EMBO/soda-nel huggingface dataset:
Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy (2012)
figf4

(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT with or without MSO. After 24 h, the cells were starved of serum, amino acids and glucose in D-PBS containing the indicated inhibitors and stimulated with amino acids for 10 min. Cell lysates were analysed for protein levels of phospho-S6K (Thr 389) and actin. Shown are representative blots of three independent experiments. (b,c) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO (0.25 M) for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy. (b) Shown are representative pictures (n=4). Scale bars, 100 μm. (c) Quantification of co-localization between mTOR and LAMP2 as seen in b. Depicted are the mean ±  s.e.m. of the percentage of co-localized pixels from four experiments.
*P0.05,
**P0.01. (d) DLD1 cells expressing FOXO3(A3)-ER were treated with or without 4-OHT, MSO and BafA1 (200 nM). After 24 h, cell lysates were analysed for protein levels of LC3 and tubulin. Shown are representative blots (n=4). (e) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetase (GS) siRNA or a non-targeting siRNA and stimulated with 4-OHT in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of glutamine synthetase, LC3 and actin. Shown are representative blots (n=2). (f) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetase siRNA or non-template (NT) control siRNA and stimulated with 4-OHT for 16 h. Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase. Shown are representative blots (n=2). (g) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h. Cell lysates were analysed for protein levels of LC3 and actin. Shown are representative blots (n=4). Uncropped images of blots are shown in Supplementary Figs S7 and S8.
Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy (2012)
figf5

(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and ER and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm. (b) Quantification of LC3-positive spots as seen in a using ImageJ software. Depicted are mean ±  s.e.m. of the number of LC3-positive spots divided by the number of DAPI-positive cells (n=4).
*P0.05. (c) MSCs expressing FOXO3(A3)-ER were treated with or without 4-OHT and BafA1 (100 nM). At the indicated time points, cell lysates were analysed for protein levels of glutamine synthetase (GS), LC3, pS6 (Ser 235/236) and actin. Shown are representative blots (n=2). (d) IL-3 was withdrawn from Ba/F3 cells in the presence or absence of BafA1 (100 nM) as indicated. Cell lysates were analysed for protein levels of glutamine synthetase, LC3 and hsp90. Shown are representative blots (n=2). (e) DLD1 cells were stimulated with l-glutamine (l-Gln) in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of LC3, p62, p-S6K (Thr 389) and actin. Shown are representative blots (n=3). (f) DLD1 cells were starved and stimulated with glutamine (5 and 10 mM). Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy, and the co-localization between mTOR and LAMP2 was quantified by ImageJ. Depicted are the means of the percentage of co-localized pixels (n=2). (g) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP–WIPI-1 and subsequently stimulated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS or medium without amino acids for 24 h. GFP–WIPI-1 puncta formation analysis was performed by confocal microscopy. Depicted are the percentages of cells positive for GFP–WIPI-1 puncta. Shown are the mean ±  s.e.m. (n=4).
*P0.05 and
**P0.01. Uncropped images of blots are shown in Supplementary Fig. S8.
Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy (2012)
figf6

(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP–ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm. (b) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP–WIPI-1 and subsequently treated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h, cells were analysed for GFP and p62 expression by confocal microscopy. Shown are representative pictures (n=3). Scale bar, 20 μm. (c) DLD1 cells expressing FOXO3(A3)-ER were treated with 4-OHT and MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h cells were analysed for endogenous Atg12 puncta formation by confocal microscopy. Shown are the mean ± s.d. (n=3).
*P0.05. (d) DLD1 cells expressing FOXO(A3)-ER were transfected with glutamine synthetase (GS) siRNA or non-template (NT) control siRNA, and stimulated with 4-OHT for 24 h with or without 3-methyladenine (3-MA). Apoptosis was determined by FACS analysis after labelling cells with annexin V (AxV)–phycoerythrin and DAPI. The graph shows relative fold versus control samples, mean ±  s.e.m. (n=3).
*P0.05.
Cardio-respiratory and phenotypicrescue of dystrophin/utrophin-deficientmice by combination therapy (2022)
Figure 1

Figure 1. Effects of dietary supplementation of glycine on the activity of PMO in adult mdx mice.

PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration.

(A) Diagram of dosing regimen for PMO supplemented with glycine orally in mdx mice. I.v. refers to intravenous injection.

(B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO in saline (PMO-S), PMO-G(IV) or PMO-G (D)(scale bar=100μm).PMO-G (IV) refers to intravenous injection of PMO in combination with intravenous injection of 5% glycine every other day for 3 weeks. PMO-G (D) represents intravenous injection of PMO supplemented with 1% glycine orally.

(C) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (IV), PMO-G (D) or PMO-S. 0.5µg, 2.5 µg, 5µg, 10µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, D-diaphragm and A-abdominal muscle.

