EMBO/soda-nel huggingface dataset:
Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy (2016)
Figure 6

Figure 6. PFT-α protects cochlea from ototoxicity without compromising and even enhancing CDDP anti-tumor efficacy in patient-derived breast cancer xenograft mice.

A ABR thresholds recorded prior to (gray plot) or at day 28 in HBCx-90 (TP53 wt) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).

B Mean ABR threshold from 4 kHz to 32 kHz derived from A. Data are expressed as mean ± SEM (before: n=20; DMSO: n=5; PFT-α: n=5; CDDP: n=5; CDDP+PFT-α: n=5). One-way ANOVA test followed by post hoc Tukey’s test (***P = 0.0002, CDDP vs. before; CDDP vs. CDDP+PFT-α.

C Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 28 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal–Wallis test followed by post hoc Dunn's test (**P ≤ 0.007, CDDP, d28 vs. DMSO, d28 or CDDP, d28 vs. CDDP+PFT-α, d28).

D Tumor growth curves alongside images of dissected tumors collected on day 28. Note the partial inhibition of growth in CDDP and CDDP+PFT- α-treated mice.

E ABR thresholds recorded prior to (gray plot) or at day 35 in HBCx-14 (TP53 mutant) bearing-mice received systemic treatment with: DMSO (black plot), PFT-α (yellow plot), CDDP (green plot), CDDP+PFT-α (pink plot).

F Mean ABR threshold from 4 kHz to 32 kHz derived from E. Data are expressed as mean ± SEM (before: n=30; DMSO: n=5; PFT-α: n=5; CDDP: n=10; CDDP+PFT-α: n=10). One-way ANOVA test followed by post hoc Tukey’s test (***P = 0.0003, CDDP vs. before or CDDP vs. CDDP+PFT-α.

G Cytocochleograms representing the percentage of surviving hair cells in three cochlear regions located at 1.1, 2.6, and 3.5 mm from the cochlear apical end. These cochleae (n=5 per group) were collected at 35 days from the beginning of treatment with DMSO (black bars), PFT-α (yellow bars), CDDP (green bars), or CDDP+PFT-α (pink bars). Data are expressed as mean ± SEM. Kruskal–Wallis test followed by post hoc Dunn's test (**P ≤ 0.008, CDDP, d35 vs. DMSO, d35 or CDDP, d35 vs. CDDP+PFT-α, d35).

H Tumor growth curves alongside images of dissected tumors collected on day 35. Note the complete disappearance of tumors at day 35 and the significantly reduced tumor regrowth up to day 70 in CDDP+PFT- α-treated mice.
Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy (2016)
Figure 7

Figure 7. PFT-α enhances CDDP-induced anti-angiogenesis and suppresses autophagy selectively in TP53 mutant tumors.

A Representative confocal images of microvessels in transversal tumor sections from HBCx-14 (TP53 mutant) tumors treated with either DMSO, CDDP or a combination of CDDP and PFT-α. The sections were immunolabeled for CD31 (red) and viewed with a 20x objective. The basal-like breast cancer cells were immunolabeled in green with an antibody against cytokeratin 5 and 8. Upper right panel is a 2D projection from the white boxed area in upper left panel. The red area corresponds to CD31 labeled endothelial area and the white area represents the lumen area. The white line shows the vessel perimeter. Scale bars: left panel=50 µm, right panels=35 µm. The tumor samples were collected at day 21.

B Histograms representing the percentage of vascular area calculated using 2D reconstruction image analysis and the formula: vessel/vascular area = area of CD31-positive objects + lumen area per field area × 100 %. The both HBCx-14 (TP53 mutant) and HBCx-90 (TP53 wt) tumors from the different treated groups were collected at day 21 (n=4 sections/tumor and 3-4 tumors/group). Data are expressed as mean ± SEM. Kruskal–Wallis test followed by post hoc Dunn's test (***P ≤0.0004, CDDP vs. DMSO or CDDP vs. CDDP+PFT-α).

C Representative confocal images of cleaved caspase 3 positive cells (red) in the transversal tumor sections and viewed with a 20x objective. The stromal compartments were immunolabeled in green with an antibody against vimentin. HBCx-14 (TP53 mutant) tumors from the different treated groups were collected at day 18. Scale bar=50 µm. c-cas-3: cleaved caspase 3.

