signaling), ATP synthase subunit alpha, ubiquinol-cytochrome C reductase core protein 2, mitochondrially encoded cytochrome C oxidase I, succinate dehydrogenase subunit B, NADH/ubiquinone oxidoreductase subunit B8 (cat. no. MS-604, MitoSciences), acyl-Coenzyme A dehydrogenase, very long-chain; cat. no. ab-155138, Abcam), acyl-Coenzyme A dehydrogenase, long-chain (cat. no. ab-82853, Abcam), hexokinase 2 (HK2; cat. no. 2857, Cell Signaling), lactate dehydrogenase A (cat. no. ABN-896, MilliporeSigma), Akt (cat. no. 2920, Cell Signaling), pAkt S473 (cat. no. 4058, Cell Signaling), GSK3α/β (5676, Cell signaling), pGSK3α/β S21/S9 (9331, Cell signaling), PDK4 (cat. no. NBP1-07047SS, Novus Biologicals), phosphofructokinase 1 (cat. no. sc-377346, Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Knock-Out

Journal: Nature Communications

Article Title: MAP4K3 mediates amino acid-dependent regulation of autophagy via phosphorylation of TFEB

doi: 10.1038/s41467-018-03340-7

Figure Lengend Snippet: Knockout of MAP4K3 promotes autophagy induction and flux. a Validation of MAP4K3 knockout (k.o.) cell lines. Wild-type (WT) and HEK293A cells gene-edited with either of two different sgRNAs (M1 and M4) were lysed, and protein lysates were immunoblotted for MAP4K3. Immunoblotting of β-actin served as a loading control. b , c Knockout of MAP4K3 promotes autophagy flux. WT HEK293A cells, M1-1 MAP4K3 k.o. cells, and M4-6 MAP4K3 k.o. cells (not shown) were cultured in complete media (CM) or subjected to amino acid starvation (– AA), and remained untreated or were treated with ammonium chloride. Protein lysates were immunoblotted for LC3 and β-actin, which served as a loading control b . The ratio of LC3-II:actin was determined by densitometry using ImageJ and normalized to WT CM, which was arbitrarily set to 1 c . One-way ANOVA with post-hoc Tukey’s test; * P < 0.05. d , e Knockout of MAP4K3 promotes autophagy induction. LC-3 immunostaining of WT HEK293A cells and three different MAP4K3 k.o. cell lines, all cultured in CM d . Quantification of LC3 puncta area per cell area was determined using ImageJ. n > 100 cells per genotype. One-way ANOVA with post-hoc Tukey’s test; ** P < 0.01, *** P < 0.001. f – h Knockout of MAP4K3 promotes autophagy flux. WT HEK293A cells and MAP4K3 k.o. cells were cultured in CM or amino acid starved, and were transfected with a GFP-mCherry-LC3 expression construct ( f ). Note the predominance of red puncta indicative of autolysosomes in MAP4K3 k.o. cells. Quantification of autophagosome number per cell was determined by counting yellow puncta GFP-mCherry-LC3-expressing cell ( g ). Quantification of autolysosome number per cell was determined by counting red puncta/GFP-mCherry-LC3-expressing cell ( h ). n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’ test; ** P < 0.01, *** P < 0.001. All experiments were performed in triplicate. Error bars = SEM. Scale bars = 10 μm

Article Snippet: Membranes were incubated overnight with primary antibodies against the following: LC3 (Novus Biologicals, NB100-2220) 1/3000; β-actin (Abcam, #ab8226) 1/10,000; Map4k3 (Cell Signaling, 9613) 1/1000; RagA (Cell Signaling, 4357) 1/1000; RagC (Cell Signaling, 5466) 1/1000; Lamtor1 (Cell Signaling, 8975) 1/1000; TFEB (Cell Signaling, 4240) 1/1000; pan 14-3-3 (Santa Cruz Biotechnology, sc-629) 1/1000; FLAG (M2) (Sigma; F1804); Raptor (24C12) (Cell Signaling, 2280) 1/1000; phospho-(Ser) 14-3-3 Binding Motif (Cell Signaling, 9601) (for detection of TFEB phosphorylation at Ser211) 1/1000; GST (Santa Cruz Biotechnology, sc-138) 1/1000, in 5% bovine serum albumin in PBS-T. Species-specific secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) or goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), diluted 1/10,000 in 5% PBS-T milk and incubated for 1 h at RT.

