EMBO/SourceData huggingface dataset:
Figure 6Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. (A+B) CD31 staining revealed that blocking of EGFL7, VEGF, or a combination of both lead to decreased tumor vessel density (n = 3; one-way ANOVA).Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. Staining for (C+D) PDGFRβ (n = 3; one-way ANOVA),Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. Staining for SMA (n = 3; one-way ANOVA)Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. Staining for Col IV (n = 3; one-way ANOVA) revealed decreased amounts of blood vessel-associated pericytes, smooth muscle cells and Col IV in the basement membrane of blood vessels upon treatment with anti-EGFL7, anti-VEGF and most significantly, a combination of both antibodies.Glioma-bearing mice were injected with anti-EGFL7 or anti-VEGF antibodies, a combination of both (Combo), or isotype negative controls. Mice were sacrificed upon showing first symptoms of disease and brains were analyzed by magnetic resonance imaging (MRI). Resulting brain tumor sections were analyzed for blood vessel density and maturation state by immunohistochemistry. This resulted in an increased intratumoral vessel permeability as measured by T1-weightened MRI analyses of extravasating Gadovist (n = 6; one-way ANOVA).Treatment with anti-EGFL7 (47 d), anti-VEGF (51 d), or a combination of both antibodies increased the median survival time of glioma-bearing Rag1-/- mice (58 d) as compared to isotype-treated controls (37 d; n = 6; log-rank test).Treatment of glioma-bearing NOD SCID mice with temozolomide (TMD) as a chemotherapeutic agent and a combination of anti-EGFL7 and anti-VEGF antibody increased the median survival time (Combo; 87 d; n = 5; log-rank test) as compared to anti-VEGF alone (80 d; n = 5; log-rank test) or isotype-treated controls (69 d; n = 5; log-rank test). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.
figf1(a) p62 localizes to the MR during cytokinesis and abscission, and is taken up together with the MR by one daughter cell. U2OS cells were fixed and stained with anti-p62 (red) and anti-MKLP1 (green) antibodies. Scale bars are 10 μm. Colocalization of p62 with MKLP1 of the MR was regularly observed in cells at this stage of cell division. The arrowheads point to MRs, which are shown enlarged in the insets. The quantification is presented in Supplementary Information Fig. S1a.(b) siRNA-mediated depletion of p62 from U2OS cells results in moderate MR accumulation, compared with a control depletion (lamin A/C). For quantification, MRs were counted in control cells and in p62-depleted cells that were negative for p62 staining. Data are means ± s.d. of three independent experiments (n, total number of cells counted; P, probability of a two-tailed paired t-test).
figf2(a) Atg8 (LC3) localizes to the MR during abscission. The upper panels show stills from time-lapse microscopy of GFP-Atg8-expressing cells. The arrowhead points to the accumulation of Atg8 at the site of the MR during abscission. The enlarged clipping on the right shows the midbody area at time-point 68 min. Scale bar is 10 μm.(b) Cytosolic MRs are found inside Atg8-positive vesicles. U2OS cells stably expressing GFP-Atg8 were fixed and stained for MKLP1 (red) and α-tubulin (blue). Scale bars are 10 μm and 1 μm for the enlarged boxed areas. Putative different stages of autophagosomes (left) and lysosomes (right) are shown.(c) Juxtanuclear MRs are found in Rab7- and LAMP2-positive lysosomes. U2OS cells stably expressing DsRed-Rab7 were fixed and stained for MKLP1 α-tubulin and DAPI (left panels); wild-type U2OS were stained for MKLP1 and LAMP2 (right panel). Scale bars are 10 μm. The insets are enlargements of the engulfed MRs. Scale bars are 1 μm.
figf3(a) Accumulation of MRs in cells treated with the PI(3)K inhibitor 3-MA. Left panels show HeLa cells treated with 3-MA (4 mM) for 3 days. Cells were fixed and stained. Images are colour-inverted for better visualization. The quantification of this experiment is shown on the upper right (mean ± s.d. of three independent experiments; n = total number of cells counted). The lower right panel shows an enlarged area of 3-MA-treated cells in true colour. Scale bars are 20 μm.(b) siRNA-mediated depletion of Atg6 (Beclin-1) and Atg7 from HeLa cells causes MR accumulations. The right panel shows the quantification of MRs per cell (mean ± s.d. of three independent experiments); the left panel the efficiency of Atg6 and Atg7 depletion.(c) Inhibition of lysosomal acidification leads to MR accumulation. Panels on the left show HeLa cells left untreated or treated with chloroquine (20 μM) or ammonium chloride (10 mM) for 3 days. Scale bar are 20 μm. Cells were fixed and stained as indicated. A quantification of this experiment is shown on the left (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).
