EMBO/soda-nel huggingface dataset:
CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation (2019)
Figure 4

Figure 4. Depletion of CCP5 rescues the angiogenesis defects caused by CEP41 deficiency.

A, B HUVECs transfected with CCP5 and/or CEP41 siRNAs were scratched and incubated to evaluate wound closure. The extent of wound closure in co-transfected cells with CCP5 and CEP41 siRNAs was compared with that in control or CEP41 siRNA-transfected cells at the indicated time points (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) is presented graphically by measuring the distance between the dotted lines at each time point in (B). Data are shown as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition). Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).

C-E The indicated siRNA-transfected HUVECs were subjected to in vitro angiogenesis assays. Tubulogenesis of HUVECs depleted for CEP41 and CCP5 was compared to that of control, CEP41 single-knockdown, or CCP5 single-knockdown cells (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at the indicated time points using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).

F, G The blood vessels in the trunks of cep41-mutant/morphant zebrafish were compared to those of zebrafish co-injected with ccp5 MOs (2 ng) at 40 hpf in (F). Asterisks and arrowheads indicate impaired ISVs and DLAVs, respectively. A, anterior; P, posterior. Scale bars, 100 μm. Quantification of the number of defective ISVs and DLAVs from data observed in equivalent fields of view (within eight somites). Data are median of three independent experiments with ≥ 20 embryos per condition. Statistical significance was determined using the Kruskal-Wallis test followed by Dunn's post hoc test (***P < 0.001).

H-J HUVECs co-transfected with CEP41 and CCP5 siRNAs were immunostained with ARL13b- and GT335-specific antibodies. The staining results were compared to those of control, single CEP41, or CCP5 siRNA-transfected cells. Representative images indicated with rectangles appear in magnified images in the lower panels. Scale bars, 20 μm. Quantification of relative intensity of GT335/ARL13b signals in the cilia (I) and ciliary length (J) in images (H) are the result of three independent experiments with ≥ 200 cells per condition (median). **P < 0.01, ***P < 0.001, ns: non-significant (Kruskal-Wallis test with Dunn's post hoc test).
CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation (2019)
Figure 5

Figure 5. Depletion of CEP41 prevents shear stress-induced endothelial cell dynamics and cilia disassembly.

A, B Control or CEP41 siRNA-transfected HUVECs cultivated under either static or shear stress states and examined in a wound healing assay in (A). Scale bars, 600 μm. Quantification of the extent of wound closure in (A) displayed graphically by measuring the distance between the lines at the indicated time points in (B). Data are presented as mean ± SD of three independent experiments (n ≥ 3 scratches per experimental condition). Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (***P < 0.01, ns: non-significant).

C-E An in vitro angiogenesis assay was performed in static or shear stress-exposed HUVECs transfected with the indicated siRNAs for 18 h in (C). Scale bars, 600 μm. Quantification of tube node number in (D) and tube length in (E) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (**P < 0.01, ***P < 0.001, ns: non-significant).

F-H Control or CEP41-deficient HUVECs cultivated under laminar shear stress and immunostained with ARL13b-specific antibodies to measure deciliation. Representative images are indicated by rectangles with numbers and shown in magnified images in the right panels. The resulting data were compared to those of cells under static conditions in (F). Scale bars, 20 μm. Quantification of ARL13b-positive ciliary cells (G) and of relative intensity of GT335/ARL13b signals in the cilia (H) in images (F) is the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (G), median (H)). *P < 0.05, ***P < 0.001, ns: non-significant (Two-way ANOVA with Tukey's post hoc test (G) and Kruskal-Wallis test with Dunn's post hoc test (H)).

I-K, Control, CEP41-, or CCP5-depleted hTERT-RPE1 cells cultured under serum starvation for 48 h and fixed at 18 h after serum retrieval for immunostaining with both ARL13b- and GT335-specific antibodies in (I). Images of the results 18 h after serum addition. Representative cells are indicated by rectangles. Scale bars, 20 μm. Quantification of ARL13b-positive ciliated cells (J) and relative intensity of GT335/ARL13b signals in the cilia (K) under either serum starvation or serum retrieval (18 h) conditions are the results of three independent experiments with ≥ 200 cells per condition (mean ± SD (J), median (K)). **P < 0.01, ***P < 0.001, ns: non-significant (One-way ANOVA with Tukey's post hoc test (J) and Kruskal-Wallis test with Dunn's post hoc test (K)).
CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation (2019)
Figure 7

Figure 7. CEP41 regulates hypoxia-induced angiogenesis by activating AURKA.