Techniques: Knock-Out, Biomarker Discovery, Western Blot, Control, Cell Culture, Immunostaining, Transfection, Expressing, Construct

Journal: Nature Communications

Article Title: MAP4K3 mediates amino acid-dependent regulation of autophagy via phosphorylation of TFEB

doi: 10.1038/s41467-018-03340-7

Figure Lengend Snippet: Phosphorylation of TFEB at serine 3 is a key determinant of TFEB cellular regulation and autophagy function. a TFEB serine 3 phosphorylation is required for mTORC1 phosphorylation at serine 211. WT HEK293A and MAP4K3 k.o. cells were transfected with TFEB-FLAG, TFEB-S3A-FLAG, or TFEB-S3E-FLAG, and Torin1 treated. FLAG immunoprecipitates were immunoblotted and TFEB serine 211 phosphorylation as a fraction of total TFEB was quantified by densitometry. b MAP4K3 phosphorylation of TFEB is required for mTORC1 phosphorylation at serine 211. WT HEK293A and MAP4K3 k.o. cells were transfected with TFEB-FLAG and either no MAP4K3 (--), WT-MAP4K3, or KD-MAP4K3. FLAG immunoprecipitates were immunoblotted, and TFEB serine 211 phosphorylation as a fraction of total TFEB was quantified by densitometry. c TFEB serine 3 phosphorylation is required for interaction with 14-3-3. TFEB k.o. cells were transfected with no TFEB (--), inducible TFEB-WT-FLAG, or inducible TFEB-S3A-FLAG, and Torin1 treated, whereas all cells received doxycycline to induce TFEB-WT or TFEB-S3A expression. Cell lysates and TFEB immunoprecipitates were immunoblotted and immunoprecipitated 14-3-3 was quantified by densitometry. One-way ANOVA with post-hoc Tukey’s test; * P < 0.05, ** P < 0.01. d TFEB serine 3 phosphorylation regulates TFEB nuclear localization. TFEB k.o. cells were transfected with inducible TFEB-WT-FLAG or TFEB-S3A-FLAG, and cultured in CM or amino-acid starved (– AA). Under amino acid deprivation, TFEB localizes to nucleus, regardless of serine 3 status; however, upon amino acid satiety, mutation of TFEB serine 3 to phospho-resistant alanine prevents retention of TFEB in the cytosol. Quantification of TFEB nuclear localization to right. n > 100 cells per condition. One-way ANOVA with post-hoc Tukey’s test; *** P < 0.001. e TFEB serine 3 phosphorylation regulates autophagy activation. TFEB k.o. cells were transfected with GFP-mCherry-LC3 and either inducible TFEB-WT-FLAG or TFEB-S3A-FLAG, and cultured in CM and doxycycline, as indicated. Note the red puncta indicative of autolysosomes in cells expressing TFEB-S3A. Autophagosome number per cell was determined by counting yellow puncta per GFP-mCherry-LC3-expressing cell, and autolysosome number per cell was determined by counting red puncta per GFP-mCherry-LC3-expressing cell. n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’s test; * P 0 < 0.05, ** P < 0.01. All experiments performed in triplicate. Error bars = SEM. Scale bars = 20 μm

Article Snippet: Membranes were incubated overnight with primary antibodies against the following: LC3 (Novus Biologicals, NB100-2220) 1/3000; β-actin (Abcam, #ab8226) 1/10,000; Map4k3 (Cell Signaling, 9613) 1/1000; RagA (Cell Signaling, 4357) 1/1000; RagC (Cell Signaling, 5466) 1/1000; Lamtor1 (Cell Signaling, 8975) 1/1000; TFEB (Cell Signaling, 4240) 1/1000; pan 14-3-3 (Santa Cruz Biotechnology, sc-629) 1/1000; FLAG (M2) (Sigma; F1804); Raptor (24C12) (Cell Signaling, 2280) 1/1000; phospho-(Ser) 14-3-3 Binding Motif (Cell Signaling, 9601) (for detection of TFEB phosphorylation at Ser211) 1/1000; GST (Santa Cruz Biotechnology, sc-138) 1/1000, in 5% bovine serum albumin in PBS-T. Species-specific secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) or goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), diluted 1/10,000 in 5% PBS-T milk and incubated for 1 h at RT.

