EMBO/soda-nel huggingface dataset:
IKKbeta is required for the formation of the NLRP3 inflammasome (2021)
Figure 6

Figure 6. IKKβ induces the formation of brighter TGN38-positive puncta after co-stimulation with LPS and nigericin.

(A-H) The experiments were performed as in Figs 5A-5H, except that the cells were fixed and prepared for immunofluorescence staining using a rabbit polyclonal TGN38 antibody, which was visualized using a secondary antibody (red). Nuclei were counterstained with DAPI (blue). Images were acquired by sequential laser scanning on a confocal microscope. Similar results were observed in many fields and obtained in three independent experiments. Representative images are shown

(I-L) As in A-H, except that iBMDM from IKKβ-CXCR3-Cre (flox/flox) (IKKβ (fl/fl) or WT mice were co-stimulated for 1 h with 100 ng/ml LPS and 10 μM nigericin or left unstimulated. Similar results were obtained in two independent experiments.

Data information: In all panels, scale bar = 50 μm.

Extended view figure legends
Acetylation of SUMO2 at lysine 11 favours the formation of non-canonical SUMO chains (2018)
Figure 1

Figure 1 Acetylation of SUMO2/3 at K11 limits the formation of SUMO chains

(a) Structure of SUMO2. Lysine residues within the flexible N-terminus (K5,7,11) are marked in magenta, lysine residue 21 is colored in teal and lysine residues involved in SUMO/SIM interactions (K33,K35,K42,K45) are colored in green. (PDB: 2N1W). The red circle indicates K11. Ac. indicates the acetylation at K11.

(b) Comparison of the amino acid sequences for SUMO1/2 and 3 and the yeast variant Smt3. Lysine residues are marked in blue. SUMO consensus motifs are marked in red. SUMO2/3 and Smt3 display a conserved consensus motif, whereas SUMO1 shows an inverted consensus motif.

(c) Substrate independent in vitro SUMOylation assay with SUMO2WT, SUMO2K11Ac or SUMO2K11Q was performed and analyzed by immunoblotting with the indicated antibodies.

(d) Substrate independent in vitro SUMOylation assay using either SUMO2WT or SUMO2K11Ac in the presence of increasing amounts of ZNF451-N. Reaction products were analyzed by immunoblotting with the indicated antibodies. Note that - when compared to (c) assays were done at reduced concentrations of E1 and E2 (see materials and methods).

(e) In vitro SUMOylation assay using a fixed amount of SUMO2WT (220 ng) with increasing concentrations of SUMO2K11Ac. Ratios ranged from 3:1 (unmodified/K11Ac) to 1:2 (unmodified/K11Ac)

(f) In vitro SUMOylation assay, using SUMO2WT or SUMO2K11Ac, with p53 as a substrate. p53 was generated by in vitro translation/transcription in a rabbit reticulocyte lysate system. SUMO chain formation on p53 was analyzed by immunoblotting with α-p53 antibodies.
Acetylation of SUMO2 at lysine 11 favours the formation of non-canonical SUMO chains (2018)
Figure 2

Figure 2 SIRT1 catalyses the deacetylation of SUMO2K11Ac

(a) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac (2.65 μM each) with recombinant SIRT1 in the presence or absence of NAD+.

(b) In vitro deacetylation of recombinant SUMO2WT, SUMO2K11Ac, SUMO2K33Ac and SUMO2K35Ac. Immunoblotting was performed with antibodies as indicated.

(c) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 (0.74 µM, 1.5 μM, 3 µM), followed by immunoblotting with α-SUMO2K11Ac.

(d) Combination of in vitro deacetylation and substrate independent in vitro SUMOylation with SUMO2WT and SUMO2K11Ac in the presence or absence of SIRT1 (1.5 μM).

(e) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1, followed by substrate dependent in vitro SUMOylation of p53.