A The control or CEP41 siRNA-transfected HUVECs were cultivated under hypoxia (1% O2) and their whole cell lysates were used for immunoblot assays of CEP41, phospho-AURKA, and AURKA. The resulting data were also compared to those of normoxic cells. Protein levels were normalized against β-ACTIN in the same blots.

B, C VEGFA (B) and VEGFR2 (C) mRNA levels were measured by qRT-PCR using cDNA from normoxic and hypoxic controls or CEP41-depleted cells. The expression of GAPDH was quantified for the normalization of those qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined with the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).

D, E Tg(kdrl:eGFP) zebrafish were injected with control- or cep41-MOs and then incubated under hypoxia at 28 hpf (a stage of high shear stress) for 2 h. They were subjected to immunostaining with phospho-AURKA-specific antibodies (red) and DAPI at 30 hpf. The insets indicate the representative areas from each immunostaining and red dots indicate activated aurka within ECs. Scale bars, 40 μm. Quantification of phospho-aurka-positive ECs (E) in equivalent fields of view for each MO-injected zebrafish in (D) are the result of three independent experiments with ≥ 20 embryos per condition. The top and bottom whiskers represent the maximum and minimum values, respectively. *P < 0.05 (Unpaired Student's t-test).

F, G Zebrafish vegfa (F) and vegfr2 (G) mRNA levels were quantified by qRT-PCR in eGFP-positive ECs of control- or cep41-MO-injected Tg(kdrl:eGFP) zebrafish subjected to either normoxia or hypoxia at 30 hpf. The expression of zebrafish β-actin was quantified for the normalization of these qRT-PCR results. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (*P < 0.05, ***P < 0.001, ns: non-significant).

H-J Control and CEP41-deficient HUVECs, transfected with expression vectors encoding nothing (MOCK), AURKA, AURKA-T288D, HIF1α, or HIF1α with CEP41 were cultivated under hypoxia and subjected to an in vitro angiogenesis assay for 18 h. Scale bars, 400 μm. Quantification of tube node number in (I) and tube length in (J) from data examined within equivalent fields of view at each time point using the ImageJ angiogenesis analyzer. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. Statistical significance was determined using the two-way ANOVA followed by Tukey's post hoc test (*P < 0.05, ***P < 0.001, ns: non-significant).
CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation (2019)
Figure 8

Figure 8. CEP41 functions upstream of HIF1α through physical interaction with it to drive hypoxia-induced angiogenesis.

A HUVECs transfected with control or CEP41 siRNAs were cultivated under hypoxia and their whole cell lysates were used for immunoblot assays of HIF1α. The HIF1α expression of hypoxic cells was compared to that of normoxic cells and the protein levels were normalized against β-ACTIN in the same blot.

B, C Co-immunoprecipitation assays using HIF1α-, and CEP41-specific antibodies were performed in HEK 293T cells expressing GFP-CEP41 under normoxia or hypoxia. An IP using IgG-specific antibodies was performed as a negative control. NOR, normoxia; HYP, hypoxia.

D The CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, or HIF1α and cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for phospho-AURKA and AURKA, and the resulting data were compared to those of normoxic and hypoxic control cells. Protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.

E, F The CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, HIF1α, or HIF1α with CEP41 and exposed to hypoxia. VEGFA (E) and VEGFR2 (F) mRNA levels were analyzed by qRT-PCR using cDNA from transfected cells. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant). NOR, normoxia; HYP, hypoxia.

G, Control or CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK) or HIF1α and treated with MG132 or not, and then the cells were cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for HIF1α, phospho-AURKA, and AURKA, and the protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.

H, I Quantification of tube node number in (H) and tube length in (I) from in vitro angiogenesis assays with HIF1α #1 or #2 siRNAs-transfected HUVECs overexpressing nothing (MOCK) or CEP41 under hypoxia. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition. Statistical significance was determined with the two-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).

J, K VEGFA (J) and VEGFR2 (K) mRNA levels were analyzed by qRT-PCR using cDNA from hypoxic HIF1α-depleted HUVECs transfected with control or CEP41 expression vectors. The results were compared to those of control and normoxic HIF1α-depleted cells. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant).

L A schematic diagram showing the molecular mechanism by which CEP41 drives hypoxia-induced angiogenesis.
Reprogramming Immunosuppressive Myeloid Cells Facilitates Immunotherapy for Colorectal Cancer (2020)
Figure 1

Figure 1. EP4 is a master regulator of IMCs.