D Representative western blot analysis using antibodies against β-actin, p53 and p21 in tumor extracts from HBCx-14 (TP53 mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO or CDDP and collected at day 18. Note the higher CDDP-induced increase of p53 and p21 expression in TP53 wt HBCx-90 tumors.

E, F Representative western blot analysis using antibodies against β-actin, p-Chk1, Beclin 1, LC3-I/II and Rab7 in tumor extracts from HBCx-14 (TP53 mutant) and HBCx-90 (TP53 wt) tumors treated with either DMSO, PFT-α, CDDP or a combination of CDDP and PFT-α. The tumor samples were collected at day 18.

G, H, I Histograms representing the levels of Beclin 1, LC3-II and Rab7 in HBCx-14 and HBCx-90 tumors treated with the different regimens (n=3-4 tumors per group, all experiments were performed in triplicate). Actin served as a loading control. Data are expressed as mean ± SEM. One-way ANOVA test followed by post hoc Tukey’s test (G: *P = 0.03, **P =0.006 , ***P ≤ 0.0004 ; H: *P =0.02, ***P = 0.0005 ; I: *P =0.03, ***P ≤ 0.0006 , CDDP vs. DMSO or CDDP vs. CDDP+PFT-α).
Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses (2021)
Figure 1

Figure 1. Neuronal Cdc50a deletion induces rapid lethality and audiogenic seizures

A. Representative confocal z-stack images of co-immunostaining for β-galactosidase (green) and CAMKII (red, upper) or GAD67 (magenta, lower) in neurons from the brains of Cdc50a knockout-first allele (Cdc50a lacZ/+) mice. Scale bar, 20 μm.

B. Survival rates of control and Cdc50a cKO mice after oil or 4-OHT injection (n = 5 for each group, P*** = 0.0003).

C. Schematic diagram of the experiment. Mice of both genotypes were sacrificed 13 days after oil or 4-OHT injection. Representative confocal z-stack images of cleaved caspase-3 (cCas3, red)- and Thy1-CreERT2, -EYFP (green)-expressing cells from control and Cdc50a cKO mice. Scale bar, 20 μm.

D. Schematic diagram of behavioral tests for measuring audiogenic seizures (0: normal behavior, 1: wild running, 2: seizure, and 3: respiratory arrest and death) following auditory stimuli.

E, F. Bar graphs showing audiogenic seizure scores following auditory stimuli (5, 10, 15, 20, 40 kHz or white noise (WN) at 70, 80, 90, or 100 dB) in control (E) and Cdc50a cKO (F) mice (n = 8 for each group).

G. Representative confocal z-stack images showing cFos (magenta)-positive neurons from control and Cdc50a cKO mice (CTX: cortex, HP: hippocampus, and IC: inferior colliculus). Scale bar, 20 μm.

H. Bar graphs showing the relative area of cFos-positive cells in the brains of control and Cdc50a cKO mice (n = 5 for each group, **P = 0.0030, n.s., not significant).

Data information: The individual dots indicate experimental replicates (3 to 5 images were taken from 2 brain slices per an animal, H). The data are the mean ± s.e.m. and were analyzed by the log-rank (Mantel-Cox) test (B) or two-tailed unpaired t-test (H).
Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses (2021)
Figure 2

Figure 2. Neuronal Cdc50a deletion induces loss of inhibitory post-synapses

A, B. Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT1 (red)-positive excitatory (A) and Gephyrin (green)- and VGAT (magenta)-positive inhibitory (B) synapses in the CTX, HP, and IC (CTX: cortex, HP: hippocampus, and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.

C, D. Bar graphs showing the numbers of excitatory (C, PSD95 and VGLUT1) and inhibitory (D, Gephyrin and VGAT) synapses in the CTX, HP, and IC of control and Cdc50a cKO mice (n = 5 for each group, for excitatory synapses: n.s., not significant, for inhibitory synapses: CTX, P*** = 0.0001, P** = 0.0023, HP, P** = 0.0038, IC, P** = 0.0023, P**** < 0.0001).

E. Representative confocal z-stack images of GABAA receptor α (red) and β (green) staining in the IC in control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.