(f) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells. Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM (10 mM) for 5h and subsequent enrichment for acetylated proteins via acetyl-lysine immunoprecipitation. As a control cells were either treated with control siRNA or siSUMO2/3. The samples were analyzed by immunoblotting with α-SUMO2/3 and α-SUMO2K11Ac antibodies. The α-SUMO2/3Ac and α-SUMO2K11Ac reactive species migrating at ~200 kDa are marked with an asterisk.
Acetylation of SUMO2 at lysine 11 favours the formation of non-canonical SUMO chains (2018)
Figure 3

Figure 3 Degradation of PML by the StUbL pathway is not affected by K11 acetylation

(a) HeLa cells constantly expressing HA-tagged PML were depleted for endogenous SUMO1/2/3 by siRNA. Subsequently, they were transfected with RGS-His tagged SUMO2T90K for 72 h. Prior to lysis, cells were treated with As2O3 for 2 h. HA-tagged PML was immunoprecipitated via a denaturing HA-IP, which was followed by LysC digestion. Peptides harboring GlyGly-remnants, corresponding to SUMOylation sites, were enriched by GlyGly-IP and the samples were analyzed by LC-MS/MS.

(b) Western blot analysis with α-RGS-His and α-HA was performed to monitor poly-SUMOylation of HA-PML. The remaining samples were measured by LC-MS/MS and final data analysis was done with MaxQuant. A K11 SUMOylated SUMO2 peptide was identified and is depicted with an Andromeda score of 49.3. Intensities for this SUMO2 peptide for the control (gray) and arsenic (purple) treated samples are blotted as a bar graph. The diagram shows a representative dataset derived from two replicates. The corresponding peptide is displayed below the graph. The SUMOylation site is marked with SUMO2 (green) and peptide fragment ions are indicated by y- (red) and b- (blue).

(c) The degradation of the PML reactive band (~ 120 kDa) was determined by immunoblotting. HeLa cells were either mock transfected or transfected with siRNA directed against SUMO1/2/3. The expression of RGS-His-tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h. Prior to lysis, cells were treated with 1 μM As2O3 for 6 h. Western blot analysis for wild-type SUMO2 and SUMO2K11Q were blotted with α-RGS-His. Immunoblotting with α-Tubulin served as a loading control. Immunoblots for knockdown controls (SUMO1 and SUMO2/3) can be found in Fig EV5B.
Acetylation of SUMO2 at lysine 11 favours the formation of non-canonical SUMO chains (2018)
Figure 4

Figure 4 Impact of K11 acetylation on SUMO chain architecture under heat stress

(a) HeLa cells were depleted for endogenous SUMO2/3 by siRNA and transfected with SUMO2T90K (WT or K11Q). Prior to lysis, cells were heat shocked at 43°C for 1 h. The lysate was digested with LysC, followed by GlyGly-enrichment. Subsequently, samples were measured by LC-MS/MS and analyzed with MaxQuant software.

(b) Immunoblotting of samples with α-RGS-His (SUMO variants) and α-PCNA as loading control. Sample preparation was performed as described in the text.

(c) Bar graph, comparing the identified SUMO-SUMO2 linkages of control and heat shock samples for SUMO2WT (gray) and SUMO2K11Q (purple). The relative abundance (y-axis) of the indicated SUMO-SUMO2 linkage sites (x-axis) are shown. The measured intensity for K11 in the SUMO2WT under control conditions was set to 100% and the relative abundance for the individual residues was calculated in relation to this value. One of two independent replicates is shown. Data from the second replicate are shown in Fig EV5C.

(d) Model depicting the possible impact of an acetylation of SUMO2 at K11 on formation of SUMO signaling. SUMO2/3K11Ac can restrict chain length by acting as a K11 chain terminator (A), SUMO2/3K11Ac can function as a binding platform for acetyllysine binding proteins (B), SUMO2/3K11Ac can trigger the formation of alternatively linked SUMO2/3 chains (C).