A. The expression of EP subtypes in bone marrow myeloid cells and colon tumor myeloid cells. The short black lines indicate individual expressions, the blue and yellow lines indicate the average expression level in each condition. The grey dotted lines represent the overall average between Ptger1 and Ptger3 or Ptger2 and Ptger4.

B. The expression of EP subtypes in CD11b+ cells isolated from tumor tissues and spleen of CT26 tumor-bearing mice by magnetic bead separation (n=3).

C. Schematic diagram of the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)-4-induced mouse monocyte DCs/macrophages differentiation assay. Cells were treated with specific antagonists of distinct EP receptors: ONO-8711, EP1; PF-04418948, EP2; L-798106, EP3; and E7046, EP4.

D. Representative flow cytometry plots revealing the effects of prostaglandin E2 (PGE2) and EP1-4 antagonists on mouse bone marrow monocyte dendritic cell (DC)/macrophage differentiation (n = 3).

E. The frequencies of F4/80+CD11C− macrophages and F4/80−CD11C+ DCs under varying treatments as analyzed by flow cytometry analysis (n = 3).

F. Schematic representation of the GM-CSF/IL-6-induced mouse bone marrow monocyte myeloid-derived suppressor cell (MDSC) differentiation assay (n = 3).

G. Representative flow cytometry plots revealing the effects of PGE2 and EP1-4 antagonists on mouse bone marrow derived-MDSC differentiation. Upper subpanel: vehicle group and PGE2 group. Lower subpanel: ONO-8711 (EP1 antagonist) group, PF-04418948 (EP2 antagonist) group, L-798106 (EP3 antagonist) group, and E7046 (EP4 antagonist) group. (n = 3).

H. The frequencies of Ly6C+Ly6G− mMDSC and Ly6CmidLy6G+ PMN-MDSCs under varied treatments as analyzed by flow cytometry analysis (n = 3).

Data information: Data are presented as mean ± SEM. (A) Kolmogorov-Smirnov test; (E, H) Two-tailed unpaired Student's t test was performed; *P < 0.05; **P < 0.01; ***P < 0.001. Exact P values and statistical tests are listed in Appendix Table S8.
Reprogramming Immunosuppressive Myeloid Cells Facilitates Immunotherapy for Colorectal Cancer (2020)
Figure 2

Figure 2. Discovery and characterization of a potent and selective EP4 antagonist, TP-16.

A. Chemical structure of TP-16.

B. Dose-effect curve of TP-16 in GloSensor™ cAMP assay in EP4- expressing 293 cells (n=3).

C. Dose-effect curves of TP-16 in PGE2-induced calcium flux assay (n=3).

D. The Schild plot of PGE2 in the presence of varying concentrations of TP-16. TP-16 shifted the dose-response curve of PGE2-induced intracellular cAMP levels in a dose-dependent manner (n=3).

E. The pA2 value and slope of the Schild plot.

F. Docked pose of TP-16 with critical residues in the putative binding pocket of human EP4 protein. EP4 is shown as a color cartoon, the residues important for the interaction are depicted in magenta sticks, and TP-16 is shown as a cyan stick figure.

G. The LIGPLOT diagram summarizes key interactions between TP-16 (purple lines) and residues that originate from EP4. T69, T76, T168 and R316 establishes hydrogen bonds with TP-16. Semicircles with radiating lines indicate non-polar interactions.

Data information: Data are presented as mean ± SEM from three independent experiments with similar results.
Reprogramming Immunosuppressive Myeloid Cells Facilitates Immunotherapy for Colorectal Cancer (2020)
Figure 3

Figure 3. EP4 antagonist TP-16 robustly suppresses the tumor growth in murine syngeneic tumor models.

A. Schematic illustration of the establishment of the murine syngeneic tumor models and drug treatment schedule. Established tumor models were orally treated daily with vehicle or TP-16 when tumor volumes reached 100-200 mm3.

B. The antitumor activities of E7046 (150 mg/kg) and TP-16 (37.5, 75, and150 mg/kg) in CT26 tumor-bearing BALB/c mice (n = 11 per group). Tumor growth curves in the CT26 model (left panel). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.

C. Representative graph showing that the antitumor activity of TP-16 is lost in CT26 tumors engrafted into BALB/c nude mice (left panel) (n = 8). Bar graphs show the individual changes in tumor growth relative to after and before drug administration (right panel). Tumor volumes reached 100-200 mm3 on day 7.

