Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: A) UMAP of scDNA and protein-seq data for pooled MNCs isolated from BM samples obtained from a CMML patient with pre-existing KRAS T58K and NRAS G12R mutations at diagnosis (n=1,826) and at BP after HMA therapy failure (n=4,001). BP was not associated with the clonal evolution of these mutations as they both had a VAF of approximately 50% at the onset of the disease. Each dot represents one cell. Cells are clustered based on immunophenotypic markers. Different colors represent cluster identity (left) or origin (right). Mono, monocytes; Ery, erythroblasts; cDC, classical dendritic cells; CD4T, CD4 + T-cells; MyHPC, myeloid hematopoietic progenitor cells; CD8T, CD8 + T-cells; NKC, natural killer cells. (B) Heatmap displaying DNA and protein reads from each sequenced cell type shown in . Colors for protein data correspond to antibody-oligonucleotide intensity signals. High protein expression is depicted in red and low protein expression is depicted in blue. DNA colors correspond to the genotypes for each individual mutation per cell read (wild-type=dark grey, mutant=red, missing=light grey) based on cluster. Percentages correspond to the frequencies of mutant reads within each cluster for a given mutation. C) UMAP of scDNA and protein-seq data for pooled MNCs isolated from BM samples obtained from a CMML patient at diagnosis (n=3,213) and at BP after HMA therapy failure (n=5,342). BP was associated with the clonal evolution of a pre-existing CBL F378Ifs mutation and the acquisition of a previously undetected CBL C384Y mutation. Each dot represents one cell. Cells are clustered based on immunophenotypic markers. Different colors represent cluster identity (left) or origin (right). Mono, monocytes; Ery, erythroblasts; DC, classical dendritic cells; CD4T, CD4 + T-cells; B-cell, B lymphocytes, myeloid hematopoietic progenitor cells; CD8T, CD8 + T-cells; NKC, natural killer cells. (D) Heatmap displaying DNA and protein reads from each sequenced cell type as shown in . Colors for protein data correspond to antibody-oligonucleotide intensity signals. Red indicates high protein expression, and blue indicates low protein expression. Colors for DNA data correspond to the genotype for each individual mutation per cell read (dark grey, wild type; red, mutant; light grey, missing) based on cluster. Percentages correspond to the frequencies of mutant reads within each cluster for a given mutation.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single Lin − CD34 + cells isolated from BM samples of 2 HDs (n=895) and 5 CMML patients (n=3,161). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; eMyHPC, early myeloid progenitor cells; dMyHPC, differentiated myeloid progenitors; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of HD (top) and CMML (bottom) Lin − CD34 + cell types among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in HSCs (left), eMyHPCs (middle), and dMyHPCs (right) from CMML samples compared with those from HD samples (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples of 3 HDs (n=9,896) and 5 CMML patients (n=9,319). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; MKP, megakaryocyte precursors; MyHPC, myeloid hematopoietic progenitor cells; Mono, monocytes; cDC, classical dendritic cells; pDC, plasmacytoid dendritic cells; Prog B, progenitor B-cells; PC, plasma cells; Pre-Ery, pre-erythrocytes; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; nCD4T, naïve CD4 + T cells; nmCD4T, naïve and memory CD4 + T cells; mCD4T, memory CD4 + T cells; nCD8T, naïve CD8 + T cells; nmCD8T, naïve and memory CD8 + T cells; eCD8T, effector CD8 + T cells; NKC, natural killer cells. Dashed lines indicate single clusters in each cell type population. (E) Distribution of HD (top) and CMML (bottom) cell types among the clusters shown in . (F) Pathway enrichment analysis of the genes that were significantly upregulated in the CMML monocyte clusters compared with those in the HD monocyte clusters shown in (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Clinical Proteomics