F. Representative current clamp traces from the IC of control and Cdc50a cKO mice; +100 pA (upper) and +400 pA (lower) currents were injected into control mice (left) and Cdc50a cKO mice (right) for 500 ms.

G. Frequency-current (F-I) curve of the IC of control and Cdc50a cKO mice. The number of spikes per 500 ms was measured and averaged for each current (each, n = 9 and 13 cells, 300 pA: *P = 0.0203, 350 pA: *P = 0.0133, 400 pA **P = 0.0098, 450 pA: **P = 0.0072).

Data information: The individual dots indicate experimental replicates (3 to 5 images were taken from 2 brain slices per an animal, C and D). The data are the mean ± s.e.m. and were analyzed by two-tailed unpaired t-test (C and D) or two-way ANOVA (G).
Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses (2021)
Figure 4

Figure 4. Neuronal Cdc50a deletion increases microglial phagocytosis of inhibitory post-synapses and prevents the loss of inhibitory post-synapses by microglial ablation

A. Representative confocal z-stack images of microglial lysosomes (CD68, green) in the IC (inferior colliculus) of control and Cdc50a cKO mice. Scale bar, 20 μm.

B. Representative confocal z-stack and colocalization images of Gephyrin (green), IBA1 (blue), and CD68 (red) in the IC of control and Cdc50a cKO mice. Scale bar, 20 μm.

C. Bar graphs showing the relative colocalization area of Gephyrin and CD68 in the IC of control and Cdc50a cKO mice (n = 3 for each group, **P = 0.0024).

D. Schematic diagram of microglial ablation in control and Cdc50a cKO mice using DMSO- or PLX3397-containing chow. DMSO- or PLX33997-containing chow was administered for 2 weeks before oil or 4-OHT injection. Bar graphs showing the audiogenic seizure scores of Cdc50a cKO mice treated with DMSO or PLX3397 after a 20 kHz/90 dB auditory stimulus (n = 7 for Cdc50a cKO;DMSO and 3 for Cdc50a cKO;PLX3397).

E. Representative confocal z-stack images of microglia (IBA1, green, upper) and inhibitory synapses (Gephyrin: green, VGAT: red, lower) in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.

F. Bar graphs showing the relative area of IBA1-positive cells in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, control;DMSO versus control;PLX3397, *P = 0.0428, control;DMSO versus Cdc50a cKO;DMSO, ***P = 0.0002, Cdc50a cKO;DMSO versus Cdc50a cKO;PLX3397, ***P = 0.0005).

G. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, P**** < 0.0001).

Data information: The individual dots indicate experimental replicates (3 to 5 images were taken from 2 brain slices per an animal, C, F and G). The data are the mean ± s.e.m. and were analyzed by two-tailed unpaired t-test (C and D) or one-way ANOVA with Tukey's test (F and G).
Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses (2021)
Figure 5

Figure 5. Mertk deficiency prevents the loss of inhibitory post-synapses and audiogenic seizures in Cdc50a cKO mice

A. Schematic diagram of the experiment. Bar graphs showing the audiogenic seizure scores of Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice after a 20 kHz/90 dB auditory stimulus (n = 4 for each group).

B. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC (inferior colliculus) of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm.

C. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice (n = 4 for each group, **P = 0.0022, ****P < 0.0001, n.s., not significant).

D. Schematic diagram of the experiment. Bar graphs showing the audiogenic seizure scores of Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice after a 20 kHz/90 dB auditory stimulus (n = 4 for each group).

E. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.

F. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice (n = 4 for each group, P**** < 0.0001, P*** = 0.0002, P** = 0.0021).

Data information: The individual dots indicate experimental replicates (3 to 5 images were taken from 2 brain slices per an animal, C and F). The data are the mean ± s.e.m. and were analyzed by two-tailed unpaired t-test (A and D) or one-way ANOVA with Tukey's test (C and F).
Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses (2021)
Figure 6

Figure 6. Microglial Mertk is required for normal pruning of PS-exposed inhibitory post-synapses

A. Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm.

B, C. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant).

D. Schematic diagram of the experiment in which Annexin V was injected intravenously (every two days) to block PS in the brains of 1-month- and 3-month-old wild-type mice. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the HP of 1-month-old wild-type mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm.

E. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of 1-month-old mice after PBS or Annexin V injection (n = 4 for each group, ***P = 0.0013, **P = 0.0090).