Techniques: Phospho-proteomics, Transfection, Expressing, Immunoprecipitation, Cell Culture, Mutagenesis, Activation Assay

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ATDC5 cells adhere more extensively to fibronectin, collagen I, and collagen IV. ATDC5 cells were screened with extracellular matrix array printed with collagen I (COL I), collagen III (COL III), collagen IV (COL IV), collagen V (COL V), collagen VI (COL VI), fibronectin (FN), vitronectin (VTN), laminin (LMN), tropoelastin (TE), and BSA as a negative control. (A) Representative bright-field images of ATDC5 cells incubated for 30 h indicated differential binding of a number of extracellular proteins. Scale bar: 40 μm. (B) Attached cell counts determined for each of the nine replicates, as well as mean and standard deviation are shown ( n = 9).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Negative Control, Incubation, Binding Assay, Standard Deviation

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Fibronectin interaction with graphene is stabilized by arginine residues. (A) Graphical rendering of the stabilized fibronectin atop the three graphene sheets with the four best arginine binders highlighted (Arg1166, Arg1369, Arg1374, Arg1403). The time evolution of the binding energy of these arginine residues with graphene is shown in the lower panel, color-coded for the amino acid residues. (B) Analogous to A but showing the data for the second studied configuration. This configuration features five arginine residue binders (Arg1166, Arg1351, Arg1379, Arg1445, Arg1493). (C) Binding energy with graphene computed for every amino acid with average binding energy above 1 kcal/mol, averaged over the 400 ns simulation. (D) Analogous to C, for the second studied configuration. The residue numbers are indicated, while the corresponding amino acid types are color-coded for both panels (C and D). (E and F) Time evolution of the fibronectin and arginine interaction energy with graphene for the two configurations. The lower plots in both panels show the fraction of arginine residue binding energy with respect to the total fibronectin-binding energy as a function of simulation time.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Binding Assay, Residue

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Mechanical properties. The measured quasi-static (A and B) and dynamic (C – E) properties of GF (hatched bars), GF coated in fibronectin (dark blue bars), and GF coated in fibronectin and cultured with ATDC5 cells (light blue bars) for 28 days. Fibronectin changed the elasticity of the composite (i.e., modulus values), but did not increase the viscoelastic properties (stress relaxation and phase shift).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Cell Culture

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Actin cytoskeleton of cells on GF and fibronectin-coated GF. Fluorescence of ATDC5 cells grown on glass-bottom tissue culture wells compared to GF, with or without fibronectin. Cell nuclei are stained blue (DAPI); Green, F-actin (Alexa Fluor 488 phalloidin); (A–D) ATDC5 cells were grown on glass-bottom tissue culture wells without (A and E) and with fibronectin (B and F); ATDC5 cells were grown on GF without (C and G) and with fibronectin (D and H). Note the prevalence of stress fibers and the absence of puncta in F and H compared to E and G, respectively. Additionally, note the relative abundance of puncta of actin which are more prevalent in the absence of fibronectin on glass-bottomed tissue culture wells as well as on GF. (A–D) Scale-bar: 50 μm. (E–H) Scale-bar: 10 μm.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Fluorescence, Staining

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ActB and Hsp90ab1 housekeeping genes. ActB and Hsp90ab1 are stably expressed by ATDC5 cells under all experimental conditions used in this study (i.e., on glass-bottom tissue culture wells, GF, and fibronectin-GF). (A) ActB and Hsp90ab1 cycle threshold levels were most consistent among all samples analyzed by qRT-PCR for candidate HKGs considered, based on pairwise analysis of variance for differences between threshold values, variance equal to 0.12. (B) Correlation analysis of cycle threshold values for Hsp90ab1 and ActB indicate a slope and an R 2 value close to 1. ( n = 15).