EV Figure legends
A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming (2020)
Figure 2

Figure 2. Activin A induces a CAF phenotype in fibroblasts

A Primary murine dermal fibroblasts, which had been treated with recombinant activin A at different concentrations, were analyzed for BrdU incorporation. N=3-4.

B Representative images of collagen gels and quantification of the extent of gel contraction (percentage of initial gel size) in the presence or absence of activin A (20 ng/ml) and/or TGF-β1 (1 ng/ml). N=3-4.

C qRT-PCR and Western blot analyses of ACTA2/αSMA expression using total RNA/lysates of fibroblasts treated with activin A or TGF-β1 for 6 h. N=3. The membrane was re-probed with GAPDH and mDia2 antibodies.

D Representative images of fibroblasts treated with activin A, TGF-β1 or vehicle (control) as in (C), stained for αSMA (green), counterstained with rhodamine-coupled phalloidin (red) and Hoechst (blue). Bar graph shows percentage of fibroblasts with stress fibers. N=3.

E Representative images of control and activin A-treated fibroblasts stained with rhodamine-coupled phalloidin; scatter plot shows quantification of filopodium length in control, activin A- and TGF-β1-treated fibroblasts. N=27-70 filopodia.

F Scratch assay of fibroblasts treated with activin A or vehicle for 24 h. N=3. The area covered by cells in the original scratch was quantified. Migration in one control culture was set to 1.

G Chemotactic migration of primary mouse fibroblasts from wt mice for 24 h in a transwell assay towards increasing concentrations of activin A or vehicle. N=3.

H qRT-PCR analysis of CAF marker genes relative to Rps29 using RNA from fibroblasts treated with activin A for 6 h. N=3.

Data information: Bar graphs show mean ± SEM. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001 (one-way ANOVA with Bonferroni post-test (A-G) or two-way ANOVA with Bonferroni post-hoc test (H)). Scale bars: 50 μm (D) and 25 μm (E). Exact P‐values are provided in Dataset EV3.
A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming (2020)
Figure 3

Figure 3. Activin A-exposed fibroblasts produce a tumor-promoting secretome and matrisome

A qRT-PCR for INHBA relative to RPL27 using RNA from primary human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h (Fb Act, clones 1 and 2) or with empty vector (Fb EV) (N=3). Fb Act and Fb EV cultures were generated from lentivirally-transduced cells upon clonal expansion of resistant single cells.

B Western blot of conditioned media (CM) of transduced fibroblasts showing mature activin βA. Ponceau S staining of the membrane was used as a loading control.

C Chemotactic migration of SCC13 cells for 24 h in a transwell assay containing CM from Fb Act (clone 2) or Fb EV. N=4. Migration in one control culture was set to 1.

D Relative colony area of SCC13 cells upon culture in CM from Fb Act (clone 2) or EV. N=3.

E Representative immunofluorescence images of de-cellularized matrix from Fb Act (clone 2) and Fb EV stained for fibronectin or collagen I.

F Relative colony area of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=3.

G Migration of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=6. The experimental setup is shown schematically above the graph.

H Fb Act (clone 2) or Fb EV cells were cultured together in 2D with SCC13 cells. After 7 day of culture SCC13 cells were identified by K14 and the number of K14-positive colony area was quantified. N=6.

I Quantification of SCC13 tumor spheroid area in single hanging drop including CM from Fb EV or Fb Act (clone 2) (including 1% FBS), DMEM/1% FBS, or DMEM/1% FBS plus 20 ng/mL recombinant activin A. The area of the spheroid formed in one Fb EV sample was set to 1. N=9-10 hanging drops.

J Representative images and quantification of K14-stained areas from 3D cultures of SCC13 cells seeded on top of either Fb Act (clone 2) or Fb EV. N=3 independent cultures, n=3 histological sections.

K Representative pictures of tumors formed in NOD/SCID mice 6-7 weeks after intradermal injection of SCC13 cells with Fb Act (clone 2) or Fb EV. Tumor volume at different time points of tumor development (6-7 weeks after injection). N=5 tumors per group.