F. Schematic diagram of the experiment in which microglial Mertk KO (CX3CR1-CreERT2;Mertkfl/fl) and control (Mertkfl/fl) mice were generated. Both 1-month-old microglial Mertk KO mice and 1-month-old control mice were injected with 4-OHT for 5 consecutive days. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm.

G. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, **P = 0.0027, *P = 0.0062).

H. Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue) and secA5 (red) in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm.

I. Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, *P = 0.0015).

Data information: The individual dots indicate experimental replicates (3 to 5 images were taken from 2 brain slices per an animal, B, C, E, G, and I). The data are the mean ± s.e.m. and were analyzed by two-tailed unpaired t-test.

EXPANDED VIEW FIGURE LEGENDS
IGSF10 regulates embryonic GnRH neuronal migration and mutations result in delayed puberty (2016)
Figure 4

Figure 4 - Expression pattern of Igsf10 mRNA in mouse and human developing brain

A-J: Igsf10 expression was not observed at E10.5 (A, sagittal section) but readily detectable at E12.5, in the NM (B and C, frontal sections) and E14.5, along the migratory path of GnRH neurons (D, frontal section). Igsf10 positive cells were negative for Isolectin b4 (marker of vasculature) at all developmental stages (E, at E14.5). Incubation with the sense probe resulted in no signal at all stages (E14.5 shown in F). Peripherin-positive cells olfactory axons were extending within the Igsf10-positive NM (G and H). Cxcl12 mRNA expression is shown in I (E14.5, frontal section). GnRH neurons are located in the MPOA by E17.5 (J, frontal section) in an Igsf10 negative area.

K-N: In human 11pcw brains, IGSF10 expression pattern was similar to that observed in mouse, with GnRH neurons interspersed in an IGSF10-positive NM (K-N, frontal sections, see also schematic in P). IGSF10 positive cells were also detected in the olfactory bulb (OB, panel O). Sense probe resulted in no specific signal (panel N).

GnRH neurons are shown by red arrowheads and Isolectin b4 by green arrowheads. NS: nasal septum. Scale bar, 250 um (panel A), 100 um (panels B, E, H), 50 um (panels C, K-O), 200 um (panels D, F, G, I, J).
IGSF10 regulates embryonic GnRH neuronal migration and mutations result in delayed puberty (2016)
Figure 5

Figure 5 - Effect of Igsf10 knockdown on GnRH neuronal migration

A: Levels of Igsf10 expression in native NIH3T3 cells, and cells stably expressing a scrambled (sh-scr) or Igsf10 (sh-Igsf10) shRNA.

B and C: migration of GN11 cells from aggregates into NIH3T3 sh-scr (B) or sh-Igsf10 (C).

D: Analysis of the migration index (see Methods).

E: RT-PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points. Expected band size: 905 bp.

F: Representative time-lapse analysis of mispair control (Control Sp-MO and Control ATG-MO) and Igsf10 morpholinos (Igsf10 Sp-MO and Igsf10 ATG-MO) injected Tg(gnrh3:EGFP) embryos and larvae, at different time-points. Images show lateral view of live larvae head (48 and 72 hpf) and dorsal view (4 and 5 dpf) of live larvae head; anterior is left. White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos. Brackets show the extension of the projections towards the hypothalamus. White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus, indicated with Δ (hpf – hours post fertilisation; dpf – days post fertilisation.) Scale bar, 100 um (panels at 48 and 72hpf in F), 150 um (panels at 4 and 5dpf in F). Panel G: Effect of Igsf10 Splice-site morpholino injection observed at 48hrs. Quantification of the percentage of embryos showing an abnormal GnRH3-neuron phenotype was significantly higher in Igsf10 Splice-site-MO (Sp-MO) injected embryos compared to relative controls. *: p=0.02422, **: p=0.00101, ***: p=0.00020, ****: p=0.00001.
Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy (2019)
Figure 1

Figure 1 - Genetic and pharmacological inhibition of Apelin impairs mammary tumor growth and tumor angiogenesis in a paracrine manner.