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Stable Transfection, Quantitative RT-PCR

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: GF supports or enhances gene expression levels. The effect of fibronectin, GF, and fibronectin in combination with GF on ATDC5 cell gene expression was investigated. Correlation analysis of relative expression levels was carried out to detect differential gene expression as a function of the cell culture substrate. The mRNA levels were compared for cells seeded on four distinct surfaces. Data points above the diagonal line indicate genes that are upregulated and data points below the diagonal line indicate genes that are downregulated. Data points falling on the diagonal line are not differentially expressed in experimental compared to control conditions. The effect of GF on gene expression is demonstrated in panels A and B. The effect of fibronectin on gene expression is demonstrated in panels C and D. (A) Relative gene expression levels in 2D cell culture conditions compared to cells grown in 3D on GF in the absence of fibronectin. (B) Relative gene expression levels in 2D cell culture conditions compared to cells grown in 3D on GF in the presence of fibronectin. (C) Relative gene expression levels in 2D cell culture conditions comparing the presence and absence of fibronectin. (D) Relative gene expression levels by cells grown in 3D on GF comparing the presence and absence of fibronectin. Genes for which expression levels met or exceeded the control are indicated in magenta, while those genes that were supported by substrate conditions are indicated by turquoise. Col2a1, a marker for chondrocyte differentiation, is shown as a diamond shape and bolded in each frame. Col2a1 is found above the diagonal line in A and B indicating upregulation as a function of 3D GF culture, and below the line in C and D, indicating downregulation as a function of fibronectin in either 2D or 3D culture. Genes included in this analysis are listed in Tables – .

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Gene Expression, Expressing, Cell Culture, Control, Marker

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding mediators of cell attachment by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Ctnnal (triangle) and Ctnnb1 (circle). (B) Relative gene expression levels of Ctnnal (gray) and Ctnnb1 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Cd44 (triangle), Ncam1 (circle), and Sgce (square). (D) Relative gene expression levels of Cd44 (gray), Ncam1 (black), and Sgce (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Itga3 (triangle), Itga5 (circle), and Itgav (square). (F) Relative gene expression levels of Itga3 (gray), Itga5 (black ) , and Itgav (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Itgb1 . (H) Relative gene expression levels of Itgb1 at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD. These genes are listed in Table with references from current literature indicating an association with chondrocyte differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Cell Attachment Assay, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding extracellular matrix proteins by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Col1a1 (circle) and Col3a1 (triangle). (B) Relative gene expression levels of Col1a1 (gray) and Col3a1 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Col2a1 (circle), Col5a1 (triangle), and Col6a1 (square). (D) Relative gene expression levels of Col2a1 (gray), Col5a1 (black), and Col6a1 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Ecm1 (circle), Emilin1 (triangle), and Tnc (square). (F) Relative gene expression levels of Ecm1 (gray), Emilin1 (black), and Tnc (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Fn (circle), Sparc (triangle), and Spp1 (square). (H) Relative gene expression levels of Fn (gray), Sparc (black), and Spp1 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (I) Time course of gene expression during chondrogenic differentiation for Thbs1 (circle), Thbs2 (triangle), and Postn (square). (J) Relative gene expression levels of Thbs1 (black), Thbs2 (white), and Postn (gray) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (K) Time course of gene expression during chondrogenic differentiation for Hapln1 (circle) and Lamb3 (triangle). (L) Relative gene expression levels of Hapln1 (gray) and Lamb3 (black) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD Table lists extracellular matrix genes with description, function, and literature citations that corroborate an upregulation during early chondrogenic differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: Expression of genes encoding matrix remodeling proteins and their endogenous inhibitors by ATDC5 cells on glass-bottom tissue culture wells, GF, and fibronectin-GF. (A) Time course of gene expression during chondrogenic differentiation for Adamts1 (circle) and Adamts2 (triangle). (B) Relative gene expression levels of Adamts1 (gray) and Adamts2 (black) at day 17 of chondrogenic differentiation in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (C) Time course of gene expression during chondrogenic differentiation for Mmp2 (triangle) and Mmp14 (circle). (D) Relative gene expression levels of Mmp2 (black) and Mmp14 (gray) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (E) Time course of gene expression during chondrogenic differentiation for Timp1 (circle), Timp2 (triangle), and Timp3 (square). (F) Relative gene expression levels of Timp1 (gray), Timp2 (black), and Timp3 (white) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. (G) Time course of gene expression during chondrogenic differentiation for Ctgf (circle) and Tgfbi (triangle). (H) Relative gene expression levels of Ctgf (gray) and Tgfbi (black) at day 17 in control 2D culture, 2D culture in the presence of fibronectin, 3D-GF, and 3D-GF coated with fibronectin. Error bars = Mean ± SD Table lists matrix remodeling genes analyzed in this study with descriptions and literature citations that have demonstrated a link between increases in gene expression and chondrogenic differentiation.