Data information: Bar graphs show mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (one-way ANOVA with Bonferroni post-test (A, I), two-way ANOVA with Bonferroni post-hoc test (K), multiple Student's t-test (G), unpaired Student's t-test (C, D, F, H)). Scale bars, 500 μm (E), 50 μm (H) or 100 μm (J). Exact P‐values are provided in Dataset EV3.
A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming (2020)
Figure 4

Figure 4. mDia2 is a direct activin A target gene in fibroblasts

A Gene Set Enrichment Analysis comparing the transcriptomes of fibroblasts isolated from pre-tumorigenic ear skin of female HPV8/Act-transgenic mice vs. HPV8/wt-transgenic mice (12-14 weeks old) to published datasets of cancer stroma or CAFs from different types of human cancers and from total Actinic Keratosis (AK) samples. Positive or negative Normalized Enrichment Scores (NES) correspond to enrichment of a given set in genes that are up- or downregulated, respectively, in response to activin A. Data are presented as pseudo-heatmap with NES magnitude color-coded as indicated in the legend; all non-grey comparisons have FDR<0.10.

B Ingenuity pathway analysis of differentially regulated genes in FACS-isolated fibroblasts from activin A transgenic mice in either a HPV8-negative or HPV8-positive background compared to respective controls, showing selected top activated functions.

C (Top) Functional enrichment of the most up-regulated genes using the Reactome database showing three unique significantly enriched terms. (Bottom) Filtered list of up-regulated genes based on the Matrisome database showing selected top up-regulated collagens, ECM regulators, and secreted factors.

D mDia2 (Diap3) expression quantified by RNA sequencing of fibroblasts isolated from ear skin. N=3 pools of 3-6 mice.

E Primary mouse fibroblasts were treated with activin A or TGF-β1 or vehicle for 6h and analyzed by qRT-PCR (N=3) and Western blot for mDia2 (Diap3) and mDia2 expression, respectively. The GAPDH loading control is identical to the one in Fig 2C, since the same membrane was probed for mDia2, αSMA and GAPDH.

F Alignment of the sequences in the first intron of mDia2 (Diap3) genes of different species that harbor a SMAD2/3 binding element (SBE) (consensus sequence highlighted in red and italics) (top). Chromatin-immunoprecipitation of lysates from activin A- or TGF-β1-treated fibroblasts using a SMAD2/3 antibody and amplification of the bound DNA with mDia2 primers (bottom). Pre-immune serum (IgG) was used as negative control, and the input chromatin is shown. N=3.

G Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX-inducible dominant-negative ActRIB mutant (dnActRIB) or an empty lentivirus (EV), treated with activin A for 2.5 h and analyzed by Western blot for mDia2 and GAPDH.

H Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act cells (clone 2) stained for mDia2 or K14 (green) (top), mDia2 (green) or MECA32/LYVE1 (red, bottom) and counterstained with Hoechst (blue).

Data information: Bar graphs show mean ± SEM. ns p>0.05, *p<0.05, **p<0.01 (one-way ANOVA with Bonferroni post-hoc test). Scale bars: 100 μm. Exact P‐values are provided in Dataset EV3.
A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming (2020)
Figure 5

Figure 5. mDia2 (DIAPH3) is overexpressed in human cancers and negatively correlates with survival

A qRT-PCR for mDia2/DIAPH3 relative to RPL27 using RNA from normal human skin (N=11), SCCs (N=9) and BCCs (N=7).

B Representative skin/tumor sections from normal human skin, SCC and BCC patients stained for mDia2/DIAPH3. Note the strong mDia2/DIAPH3-positivity in the tumor stroma. Scale bars: 100 μm. D: Dermis, E: Epidermis: T: Tumor.