D. Single-cell suspensions of tumor tissues from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).

E. Growth curves (left panel) and relative change after 18 days treatment (right panel) in colon cancer (MC38) xenograft tumors in C57BL/6 mice (n = 7). Tumor volumes reached 100-200 mm3 on day 7.

F. Single-cell suspensions of tumor tissues from MC38 tumor-bearing C57BL/6 mice treated with vehicle or TP-16 for 2 weeks were analyzed for immune cell infiltration by flow cytometry analysis. Representative flow cytometry and dot plots of intratumoral frequencies of CD8+ T cells (n = 5).

G. The representative bioluminescence images (left panel) and quantitative growth curves (right panel) of an orthotopic colorectal cancer mouse model, CT26-Luc model (n = 4).

Data information: Data are presented as mean ± SEM. (B) One-way analysis of variance (ANOVA), Tukey's multiple comparison test; *P < 0.05. (C-G) Two-tailed unpaired Student's t test was performed; *P < 0.05; **P < 0.01. Exact P-values and statistical tests are listed in

Appendix Table S8.

Source data are available online for this figure.
Reprogramming Immunosuppressive Myeloid Cells Facilitates Immunotherapy for Colorectal Cancer (2020)
Figure 4

Figure 4. EP4 antagonist TP-16 reprograms tumor associated myeloid cells (IMCs) and enhances antitumor immunity.

A, B. Tumors from CT26 tumor-bearing BALB/c mice treated with vehicle or TP-16 for 2 weeks were harvested, and tumor single-cell suspensions were analyzed for tumor-associated myeloid cells by flow cytometry analysis. Representative graphs and quantification of F4/80+MHC-II+ immunosuppressive myeloid cells (IMCs)-M1 macrophages (A) and F4/80+CD206+ IMCs-M2 macrophages (B) gated on CD45+CD11b+ myeloid cells (n = 5).

C. Representative flow cytometry analysis and quantification of Ly6C+Ly6G- IMCs-monocytic myeloid-derived suppressor cell (mMDSC) and Ly6CmidLy6G+ IMCs-PMN-MDSC frequencies gated on CD45+CD11b+ myeloid cells (n = 5).

D-G. Relative expression of selected M1, M2, and MDSC markers on flow-sorted M1 macrophages (D), M2 macrophages (E), mMDSCs (F), and PMN-MDSCs (G) from CT26 tumors. Data are normalized to β-actin expression levels (n = 5).

H-K. Representative flow analysis and quantification of CD8+ T cell activation markers: Granzyme B (H), IFN-γ (I), TNF-α (J), and PD-1 (K) (n = 5).

Data are presented as mean ± SEM. (A-K) Two-tailed unpaired Student's t test; *P < 0.05; **P < 0.01; ***P < 0.001. Exact P-values and statistical tests are listed in Appendix Table S8.
Reprogramming Immunosuppressive Myeloid Cells Facilitates Immunotherapy for Colorectal Cancer (2020)
Figure 5

Figure 5. EP4 antagonist TP-16 reverses the immunosuppressive effects

of PGE2 on myeloid differentiation and activation.

A, B. M-CSF induced mouse bone marrow derived macrophages (BMMs) were stimulated with 50 ng/mL mouse recombinant IFN-γ (A) or 20 ng/mL mouse recombinant IL-4 (B) in the absence or presence of PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected M1 markers (Cxcl10 and Tnfa) and M2 markers (Arg-1 and Ym-1) were measured by q-PCR. Data are normalized to β-actin expression levels (n=3).

C. Bone marrow cells were cultured with 40 ng/mL GM-CSF/IL-6 or co-stimulated with PGE2 ± varying concentrations of TP-16 for 6 days. Representative quantification of Ly6C+Ly6G− monocytic myeloid-derived suppressor cells (mMDSCs) and Ly6CmidLy6G+ PMN-MDSCs (n = 3).

D. Bone marrow cells were treated with GM-CSF/IL-6 for 6 days to induce MDSCs and thereafter, with PGE2 ± varying concentrations of TP-16 for 12 hr. The mRNA levels of selected MDSC markers (Arg-1, Ptgs2, Il-4, and Il-10) were measured by qPCR. Data are normalized to β-actin expression levels (n=3).