(D) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (i.v., n=4), PMO-G (D25 and D50, n=3) and PMO-S (n=3) (*P<0.05,**P<0.001; One way-ANOVA post hoc Student-Newman-Keuls test).

Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.
Cardio-respiratory and phenotypicrescue of dystrophin/utrophin-deficientmice by combination therapy (2022)
Figure 2

Figure 2. Evaluation of PMO supplemented with glycine orally in DKO mice. PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (A) Muscle function was assessed to determine the physical improvement with grip strength test (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). (B) Measurement of bodyweight changes of DKO mice treated with PMO-G or PMO-S (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). (C) Measurement of TA muscle mass of DKO mice treated with PMO-G or PMO-S (n = 3, *P < 0.05; one-way ANOVA post-hoc Student-Newman-Keuls test). (D) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm). (E, F) Western blot (D) and quantitative analysis (E) for the dystrophin protein in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle and D-diaphragm. 2.5 µg, 5µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.
Cardio-respiratory and phenotypicrescue of dystrophin/utrophin-deficientmice by combination therapy (2022)
Figure 3

Figure 3. Systemic investigation of combinatorial effects of PMO with dietary supplementation of glycine and metformin in DKO mice.

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction (PMO-G) and metformin daily via drinking water (PMO-GM).

(A) Diagram of dosing regimen for PMO supplemented with glycine and metformin orally in DKO mice. I.v. refers to intravenous injection.

(B) Measurement of Ejection Fraction (EF) and Fractional Shortening (FS) in treated DKO mouse hearts (n=3) and C57BL/6 mice (n=6) with echocardiography (*P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test).

(C) Morphological examination of treated DKO mouse hearts with H&E staining (scale bar=800μm). Boxed areas are magnified. (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test)

(D-F) Immunohistochemistry for immunoglobin G (IgG) in treated DKO mouse hearts (D, scale bar=100 μm). Muscle function was evaluated to determine the physical improvement with grip strength (E) or running wheel tests (F) for treated DKO (n=3) or C57BL/6 mice (n=4) (*P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test).

(G) Immunohistochemistry for dystrophin expression in body-wide muscles from treated DKO mice (scale bar=100 μm).

(H) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3). 5µg, 10µg and 20µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control.

Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.
Cardio-respiratory and phenotypicrescue of dystrophin/utrophin-deficientmice by combination therapy (2022)
Figure 4

Figure 4. Long-term combinatorial effects of PMO with dietary supplementation of glycine and metformin (PMO-GM) in DKO mice.

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water.

(A) Diagram of long-term dosing regimen for PMO supplemented with glycine and metformin orally in DKO mice. I.v. refers to intravenous injection.

(B) Survival rate of DKO mice treated with PMO-GM (n=6).The number of untreated DKO controls is 15.

(C) Measurement of kyphosis index of DKO mice treated with PMO-GM (scale bar=5mm) and wild-type C57BL/6 mice (scale bar=8mm)(n=3, *P<0.05; One way- ANOVA post hoc Student-Newman-Keuls test).

(D) Measurement of tidal volume in treated DKO mice with a cardio-respiratory gated technique (n=3, *P<0.05; one-way ANOVA post hoc Student-Newman-Keuls test). (E-F) Examination of EF and FS (E) and stroke volume and cardiac output (F) in treated DKO mouse hearts (n=3) and C57BL/6(n=5) with echocardiography (*P<0.05, **P<0.001; one-way ANOVA post hoc Student-Newman-Keuls test).

(G) Masson trichrome's staining for cardiac fibrosis and quantitative analysis of fibrotic areas in treated DKO mouse hearts (scale bar=800 μm). Boxed areas are magnified (n=3, **P<0.001;One way-ANOVA post hoc Student-Newman-Keuls test).

(H) Measurement of serum creatine kinase-MB (CK-MB) levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test).

(I-J) Muscle function was assessed to determine the physical improvement with grip strength (I) or running wheel tests (J) in treated DKO (n=4) and C57BL/6 mice (n=6) (*P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test).

Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.
Cardio-respiratory and phenotypicrescue of dystrophin/utrophin-deficientmice by combination therapy (2022)
Figure 5

Figure 5. Molecular correction and pathological assessment of DKO mice treated with long-term repeated administration of PMO-GM.

PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO introduction, followed by 50 mg/kg/month for 5 months with dietary supplementation of glycine for 7 consecutive days per month prior to PMO introduction, in combination with metformin daily via drinking water.

(A) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm).

(B) Western blot and quantitative analysis for dystrophin protein in treated DKO mice (n=3).5µg, 10µg and 25µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control.

(C) Re-localization of DAPC components in treated DKO mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar=50μm). The arrowheads point to identical myofibers.

(D) Measurement of serum CK levels in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test).