C Expression levels of mDia2/DIAPH3 in tumor and normal tissues based on TCGA cancer data (TCGA data version 20141017, http://firebrowse.org/). Gene expression levels were presented as normalized log2 RSEM (RNA-Seq by Expectation-Maximization) index from the database. BLCA: Bladder Urothelial Carcinoma (N = 408 & 19 tumor and normal tissue samples, respectively), BRCA: Breast Invasive Carcinoma (N = 1100 & 112), CESC: Cervical SCC (N = 306 & 3), ESCA: Esophageal SCC (N = 185 & 11), HNSC: Head-Neck SCC (N = 522 & 44), LIHC: Liver Hepatocellular Carcinoma (N = 373 & 47), LUSC: Lung SCC (N = 501 & 51), PAAD: Pancreatic Adenocarcinoma (N = 179 & 4), PRAD: Prostate Adenocarcinoma (N = 496 & 50), READ: Rectum Adenocarcinoma (N = 167 & 10), STES: Stomach and Esophageal Carcinoma (N = 600 & 46), UCEC: Uterine Corpus Endometrial Carcinoma (N = 546 & 35). Box plot shows relative mDia2/DIAPH3 expression in human tumor (red) and normal (blue) tissue. Boxes indicate the interquartile range, whiskers indicate data points within 1.5 times interquartile range, dots outside the whiskers indicate outliers, and the central line indicates median.

D-G Relative gene expression of mDia2/DIAPH3 and INHBA in the stroma of intrahepatic cholangiocarcinoma, breast and colorectal cancers vs. stroma of respective normal tissues based on datasets GSE45001 (D) (N = 10 normal tissue & 10 cancer samples, GSE9014 (E) (N = 4 & 13), and GSE35602 (F) (N = 6 & 53), and in CAFs from breast cancer samples vs. normal breast fibroblasts (GSE29270 dataset) (G) (N = 5 & 23). NS: Normal stroma, CS: Cancer stroma, NF: Normal fibroblasts, CAF: Cancer-associated fibroblasts). The expression values of the datasets were obtained using GEO2R tool in the GEO database.

H Kaplan-Meier survival curves based on datasets from GEO, EGA and TCGA, demonstrating that high expression of both mDia2/DIAPH3 and INHBA in patients with liver, stomach and breast cancer correlates with poor overall survival. Data and statistical analyses were collected from KMPlotter.

Data information: Graphs show mean ± SEM. p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, unpaired Student's t-test (D-G) and one-way ANOVA with Bonferroni post-hoc test (A). Exact P‐values are provided in Dataset EV3.
A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming (2020)
Figure 6

Figure 6. Knock-down of mDia2 in fibroblasts suppresses squamous cell malignancy and tumor growth via p53

A Immortalized mouse fibroblasts were transduced with a lentiviral vector expressing different mDia2-targeting shRNAs (shmDia2 #1-3) or empty vector (sh-EV). The knock-down was confirmed by qRT-PCR (N=3), Western blot and immunofluorescence.

B,C Ki67 staining (B) and migration in scratch assays (C) of mDia2 knock-down and control mouse fibroblasts. N=3.

D qRT-PCR for mDia1, mDia3, Fmn1 and Fmn2 and several CAF genes relative to Rps29 using RNA from control (sh-EV) and mDia2 knock-down (sh-mDia2) fibroblasts. N=3.

E Representative images of mDia2 knockdown and control mouse fibroblasts stained for p53 (left), and quantification of cells showing accumulation of p53 in the nucleus (right). N=3.

F,G Representative Western blots for p53, vinculin and histone H1 using nuclear and cytoplasmic fractions and total lysate of mDia2 knock-down (F) or activin A-treated fibroblasts (6h, 20 ng/ml) (G) and control mouse fibroblasts and quantification of the nuclear p53/histone H1 ratio. Vinculin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. N=6.

H Representative Western blots for p53, mDia2 and GAPDH using total lysates of mDia2 knock-down and control mouse fibroblasts treated for 24 h with pifithrin-α (PFT-α) (15 μM).