E, F. In vitro T cell suppression activity of CT26-tumor infiltrating CD11b+ myeloid cells collected at 2 weeks post-treatment with vehicle or 75 mg/kg TP-16. CD8+ T cells were labeled with CFSE and then stimulated with CD3/CD28 antibodies for 3 days in the absence or presence of tumor-infiltrating CD11b+ myeloid cells from vehicle- or 75 mg/kg TP-16-treated CT26 tumors. Representative flow cytometry histograms (E) and percentages (F) of proliferating CD8+ T cells when plated in a ratio of 2:1, 1:1, and 1:2 CD8+ T cells to CD11b+ myeloid cells (n = 3).

Data are presented as mean ± SEM. (A-D, F) One-way analysis of variance (s), Tukey's multiple comparison; *P < 0.05; **P < 0.01; ***P < 0.001. Exact P-values and statistical tests are listed in Appendix Table S8.
Reprogramming Immunosuppressive Myeloid Cells Facilitates Immunotherapy for Colorectal Cancer (2020)
Figure 6

Figure 6. EP4 antagonist TP-16 improves the efficacy of immune checkpoint blockade and induces a durable antitumor immune response in CT26 tumor-bearing mice.

A. The growth of CT26 tumor volume when treated with vehicle, TP-16 (75 mg/kg, p.o., daily), anti-PD-1 antibody (50 μg, i.p., twice weekly) or their combination (left) and percent change in tumor volumes between days 0 and 20 (right) (n=12).

B. BALB/c mice with subcutaneous CT26 tumors were treated with vehicle, TP-16 (75 mg/kg, p.o., daily), PD-1 antibody (50 μg, i.p., twice weekly) or combined therapies for 2 weeks. Animal survival (time to tumor burden reaching 2000 mm3) was analyzed by the Kaplan-Meier method using GraphPad Prism (n=10). P values were calculated using the Log-rank test.

C. Representative immunofluorescence staining images of tumor sections from CT26 tumor-bearing mice stained for CD8 and DAPI. Scale bars, 50 μm.

D. Representative immunostaining images of tumor sections from CT26 tumor-bearing mice stained for p-STAT3, p-AKT and PD-L1. Scale bars, 100 μm.

E. Transcriptome profile changes mediated by the synergy of TP-16 and anti-PD-1 analyzed using Agilent SurePrint G3 Mouse GE V2.0 Microarray. Venn diagrams depicting the number of changed genes (FC ≥ 2 or FC ≤ −2, P ≤ 0.05) compared with vehicle control following TP-16, anti-PD-1 antibody, or TP-16 and anti-PD-1 combined therapy.

F. The heatmap of T cell activation biomarkers for RNA-seq analysis. The unit of heatmap scale is expression value after centering and scaling by genes.

Data are presented as mean ± SEM. (A) One-way analysis of variance One-way analysis (ANOVA), Tukey's multiple comparison; **P < 0.01; ***P < 0.001. (B) Log-rank test; *P < 0.05; **P < 0.01; ***P < 0.001. Exact P values and statistical tests are listed in Appendix Table S8.

Source data are available online for this figure.
Reprogramming Immunosuppressive Myeloid Cells Facilitates Immunotherapy for Colorectal Cancer (2020)
Figure 7

Figure 7. TP-16 render anti-PD-1 antibody effective in AOM/DSS-induced colorectal cancer model.

A. Schematic diagram of the establishment of azoxymethane/dextran sodium sulfate (AOM/DSS)-induced colorectal cancer model and drug therapy regimen. AOM/DSS-induced colorectal tumor mice were treated with vehicle, TP-16 (75 mg/kg, po, daily), PD-1 antibody (50 μg, ip, twice weekly), or TP-16 and anti-PD-1 combined. The endpoint was day 37 from treatment initiation.

B-D. Representative resected colons (B), total number of colon tumors per mouse (C, left) and tumor size distribution (C, right), and colon length (D) after 5 weeks of indicated treatment (n=7).

E. Representative hematoxylin and eosin (H&E) staining and immunostaining for CD8, p-STAT3, p-AKT, and ARG-1 of tumors treated with vehicle, TP-16, anti-PD-1 antibody, or TP-16 and anti-PD-1 combined. Scale bars, 500 μm (left images), 50 μm (right images).

F. Quantitative polymerase chain reaction (q-PCR) analysis of the mRNA level of genes associated with immunosuppression (n = 6).

Data are presented as mean ± SEM. (C, left panel) One-way analysis of variance (ANOVA), Tukey's multiple comparison; **P < 0.01; ***P < 0.001. (C, right panel) Two-tailed unpaired Student's t test; *P < 0.05; **P < 0.01; ***P < 0.001. (F) Exact P values and statistical tests are listed in Appendix Table S8.

Source data are available online for this figure.

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