figf4(a) Cell-cycle lengths of different cell lines correlate well with the average amount of MRs. Indicated cell lines were grown on coverslips, fixed and MRs were counted after staining with anti-MKLP1 antibodies. Cell-cycle lengths were assessed as described in the Methods. Data are mean ± s.d. of three independent experiments.(b) The degree of MR persistence depends on the time and not on cell-cycle duration. HeLa cells were either left untreated or were arrested in S-phase with hydroxyurea (HU, 5 mM). Cells were fixed and stained as indicated. Scale bars are 20 μm. The right panel shows quantification of MR counting (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).
figf5(a) Fibroblasts of individuals with mucolipidosis II (I-cell disease; upper right panel) or Hurler syndrome (lower right panel) were compared with parental control cells (left panels). The alleles are indicated. Cells were fixed and stained with the indicated antibodies and DAPI to visualize DNA. Arrowheads point to the accumulated MRs. Scale bars are 20 μm. (b) Quantification of a (mean ± s.d. of three independent experiments; n, total number of cells; P, probability of a two-tailed paired t-test).
Figure 1(G) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the lower row. Scale bar, 25μM. (H) Quantification of ∆FosB+ nuclei in cultured MSNs after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(I) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs exposed to consecutive stimulation, with different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the right column. Scale bar, 25μM. (J) Quantification of ∆FosB+ nuclei in cultured MSNs after different 1st and 2nd treatments with activin A (Act), SKF81297 (SKF) or their combination as indicated. Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(K) Representative immunofluorescence staining of ∆FosB (green) in cultured MSNs extracted from mutant mice lacking ALK4 in GABAergic neurons (Gad67Cre;Alk4fl/fl) and control mice (Alk4fl/fl) after exposure to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with MAP2 and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (L) Quantification of ∆FosB+ nuclei in cultured MSNs from mutant and control mice after the indicated treatments (same treatment in both treatment days). Results are expressed as average ± SEM of % of MAP2+ cells displaying ∆FosB in the nucleus. N=6. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.
Figure 3(D) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in HeLa cells exposed to consecutive stimulation (same treatment in both treatment days) as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (E, F) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in HeLa cells after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(G) Representative images of RNA-PLA (green) between PCBP1 (left) or Smad3 (right) and sequences in exon 4 (pE4) of FosB mRNA, respectively, in cultured MSNs exposed to consecutive stimulation as indicated. Counterstaining with phalloidin and DAPI is shown in the second and fourth columns. Scale bars, 20μM. (H, I) Quantification of RNA-PLA between PCBP1 (E) or Smad3 (F) and pE4 in cultured MSNs after the indicated treatments. Results are expressed as average ± SEM of number of RNA-PLA dots per cell. Only nuclear dots were quantified in this analysis. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.
Figure 4(D) Representative immunofluorescence staining of ∆FosB (red) in cultured MSNs treated with scrambled and shPCBP1 lentiviruses and exposed to consecutive stimulation (same treatment in both treatment days) with activin A (Act), SKF81297 (SKF) or their combination as indicated. Counterstaining with green fluorescent protein (GFP, expressed by the lentiviruses) and DAPI is shown in the second and fourth rows. Scale bar, 25μM. (E) Quantification of ∆FosB+ nuclei in cultured MSNs treated with scrambled and shPCBP1 lentiviruses followed by the indicated treatments. Results are expressed as average ± SEM of % of GFP+ cells displaying ∆FosB in the nucleus. N=4. Data information: *, p<0.05; **, p<0.01;***, p<0.001; two-way ANOVA followed with Tukey's multiple comparisons test in E). N denotes biological replicates.