I Representative p53 immunofluorescence images of mDia2 knock-down mouse fibroblasts treated with PFT-α or vehicle (DMSO) (top) and quantification of cells showing accumulation of p53 in the nucleus (bottom). N=3.

J qRT-PCR for mDia2, Postn, Mmp13, Fn1, Spp1 and Acta2 relative to Rps29 using RNA from mDia2 knock-down and control fibroblasts treated with PFT-α or vehicle. N=3.

K Colony size of SCC13 Act cells (clone 2) after co-culture with either immortalized mDia2 knockdown (sh-mDia2) or control (sh-EV) mouse fibroblasts. N=4.

L Representative pictures of 5-week-old tumors formed upon intradermal co-injection of SCC13 Act cells (clone 2) with sh-EV or sh-mDia2 knock-down immortalized mouse fibroblasts. Graph shows tumor volume at different timepoints of tumor development. N=3-5 tumors per group.

M,N Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act (clone 2) co-injected with sh-EV or sh-mDia2 fibroblasts, stained for E-cadherin (green) and periostin or p53 (red), counterstained with Hoechst (blue).

Data information: Bar graphs show mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, unpaired Student's t-test (B, E-G, I and K) and one-way ANOVA with Bonferroni post-hoc test (A, C), two-way ANOVA with Bonferroni post-test (D, J, L). Scale bars: 100 μm. Exact P‐values are provided in Dataset EV3.
A paracrine activin A-mDia2 axis promotes squamous carcinogenesis via fibroblast reprogramming (2020)
Figure 7

Figure 7. Inhibition of activin A-mDia2 signaling suppresses squamous tumor growth

A Tumor volume at different time points after co-injection of SCC13 cells with EV or dnActRIB fibroblasts. N=5 tumors.

B Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co-injected with EV or dnActRIB fibroblasts stained for K14 (left) or PDGFRα (right) (green) and mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.

C SCC13 EV or SCC13 Fst cells were injected into the ear skin of NOD/SCID mice and tumor weight was determined 12 weeks after injection. N=9-10 tumors.

D Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst cells stained for K14 or mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.

E Tumors formed by SCC13 Act cells were treated ex vivo for 6h with follistatin (FST) (50 ng/ml) and/or SMIFH2 (10 μM), or with activin A (20 ng/ml) and stained for Ki67. N=3 tumor explants, n=4 histological sections.

F Representative images of spheroids formed by SCC13 Act cells labeled with PKH67 (green) with primary human fibroblasts embedded in collagen gels and graph showing increase in the spheroid area between day 0 and day 3. Spheroids were either treated with FST (50 ng/ml) or SMIFH2 (10 μM) or left untreated. N=6-8 tumor spheroids.

G Increase in A431 tumor volume (mm3) compared to day 0 (time when the tumors were first injected with FST, approximately 3-4 weeks after injection of the cells) upon treatment of the tumors with FST (100 ng per injection) or vehicle (PBS). N=3-5 tumors per group. Representative immunofluorescence images the FST or vehicle-treated tumors, stained for mDia2 (green) and K14 (red), counterstained with Hoechst (blue).

H Proposed role of activin A secreted by cancer cells in inducing a pro-tumorigenic CAF phenotype in fibroblasts. Activin A signaling (blocked by follistatin or dnActRIB (not shown in the figure) induces upregulation of mDia2 (inhibited by SMIFH2), which in turn contributes to tumor development by reducing the nuclear p53 pool and promoting the expression of CAF marker genes, fibroblast proliferation and migration.

Data information: Bar graphs show mean ± SEM. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (unpaired Student's t-test (B-D), one-way ANOVA with Bonferroni post-test (E, F), two-way ANOVA with Bonferroni post-test (A, G)). Scale bars: 100 μm. Exact P‐values are provided in Dataset EV3.

Expanded View Figure Legends