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single Lin − CD34 + cells isolated from BM samples of 5 CMML patients at diagnosis (n=1,840) and at BP after HMA therapy failure (n=1,711). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; eMyHPC, early myeloid hematopoietic progenitor cells; dMyHPC, differentiated myeloid hematopoietic progenitor cells; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of Lin − CD34 + cell types at diagnosis (top) and BP (bottom) among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in HSCs (left) and dMyHPCs (right) at the time of BP after HMA therapy failure compared with those at diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) UMAP of scATAC-seq data for pooled Lin − CD34 + cells isolated from BM samples obtained from a CMML patient at diagnosis (n=2,027) and at BP after HMA therapy failure (n=2,895). Each dot represents one cell. Different colors represent the cluster identity (left) or sample of origin (right). HSC, hematopoietic stem cells; MyHPC, myeloid progenitor cells; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. (E) Pathway enrichment analysis of genes whose distal elements were enriched in open chromatin regions in HSCs (cluster 1, shown in ) at the time of BP as compared with those at diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples of 6 CMML patients at diagnosis (n=16,372) and at BP after HMA therapy failure (n=19,541). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample of origin (right). MyHPC, myeloid hematopoietic progenitors; My/MoP, myelo/monocytic progenitors; Mono, monocytes; cDC, classical dendritic cells; pDC, plasmacytoid dendritic cells; MKP, megakaryocyte precursors; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; B-cell, B lymphocytes; PC, plasma cells; nCD4T, naïve CD4 + T cells; mCD4T, memory CD4 + T-cells; eCD8T, effector CD8 T-cells, NKC, natural killer cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of MNC types at diagnosis (top) and (bottom) among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in the monocytic populations shown in the time of BP after HMA therapy failure compared with those at the time of diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) Numbers of live Lin − CD34 + CD38 − HSCs and Lin − CD34 + CD38 + MyHPCs from CMML patients with BP after treatment with vehicle or 20 nM AMG-176 (n=4) for 48 h. Lines represent means ± SDs. Statistical significance was calculated using a two-tailed Student’s t- test (*** P <0.001; **** P <0.0001). (E) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples obtained from a representative CMML patient at the time of BP after HMA therapy failure (n=6,209) and after the failure of venetoclax-based therapy (n=6,795). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or the sample of origin (right). HSC, hematopoietic stem cells; MyHPC, myeloid hematopoietic progenitor cells; My/MoP, myelo/monocytic progenitors; Mono, monocytes; Ery/MkHPC, erythroid/megakaryocytic hematopoietic progenitor cells; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; Pre-E, pre-erythrocytes; mCD8T, memory CD8 + T cells; eCD8T, effector CD8 + T cells; NKC, natural killer cells. (F) Pathway enrichment analysis of the genes that were significantly upregulated in MyHPCs at the time of venetoclax failure compared with those at the time of HMA therapy failure (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (G) Distribution of myeloid cell types among the myeloid compartments at HMA therapy (top) and venetoclax-based therapy (bottom) failure. (H) Proposed working model of RAS pathway–mutated CMML initiation and progression after HMA and venetoclax-based therapies. Compared with physiological adult hematopoiesis (top left), RAS pathway–mutated CMML HSPCs undergo proliferation and monocytic differentiation in response to inflammatory responses while maintaining an intact apoptotic program. Inflammatory reprograming is exacerbated in downstream monocytic populations, which contributes to disease maintenance (bottom left). At BP after HMA therapy failure, RAS pathway–mutated CMML HSCs undergo epigenetic reprogramming and drive the expansion of downstream MyHPCs. MyHPCs and downstream monocytes rely on NF- K B signaling–mediated anti-apoptotic pathways to maintain survival and suppress the immune microenvironment (bottom right). NF- K B signaling–mediated survival pathway activation persists after venetoclax therapy and leads to treatment resistance and failure (top right).

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery, Clinical Proteomics, Two Tailed Test, Activation Assay

Journal: Nature communications

Article Title: Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

doi: 10.1038/s41467-020-17402-2

Figure Lengend Snippet: Fig. 1 LPS increases glycolysis as well as lactate production by BM neutrophils. a Flow cytometry quantitative analysis of 2-NBDG-glucose uptake by BM neutrophils (CD11bhighLy6Ghigh cells; n = 6) 4 h following i.p. administration of LPS in vivo in wild-type (WT) mice. b Gene expression of glycolytic enzymes in sorted BM neutrophils from WT mice following LPS treatment (n = 3, PBS; n = 5, LPS). On each box, the bottom, middle, and the top edges indicate the 25th, 50th, and 75th percentiles, respectively. The whiskers extend to the most extreme data points. c Quantitative analysis and representative histogram plot showing mean fluorescent intensity (MFI) of ROS production in BM neutrophils following LPS administration (n = 9). d Percentage of HIF-1α+ neutrophils in the BM following LPS administration (n = 11). e Quantitative analysis and representative histogram plot of LDHA expression in BM neutrophils following LPS treatment (n = 7). ***p(0.0003). f BM lactate levels in WT mice treated with PBS, LPS, or LPS followed by α-Ly6G Ab (n = 7). g Lactate levels released from isolated BM neutrophils treated ex vivo with PBS or LPS (120 ng/ml; n = 4 mice).**p(0.0063). h MCT4, MCT1, and GPR81 (yellow) distribution on BM CD11b+ (green)/Ly6G+ (red) neutrophils visualized and quantified by ImageStream analysis. Images are from one representative experiment out of three. Scale bar indicates 7 μm. i Quantitative analysis of MCT4 expression on BM neutrophils 4 h following LPS administration (n = 7). j A scheme of the proposed mode of action of LPS in lactate production by BM neutrophils. Data are represented as mean ± SEM from 3 to 4 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, Student’s two-tailed unpaired t test (a, c–e, g, i), one-way ANOVA with Tukey’s post hoc test (f, h) or two-way ANOVA with Tukey’s post hoc test (b). See also Supplementary Fig. 1.