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Expressing, Gene Expression, Control

Journal: ACS Applied Materials & Interfaces

Article Title: Prechondrogenic ATDC5 Cell Attachment and Differentiation on Graphene Foam; Modulation by Surface Functionalization with Fibronectin

doi: 10.1021/acsami.9b14670

Figure Lengend Snippet: ECM Genes Expressed during Chondroprogenitor Cell Differentiation on GF

Article Snippet: Bovine fibronectin protein solution was obtained from R & D Systems (Biotechne Corporation, Minneapolis, MN, U.S.A.) and diluted to a concentration of 100 μg/mL in Ca 2+ - and Mg 2+ -free phosphate-buffered saline (PBS).

Techniques: Cell Differentiation, Binding Assay, Activity Assay, Membrane

Journal: Analytical chemistry

Article Title: Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry

doi: 10.1021/acs.analchem.2c02709

Figure Lengend Snippet: Workflows showing absolute quantitation for a) multiple proteins in a protein mixture, b) HCPs in mAb , and c) mAb deamidation by CMS.

Article Snippet: Host cell proteins (HCPs) are a major class of impurities derived from biotherapeutics production processes.

Techniques: Quantitation Assay

Journal: bioRxiv

Article Title: PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

doi: 10.1101/2020.09.09.289009

Figure Lengend Snippet: (A) A protein interaction network of SPG11 illustrating novel protein interactors identified by mass spectrometry in this study (orange edges), literature-derived protein interactors from PINOT and BioPlex 3.0 (blue edges), and an interactor common to both our novel dataset and literature-derived data (green edge). The edge thickness positively correlates with protein interaction confidence based on distinct method detection strategies used and the number of specific documented reports. (B) The subcellular localisation of the interactors resulted from our AP/MS analysis and of SPG11 is visualised, with each colour corresponding to a different location block. The collection of subcellular location data in the form of Gene Ontology cellular component terms was performed through AmiGO and the grouping of each term into location blocks with an in-house grouping protocol.

Article Snippet: The sequence of human SPG11 was cloned into a pReceiver-M12 Expression Clone vector (GeneCopoeia).

Techniques: Mass Spectrometry, Derivative Assay, Protein-Protein interactions, Blocking Assay

Journal: bioRxiv

Article Title: PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

doi: 10.1101/2020.09.09.289009

Figure Lengend Snippet: (A) 3xFlag-SPG11 overexpressed in HEK293T cells pulls down endogenous 14-3-3 proteins. (B) Immunocytochemical evaluation of 3xFlag-SPG11 subcellular compartmentalisation in HeLa cells reveals predominant perinuclear localisation, partially overlapping with the ER marker calnexin, and sporadic enrichment at the cell periphery in protrusions-like structures. (C) Co-staining of 3xFlag-SPG11 and 14-3-3s suggests that SPG11 only overlaps with a subset of 14-3-3s, as confirmed via pull-down assays between 3xFlag-SPG11 and recombinant 14-3-3 isoforms purified by IMAC (D). Scale bars=20μm.

Article Snippet: The sequence of human SPG11 was cloned into a pReceiver-M12 Expression Clone vector (GeneCopoeia).

Techniques: Marker, Staining, Recombinant, Purification

Journal: bioRxiv

Article Title: PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

doi: 10.1101/2020.09.09.289009

Figure Lengend Snippet: (A) Schematic of the putative 14-3-3 binding sites predicted by 14-3-3-Pred. Ser1955, highlighted in light blue, was also reported to be a phosphorylated residue by PhosphoSitePlus ® , and confirmed by phospho-peptide enrichment experiments performed in this work. (B) Binding affinity of 14-3-3s for phosphorylated SPG11-Ser1955 was confirmed through generation of phospho-deficient S1955A-mutant by site-directed mutagenesis. The non-phosphorylatable mutant displays ~ 50% reduction in binding as compared to the WT counterpart.

Article Snippet: The sequence of human SPG11 was cloned into a pReceiver-M12 Expression Clone vector (GeneCopoeia).