Figure 6(A-F) RT-qPCR analysis of FosB (A, D), c-Fos (B, E) and ∆FosB (C, F) mRNAs in NAc of Gad67Cre;Alk4fl/fl (A-C) mutant mice 1 hour (A-C) or 24 hour (D-F) after the second saline (Sal) or cocaine (Coc) injection of TIPS. Results are expressed as average ± SEM of fold increase over the saline group (Sal/Sal) of Gad67Cre mice. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(G) Representative immunofluorescence staining of ∆FosB in Nac of control (Gad67Cre) and mutant (Gad67Cre;Alk4fl/fl) mice, 24 hours after the second injection of the TIPS protocol. Scale bars 20 μm. (H) Quantification of ∆FosB positive cells 24 hours after the second injection of TIPS protocol in the Nac of Gad67Cre and Gad67Cre;Alk4fl/fl mice. Results are expressed as average ± SEM of the number of ∆FosB+ cells per mm2. At least 10 images from both left and right hemispheres were analyzed per mouse and condition. N=5. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.(I) Western blot analysis of FosB and ∆FosB in in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Blots were probed with an antibody that recognizes both FosB (upper band, 48kDa) and ∆FosB (lower band, 38 kDa). Lower blot shows reprobing with anti-β-actin. (J, K) Quantification of FosB (J) and ∆FosB (K) levels in NAc of Gad67Cre and Gad67Cre;Alk4fl/fl mice 24 hour after the second injection of TIPS. Results are expressed as average ± SEM of fold increase of FosB and ∆FosB over the saline group (Sal/Sal) of Gad67Cre mice normalized to β-actin levels. N=4s. Data information: *, p<0.05; **, p<0.01;***, p<0.001; one-way ANOVA followed with Tukey's multiple comparisons test. N denotes biological replicates.
Figure 1A A neutral red-stained mouse pancreas is excised and digested with collagenase. Islets are then separated by hand picking and further digested into single islet cells. Scale bars: 10 mm (left image); 200 µm (middle image); 60 µm (right image).B Epidydimal fat pads are isolated and processed mechanically and enzymatically. The obtained mixture, containing MVF and ECM, is filtrated for the purification of MVF. Scale bars: 20 mm (left image); 25 µm (right image).C Western blots of CD31, α-SMA and GAPDH in whole cell extracts of eEND, HDMEC, HUVEC and MVF.D Immunofluorescence stainings of CD31 / α-SMA and CD31 / PDGFR-β in MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 20 µm.F PI and PI+MVF on days -5, -3 and 0. Scale bar: 200 µm.G Surface morphology of FI, CI, PI and PI+MVF on day 0 as visualized by scanning electron microscopy. Scale bar: 50 µm.
Figure 2A Neutral red and trypan blue stainings of FI, CI, PI and PI+MVF. CI incubated for 24 h with 0.2% H2O2 served as control. Scale bar: 100 µm.B Representative flow cytometric scatterplots of propidium iodide / annexin V-stained cells from PI+MVF and CI+H2O2 (positive control). C Quantitative analysis of propidium iodide / annexin V-stained cells from FI, CI, PI, PI+MVF and CI+H2O2 subdivided in necrotic, necroptotic, apoptotic and vital cells in % of total cell number (n = 3 each). D Schematic illustration of the mitochondrial ROS-sensor mito-roGFP2-Orp1. The fluorescence maximum of this sensor shifts from 488 nm to 405 nm with increasing mitochondrial H2O2 levels, as shown by representative fluorescence images of FI. Scale bar: 70 µm.E Quantitative analysis of the ROS sensor ratio (405 nm / 488 nm) of FI, CI, PI and PI+MVF (n = 25 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.F Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from FI, CI, PI and PI+MVF. G Quantitative analysis of HO-1 expression in FI, CI, PI and PI+MVF (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. H Representative immunofluorescence stainings of HO-1 / insulin and HO-1 / CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 50 µm.I Representative Western blot analysis of β-actin and HO-1 in whole cell extracts from PI and MVF.
Figure 3A Representative immunofluorescence stainings of insulin / glucagon and insulin / somatostatin in FI, CI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 60 µm. B-D Quantitative analysis of insulin- (B), glucagon- (C) and somatostatin-positive cells (D) in FI, CI, PI and PI+MVF in % of all islet or organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. E Western blot analysis of β-actin and insulin in whole cell extracts from FI, CI, PI and PI+MVF.Insulin secretion (µU/mL) (normalized to DNA content) of FI, CI, PI and PI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test as well as an unpaired Student's t-test were used for statistical analysis. *P < 0.05 vs. insulin secretion at 1.1 mM glucose within each individual group; +P < 0.05 vs. insulin secretion of PI at 16.5 mM glucose.