Article Snippet: Flushed BM cells (from WT and mutated mice) were resuspended in 200 μl MACS buffer (2 mM EDTA, 0.5% bovine serum albumin in PBS) and BM neutrophils were isolated according to the protocol of “Neutrophil isolation kit, mouse” by Miltenyi.

Techniques: Flow Cytometry, In Vivo, Gene Expression, Expressing, Isolation, Ex Vivo, Two Tailed Test

Journal: Nature communications

Article Title: Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

doi: 10.1038/s41467-020-17402-2

Figure Lengend Snippet: Fig. 3 Lactate production by neutrophils requires NOX/ROS signaling. a Percentage of BM HIF-1α+ neutrophils in WT (n = 9) and gp91phox−/−(n = 7) mice treated with either PBS or LPS. **p(WT + LPS vs. gp91phox−/−+LPS) = 0.0012. b Quantitative analysis and representative histogram plot showing LDHA expression in BM neutrophils from WT (n = 5) and gp91phox−/−(n = 4) mice. **p(WT + PBS vs. WT + LPS) = 0.0054; *p(WT + LPS vs. gp91phox−/−

Article Snippet: Flushed BM cells (from WT and mutated mice) were resuspended in 200 μl MACS buffer (2 mM EDTA, 0.5% bovine serum albumin in PBS) and BM neutrophils were isolated according to the protocol of “Neutrophil isolation kit, mouse” by Miltenyi.

Techniques: Expressing

Journal: Nature communications

Article Title: Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

doi: 10.1038/s41467-020-17402-2

Figure Lengend Snippet: Fig. 7 Salmonella increases lactate production by inflammatory BM neutrophils. a, b Lactate levels in (a) BM or (b) blood from WT mice treated with PBS or Salmonella (n = 5). a ***p(0.0004); b **p(0.0081). c Quantitative analysis of ROS production in BM neutrophils from WT (n = 7) vs. gp91phox−/−

Article Snippet: Flushed BM cells (from WT and mutated mice) were resuspended in 200 μl MACS buffer (2 mM EDTA, 0.5% bovine serum albumin in PBS) and BM neutrophils were isolated according to the protocol of “Neutrophil isolation kit, mouse” by Miltenyi.

Techniques:

Journal: Nature communications

Article Title: Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

doi: 10.1038/s41467-020-17402-2

Figure Lengend Snippet: Fig. 8 Mechanisms of lactate-induced neutrophil mobilization from the BM. Our model suggests that enhanced lactate-produced by BM neutrophils during bacterial infection induces neutrophil mobilization by modulating metabolic signaling in BM endothelial cells. LPS binds TLR4 expressed on neutrophils that directly activates NADPH oxidase (NOX) and enhances glucose uptake via a glucose transporter 1a. Glucose in turn, is converted to pyruvate by the glycolysis pathway 1b. NOX activity leads to ROS production (2) which elevates HIF-1α expression. HIF-1α in turn, induces downstream expression of LDHA that converts pyruvate to lactate 3a. HIF-1α also up-regulates the lactate transporter MCT4 3b to allow lactate release (4). Under steady-state conditions, surface VE–cadherin on endothelial cells (ECs) is highly expressed, which maintains the endothelial barrier integrity with low permeability (a). During inflammation (b), lactate released from BM neutrophils binds to GPR81 on sinusoidal BM endothelial cells 5a, activates Gi protein and thereby reduces cAMP/Epac1 activity 5b. Consequently, lactate via GPR81 signaling decreases surface VE–cadherin expression (5c), leading to higher BM vascular permeability (6). In addition, lactate elevates CXCL1 and G-CSF levels (produced by different cells including ECs50,52) also in a GPR81- independent manner 7a with more moderate increases in CXCL2 levels in WT mice (most probably produced by BM neutrophils50). Lactate-induced elevation of CXCL1, CXCL2, and G-CSF downregulates surface CXCR2 expression on PB neutrophils 7b facilitating neutrophil mobilization (8). Taken together, LPS-induced lactate promotes rapid neutrophil mobilization from the BM to the blood (8) preferentially via BM GPR81/VE–cadherin-dependent (5–6) and also by GPR81-independent (7) pathways.

Article Snippet: Flushed BM cells (from WT and mutated mice) were resuspended in 200 μl MACS buffer (2 mM EDTA, 0.5% bovine serum albumin in PBS) and BM neutrophils were isolated according to the protocol of “Neutrophil isolation kit, mouse” by Miltenyi.

Techniques: Produced, Infection, Activity Assay, Expressing, Permeability

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