(E-F) Measurement of serum indices from treated DKO mice to reflect liver (E) and kidney (F) functions (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman- Keuls test).

(G) Morphological examination of liver and kidney from treated DKO mice (scale bar=50 μm).

Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified. Source data are available online for this figure.

Expanded View Figure Legends
RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells (2020)
Figure 2

Figure 2. GTP- and GDP-RhoJ differentially associate with PlexinD1 and VEGFR2.

Confocal (left three columns) and super-resolution (right-most columns) images of subcellular distributions of ectopic RhoJ (red), endogenous VEGFR2 (green) and PlexinD1 (left, white; right, blue) in cultured HUVECs 6 h after transfection with the indicated RhoJ constructs. Note the colocalization of WT-RhoJ and CA-RhoJ (Q79L) with PlexinD1 in perinuclear endosomes and DN-RhoJ (T35N) with VEGFR2 in cytoplasmic and nuclear puncta. Light-blue asterisks in the PlexinD1 panels indicate nuclei. See also Appendix Fig S2 for the full images of super-resolution microscopy. Scale bar, 5 µm (left); 1 µm (right).

Proportion of ectopic RhoJ that colocalized with endogenous VEGFR2 and/or PlexinD1. n = 10 cells per group. Data represent mean.

Co-immunoprecipitation (co-IP) from 293T cells 48 h after transfection. WT- and CA-RhoJ associated with PlexinD1, while DN-RhoJ associated with VEGFR2.

Source data are available online for this figure.
RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells (2020)
Figure 3

Figure 3. Sema3E induces EC contraction by releasing RhoJ from PlexinD1.

Schematic representation of full-length PlexinD1 (PD1) and its deletion mutants.

Co-IP from 293T cells 48 h after transfection. RhoJ-PlexinD1 binding was disrupted by deleting the RBD or ECD of PlexinD1 and by 30-min stimulation with Sema3E. Arrows indicate PD1 Full (upper, 250 kDa) and PD1△RBD (lower, 1.7 kDa smaller than PD1 Full).

PlexinD1 internalization in HUVECs 30 min after Sema3E stimulation. Scale bar, 10 µm.

Super-resolution images of subcellular distributions of ectopic WT-RhoJ (red) and endogenous PlexinD1 (green) in HUVECs 24 h after transfection. RhoJ dissociated from internalized PlexinD1 30 min after Sema3E stimulation. Scale bar, 1 µm.

Labeling of siRNA-transfected HUVECs with phalloidin (white) 30 min after Sema3E stimulation. Scale bar, 50 µm.

Quantification of areas of individual HUVECs. n > 100 cells per group. Data represent mean ± SEM. ***p < 0.001; NS, not significant, by Tukey-Kramer test.

A model of Sema3E-induced cell contraction.

Source data are available online for this figure.
RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells (2020)
Figure 4

Figure 4. RhoJ facilitates Sema3E-indcued reverse EC migration via activation of VEGFR2 and p38 MAPK.

Co-IP from HUVECs. Blots for β-actin shown in (A) were used to confirm equal loading in (B), as the same cell lysates were analyzed in both experiments.

Immunoblots in HUVECs.

Immunoblots in siRNA-transfected HUVECs.

Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.

Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after Sema3E stimulation. Left panels show immunolabeling of VEGFR2 (green), PlexinD1 (red), and Nrp1 (white), with light-blue asterisks indicating nuclei. Middle panels show magnified views of white boxes in the left panels. Right panels represent fluorescence intensity profiles along 5 μm arbitrary lines in the middle panels. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in PlexinD1-positive vesicles. n = 10 cells per group.

Trajectory plots (left) and rose plots (right) of HUVECs pre-treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h. Red trajectories indicate cells with displacement to the negative y-axis at the end of analyses. P values for the Rayleigh test represent non-random distributions of cell endpoints. The graphs show FMI along the y- and x-axes during cell migration. n = 90 cells per group.

A model of Serma3E-induced reverse cell migration.

Data information: Data represent mean ± SEM (D and F) and mean (E). *p < 0.05; ***p < 0.001; NS, not significant, by Tukey-Kramer test (D) and Student's t-test (E and F).

Source data are available online for this figure.
RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells (2020)
Figure 5

Figure 5. RhoJ sustains VEGF signals by preventing degradation of internalized VEGFR2.

Super-resolution images of receptor colocalization in siRNA-transfected HUVECs 30 min after VEGF stimulation. VEGFR2 (green), PlexinD1 (red), and Nrp1 (white) are shown as in Fig 4E. Scale bar, 10 µm. The graph shows the proportion of receptor complexes in the PlexinD1-positive vesicles. Data of "no stimuli" are the same from Fig 4E. n = 10 cells per group.

Co-IP from siRNA-transfected HUVECs. The graph shows relative levels of PlexinD1-VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.