(A) Kaplan Meier plot for survival in NeuT;Apln+/+ (n=11) and NeuT;Apln-/- (n=10) mice with mammary cancer after tumor onset. *P=0.0185; Log rank test. Mice were sacrificed when the tumor size reached 1cm3, following ethical guidelines. (B) Tumor volumes, followed over time, of control mammary tumor E0771 cells (shRenilla) and Apelin-depleted (shApln) E0771 cells orthotopically-injected into both syngeneic C57BL/6J Apln+/+ and Apln-/- mice (5x105 cells/mouse), respectively. Tumor volumes were determined using calipers and are shown as mean tumor volumes ± S.E.M. Data shown is pooled from 2 independent experiments. Apln+/+;shRenilla (n=18), Apln+/+;shApln (n=17), Apln-/-;shRenilla (n=14), Apln-/-;shApln (n=15); ** P<0.01, *** P<0.001; two-way ANOVA. (C) Mean percentages (± S.E.M.) of CD31+ area in E0771 shRenilla (n=3) or shApln (n=3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. (D) Mean percentages (± S.E.M.) of extravasated Dextran in E0771 shRenilla (n=9) or shApln (n=12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln+/+ or Apln-/- mice, respectively. **P<0.01; t test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 μm (E) Mean counts (± S.E.M.) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n=6) or shApln (n=4) into C57BL/6J wild-type mice (5x105 cells/mouse). *P<0.05; t test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; Scale bars = 200 μm (upper panels) and 50 μm (lower panels). (F) Mean percentage (± S.E.M.) of tumor-infiltrating immune cells normalized to CD45+. E0771 shRenilla (n=8) or shApln (n=6) were orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice (5x105 cells/mouse), respectively and tumors were harvested day 25 post-injection. *P<0.05, **P<0.01; t test. All immune cell populations were gated for viable CD45+ cells and then further defined as: CD8 T cells (Thy1.2+, CD8+), CD4 T cells, (Thy1.2+, CD4+), Inflammatory Monocytes (Ly6C+, Cd11b+, Ly6G-), PMN-MDSCs (Ly6G+, Cd11b+), natural killer cells (Thy1.2-, Ly6G-, NK1.1+), natural killer T cells (Thy1.2+, CD4-, CD8-, NK1.1+) and peripheral dendritic cells (Ly6G- ,Ly6C+, Cd11b- , PDAC1+, B220+).
Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy (2019)
Figure 2

Figure 2 - Apelin deletion delays VEGF-induced blood vessel sprouting.

(A) Left - Heatmap of RNA-Seq transcriptome analysis of CD31+/CD105+ endothelial cells sorted from tumors established by E0771 shRenilla (n=6) or shApln (n=3) cells orthotopically injected into C57BL/6J Apln+/+ or Apln-/- mice, respectively. Tumors were harvested day 25 post-injection and genes displayed are significantly deregulated at the adjusted p-value cut-off of 0.05. Right - Ingenuity Pathway Analysis for biological processes predicted to be decreased downstream of the differentially expressed genes. (B) Quantification of vessel sprouts (mean values ± S.E.M.) upon VEGF treatment (30ng/mL) of embryoid bodies (EB) derived from murine ES cells (mESCs) with sense integrations in the Apelin gene (Apln STOP) of the splice acceptor described in Appendix Figure S1B or Cre-reverted antisense (Apln GO) sister cells. Apln GO (Day 2 n=6; Day 4 n=40, Day 6 n=54, Day 8 n= 32, Day 10 n=35, Day 11 n=31), Apln STOP (Day 2 n=4; Day 4 n=15, Day 6 n=31, Day 8 n= 28, Day 10 n=32, Day 11 n=37); ** P<0.01, *** P<0.001, two-way ANOVA. Representative brightfield images and automated analysis of vessel sprouts by Definiens software are shown in the bottom panels. Scale bars = 200 μm (C) Differentially expressed genes using RNAseq transcriptome analysis of CD31+ endothelial cells (ECs) sorted from sprouting EBs from Apelin STOP cells stimulated with VEGF (30ng/mL) and DMSO (-Apln) and repaired Apelin GO sister cells stimulated with VEGF and Apelin (1000nM) (+Apln). VEGF target genes and angiogenesis-related genes, predicted by Ingenuity Pathway Analysis (IPA) software, are indicated by bars on the upper axis of the heatmap. GO terms were analyzed by DAVID on-line software.