Figure 4A Representative immunofluorescence stainings of insulin and CD31 in CI, FI, PI and PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm. B Quantitative analysis of CD31-positive cells in FI, CI, PI and PI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI. C Representative Western blot analysis of CD31 and β-actin in whole cell extracts from CI, FI, PI and PI+MVF.D Quantitative analysis of CD31 expression in FI, CI, PI and PI+MVF in % of FI (n = 3). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.E Confocal microscopic images of CD31 and cell nuclei (blue) in PI+MVF, demonstrating endothelial cells (arrowheads). Scale bar: 100 µm.F Representative immunofluorescence staining of Ki67 and CD31 in PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark endothelial cells expressing Ki67. Scale bar: 100 µm.G Quantitative analysis of CD31 / Ki67-positive cells in FI, CI, PI and PI+MVF in % of all endothelial cells (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.H Flow cytometric analysis of BrdU-positive cells from PI+MVF treated with DMSO (vehicle) or linsitinib during the entire fusion process (n = 5 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.I Representative Western blot analysis of pAkt, Akt, pERK1/2 and ERK1/2 from MVF cell extracts. MVF were treated with vehicle or insulin for 3 h. Quantitative analysis of pERK1/2 / ERK1/2 (J) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.Quantitative analysis of pAkt / Akt (K) ratio in PI+MVF treated with vehicle or insulin for 3 h. Data are expressed as relative intensities (n = 3 each). Mean ± SD. Unpaired Student's t-test was used for statistical analysis. *P < 0.05 vs. vehicle.L Representative bright field image of angiogenic sprouting from PI+MVF on 3 day of the sprouting assay. Scale bar: 150 µm. Insert: Immunofluorescence staining of insulin (red) and CD31 (green) of sprouted PI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 30 µm.M Quantitative analysis of sprouting area of FI, CI, PI and PI+MVF in % of the initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.
Figure 5B Bright field images of hFI, hCI, hPI and hPI+MVF. Scale bar: 100 µm.C Representative immunohistochemical stainings of insulin and CD31 in hFI, hCI, hPI and hPI+MVF. Cell nuclei are stained with Hoechst 33342 (blue). Scale bar: 100 µm.D-G Quantitative analysis of insulin-(D), glucagon-(E), somatostatin-(F) and CD31-positive cells (G) in hFI, hCI, hPI and hPI+MVF in % of all islet or islet organoid cells (n = 20 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.H Representative bright field images of angiogenic sprouting of hFI, hCI, hPI and hPI+MVF on day 3 of the sprouting assay. Scale bar: 150 µm.I Quantitative analysis of sprouting area of hFI, hCI, hPI and hPI+MVF in % of initial size (day 0) (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. hFI; #P < 0.05 vs. hCI; +P < 0.05 vs. hPI.J Quantitative analysis of C-peptide secretion (pg/mL) (normalized to DNA concentration) of hFI and hPI+MVF exposed to 1.1 mM and 16.5 mM glucose (n = 3 each). Mean ± SD.
Figure 6B Observation window of a dorsal skinfold chamber with 8 neutral red-stained transplanted FI on day 0. Scale bar: 5 mm.C Take rate (% of transplanted islets) on day 14 after islet transplantation into the dorsal skinfold chamber (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. +P < 0.05 vs. PI.D Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 0 and 14. FITC-labeled dextran is used for the visualization of blood-perfused microvessels. Grafts are marked by broken lines. Scale bar: 150 μm.E, F Quantitative analysis of the functional capillary density (cm/cm-2) (E) and revascularized area (mm2) (F) of transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14 (n = 8 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.G Representative intravital fluorescent microscopic images of FI, CI, PI and PI+MVF within the dorsal skinfold chamber on day 14. Rhodamine 6G is used to visualize blood perfused vital endocrine cells (bright signals). Scale bar: 150 µm.H Quantitative analysis of the rhodamine 6G-positive area (% of islet size) within transplanted FI, CI, PI and PI+MVF on day 0, 3, 6, 10 and 14. Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.
Figure 7A Representative immunofluorescent stainings of insulin / glucagon, insulin / somatostatin and insulin / CD31 in FI, CI, PI and PI+MVF on day 14 after transplantation. Cell nuclei are stained with Hoechst 33342 (blue). Arrows mark intra-islet endothelial cells. Scale bar: 70 µm. B-E Quantitative analysis of insulin- (B), glucagon- (C), somatostatin- (D) and intra-islet CD31-positive cells (E) within FI, CI, PI and PI+MVF on day 14 after transplantation (n = 10 each). Mean ± SD. One-way ANOVA and Tukey's multiple comparisons post hoc test were used for statistical analysis. *P < 0.05 vs. FI; #P < 0.05 vs. CI; +P < 0.05 vs. PI.