Techniques: Binding Assay, Residue, Mutagenesis

Journal: bioRxiv

Article Title: PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

doi: 10.1101/2020.09.09.289009

Figure Lengend Snippet: (A) Summary of the putative kinases phosphorylating SPG11 on Ser1955, with relative confidence score, as obtained from NetPhos 3.1. The PKA consensus sequence is also indicated and aligned against the SPG11 motif containing Ser1955. (B) Endogenous SPG11 was pulled down from genome-edited monoclonal lines taking advantage of the 3xFlag tag sequence inserted at the endogenous locus. 15 minutes treatment with Forskolin/IBMX increases binding affinity for η-14-3-3, suggesting that phosphorylation by PKA on Ser1955 is the signal regulating interaction (n=2). (C) PLA experiments confirmed (i) interaction between SPG11 and 14-3-3s at the endogenous level and (ii) increased binding in the presence of PKA activation. (PLA reactions were performed between rabbit α-Flag and mouse α-pan-14-3-3 antibodies. Average background signal detected in naïve cells, employed as a negative control for the reaction, was subtracted to all measurements; n=2, ~125 cells counted/condition).

Article Snippet: The sequence of human SPG11 was cloned into a pReceiver-M12 Expression Clone vector (GeneCopoeia).

Techniques: Sequencing, Binding Assay, Phospho-proteomics, Activation Assay, Negative Control

Journal: bioRxiv

Article Title: PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

doi: 10.1101/2020.09.09.289009

Figure Lengend Snippet: (A) Cell surface biotinylation assay showing endogenous SPG11 internalisation upon 15 minutes treatment with Forskolin, suggesting that PKA activation is the stimulus to initiate SPG11 trafficking towards intracellular compartments. (B) Representative images of HeLa cells transfected with WT or S1955A-SPG11 and treated with Forskolin/IBMX for 15 and 90 minutes show that only the WT protein is able to respond to PKA activation, suggesting that the mechanism is mediated by 14-3-3 binding. White arrowheads indicate SPG11 enrichment at protrusion-like structures, which is reduced after 15 minutes treatment with Forskolin/IBMX. Scale bar=20μm.

Article Snippet: The sequence of human SPG11 was cloned into a pReceiver-M12 Expression Clone vector (GeneCopoeia).

Techniques: Cell Surface Biotinylation Assay, Activation Assay, Transfection, Binding Assay

Journal: bioRxiv

Article Title: PKA-mediated phosphorylation of SPG11/spatacsin regulates binding with a subset of 14-3-3 proteins

doi: 10.1101/2020.09.09.289009

Figure Lengend Snippet: Canonical motifs for interactions with binding partners and transmembrane domains that have been predicted or identified by other groups ( ; ) and confirmed by in silico data from this study are summarised. The AP5-interacting region and the spatacsin C domain, which shares sequence and structure similarity with the Vps16 protein, identified in the study by Patto and O’Kane are also included, together with the phospho-peptide responsible for the binding of 14-3-3s identified in this study.

Article Snippet: The sequence of human SPG11 was cloned into a pReceiver-M12 Expression Clone vector (GeneCopoeia).

Techniques: Binding Assay, In Silico, Sequencing

Journal: Nutrients

Article Title: Vitamin C: A Novel Regulator of Neutrophil Extracellular Trap Formation

doi: 10.3390/nu5083131

Figure Lengend Snippet: Autophagy signaling is induced in Vitamin C deficient neutrophils. ( A ) Real time QPCR for ATG3, ATG5, ATG6, ATG7, and ATG8 mRNA from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice, ( N = 6 for each group, * p < 0.05). ( B ) Representative Western blot for expression of LC3B-I and LC3B-II from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice. Densitometry of LC3B-II/actin from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice ( N = 6 for each group, * p < 0.05). ( C ) Representative Western blot for expression of p62 and actin from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice. Densitometry of normalized p62 expression from peritoneal PMNs of VitC sufficient and deficient Gulo −/− mice ( N = 6 for each group, ns p = 0.3).

Article Snippet: Purified rabbit polyclonal antibodies to LC3B (L7543, Sigma-Aldrich), cleaved caspase-3 (#9661, Cell Signaling), caspase-3 (#9662, Cell Signaling), p62/SQSTM1 (NBP1-48320, Novus Biologicals), NFκB p65 (sc-109, Santa Cruz Biotechnology), Lamin B (sc-6216, Santa Cruz Biotechnology), and actin (sc-1616, Santa Cruz Biotechnology) were used in this study.

Techniques: Western Blot, Expressing