Immunoblots in siRNA-transfected HUVECs. Blots for β-actin shown in (C) were used to confirm equal loading in (D), as the same cell lysates were analyzed in these experiments. The graph shows changes of total VEGFR2 protein levels as a percentage of the values at 0 min. n = 3 per group.

Immunoblots in siRNA-transfected HUVECs. Note the early and transient activation of PLCγ, Erk1/2, and Akt, but not p38 MAPK, in RhoJ-knockdown ECs.

Immunoblots for ectopic RhoJ and endogenous VEGFR2 in HUVECs 12 h after transfection of RhoJ-expressing vectors. The graph shows protein levels of VEGFR2 relative to those of the control set as 100%. n = 3 per group.

A model of VEGF-induced forward cell migration.

Data information: Data represent mean (A) and mean ± SEM (B, C, and E). *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, by Student's t-test (A) and Tukey-Kramer test (B, C, and E).

Source data are available online for this figure.
RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells (2020)
Figure 6

Figure 6. RhoJ promotes directional EC migration in sprouting vessels.

Kinetic analyses of EC migration in an ex vivo aortic ring angiogenesis model. Upper panels are snapshots of time-lapse imaging in which EC nuclei are labeled by SYTO dye (green). In analyzed regions framed by dotted lines, the centroids of the first five EC nuclei from the tip at 0 min are pseudocolored in magenta and those of the remaining EC nuclei are shown in cyan. Initial discrete labeling becomes intermixed over time (see also Movie EV1). Lower panels show trajectory analyses representing normalized positional changes of individual ECs during vessel elongation. Scale bar, 100 µm.

Quantification of the kinetics of EC migration. Values of forward and reverse migration for RhojWT/WT are normalized to 1. n = 33 vessel branches per group.

Quantification of vessel elongation (left graph). n = 33 vessel branches per group. Scatter plot shows relationship between "vessel elongation" and "reverse migration" in RhojGFP/GFP aortic rings as evaluated by a Pearson correlation coefficient.

Schematic representation for construction of Rhoj mutant mice. Cre-loxP-mediated genetic recombination of the Rhoj-flox allele generates the Rhoj-KO allele by excising the RhoJ cDNA-polyA cassette inserted into exon 1 of the Rhoj gene.

Labeling for YFP/GFP (green), CD31 (red), and ETS transcription factor ERG (blue) in retinal vascular fronts of P5 CAG-MerCreMer:R26R-EYFPflox/WT and CAG-MerCreMer:Rhojfloxflox mice after intraperitoneal (i.p.) injections of 10 µg of 4-hydroxytamoxifen (4OHT) at P1. Scale bar, 50 µm.

Relative contribution of YFP/GFP-positive ECs to the tip positions. n = 16 per group. Values for CAG-MerCreMer:R26R-EYPflox/WT are normalized to 1.

Data information: Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, by Student's t-test (B and C) and Mann-Whitney U-test (F).
RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells (2020)
Figure 7

Figure 7. RhoJ-deficiency delays vascular development and suppresses pathological angiogenesis.

Labeling for GFP (green) and CD31 (red) in retina of P4 RhojGFP/GFP mouse. Scale bar, 200 µm (left); 20 µm (right).

Labeling for EdU (green), ERG (red), and CD31 (gray) in retinas of P4 RhojWT/WT and RhojGFP/GFP mice 2 h after i.p. EdU injections. Scale bar, 20 µm. The graph shows EC proliferation index in retinal capillaries behind the angiogenic fronts. n = 12 per group.

Labeling for CD31 in retinas of P4 and P7 RhojWT/WT and RhojGFP/GFP mice. Scale bars, 500 µm.

Morphometric analyses of retinal vessels in RhojWT/WT (P4, n = 18; P7, n = 8) and RhojGFP/GFP (P4, n = 16; P7, n = 11) mice.

Labeling for CD31 in retinas of P4 Rhojflox/flox and Pdgfb-iCreERT2:Rhojflox/flox (RhojiΔEC) mice after single i.p. injection of 100 µg of 4OHT at P1. Scale bar, 500 µm.

Morphometric analyses of retinal vessels in P4 Rhojflox/flox (n = 7) and Rhoji∆EC (n = 6) mice.

Relative expression levels of Vegfa and Sema3e mRNA from WT mouse retinas. n = 3 per group.

Labeling for GFP (green) and CD31 (red) in retina of P18 OIR-RhojGFP/WT mouse. Scale bar, 200 µm.

Labeling for CD31 in retinas of P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice after daily i.p. injection of 200 µg of 4OHT from P12. Scale bar, 500 µm.

Quantification of areas of neovascular tufts in P18 OIR-Rhojflox/flox and OIR-Rhoji∆EC mice. n = 6 per group.

Data information: Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant, by Mann-Whitney U-test (B, D, F, and J) and Student's t-test (G).

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