Fig 1: Galectin-3 associates with and induces activation of VEGFR2. (A) Confluent HRECs were lysed using RIPA lysis buffer. HREC lysate was incubated with Gal3-conjugated beads, washed, and eluted either with a non-competitive sugar (0.1 mM sucrose) or a competitive sugar (0.5 mM lactose). Bound VEGFR2 was eluted once it was competed off of the beads by lactose (B) Serum-starved HRECS were stimulated with 50 μg/ml Gal3 or 10 ng/ml VEGF for 2 h with or without sunitinib then total RNA was isolated and reverse transcribed into cDNA. E-selectin, VCAM-1, and HPRT1 mRNA expression were analyzed using qPCR. One-way ANOVA analysis was applied, ∗P < 0.05, ∗∗P < 0.01, ****P < 0.0001, n = 3.
Fig 2: Galectin-3 is not required for VEGF-induced VEGFR2 activity. (A) HRECs transfected with siRNA targeting Gal3 (siGal3) demonstrated over 90% mRNA knockdown of Gal3 compared to cells transfected with control siRNA (siCtrl), analyzed by reverse-transcription and qPCR assay. Student T-test was applied, ∗P < 0.05, n = 3. (B) Lysate from HRECs transfected with siGal3 or siCtrl was analyzed by western blot and probed with an antibody against Gal3, which resulted in over 90% Gal3 protein reduction. Student T-test was applied, ∗P < 0.05, n = 3. (C) siCtrl and siGal3 transfected HRECs were stimulated with 10 ng/ml of VEGF for 0, 1, 5, 10, 15, and 30 min. Cell lysates were collected and analyzed by western blot for tubulin, VEGFR2, and phospho-VEGFR2 (pVEGFR2 Y1175). Protein levels for pVEGFR2 were quantified using ImageJ and normalized to total VEGFR2 and α-tubulin. Ordinary one-way ANOVA was applied for comparisons within groups, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 3. (D) Confluent HRECs, transfected with siCtrl or siGal3, were scratched and stimulated with 10 ng/ml of VEGF. Images were taken immediately after scratching and 15 h later. Cell migration was quantified using ImageJ and normalized to the non-treatment control group. Scale bar represents 500 μm. Student T-test was applied, ∗∗P < 0.01, ∗∗∗P < 0.001, ****P < 0.0001, n = 10. (E) HRECs transfected with siCtrl and siGal3 were stimulated with 10 ng/ml VEGF. Cell surface proteins were labeled with NHS-SS-biotin and isolated from cell lysate using avidin agarose beads. Total VEGFR2 and CD31 protein levels were analyzed by western blot. Student T-test was applied, ∗P < 0.05, n = 3.
Fig 3: Galectin-3 requires VEGF to induce VEGFR2 activity. (A) Ranibizumab (10 μg/ml) was added to neutralize endogenous VEGF and HRECs were stimulated with 50 μg/ml of Gal3 for 0, 5, 10, 30, and 60 min. Cell lysates were collected and pVEGFR2 Y1175 (pR2), VEGFR2 (TR2), and α-tubulin protein levels were analyzed by western blot. Ordinary one-way ANOVA was applied. ∗P < 0.05, n = 3. (B) Confluent HRECs were scratched and stimulated with 50 μg/ml of Gal3 with 10 μg/ml of ranibizumab or control IgG. Images were taken immediately after scratching and at 15 h later. Scale bar represents 500 μm. Cell migration was quantified using ImageJ. Student T-test was applied, ∗P < 0.05, n = 6.
Fig 4: Downregulated MYOF expression causes lysosomal dysfunction during the adipogenic differentiation of hMSCs. a Relative mRNA expression of adipogenic markers was detected using qRT–PCR after APPL1 overexpression or MYOF knockdown. b hMSCs adipogenesis was revealed with ORO staining and quantification after APPL1 overexpression or MYOF knockdown. c Protein levels of adipogenic markers were detected using Western blot analysis after APPL1 overexpression and MYOF knockdown. Quantification of the data is shown in the right panel. d After MYOF knockdown, the protein levels of lysosome markers (LAMP1) were analysed using Western blotting. Quantification of the data is shown in the lower panel. e The lysosomes of hMSCs with or without MYOF knockdown that were cultured in the control or adipogenic induction medium were stained with LysoTracker and revealed by immunofluorescence staining. Quantification of LysoTracker is shown in the lower panel. f Time course of LysoTracker staining in siControl and MYOF knockdown hMSCs following treatment with LLOMe for 0, 0.5, 1 and 2 h. Quantification of LysoTracker is shown in the right panel. g Immunofluorescence staining for GAL3 (green) and LAMP2 (red) along with adipogenic induction, and the nucleus was stained with DAIP (blue). Quantification of the data is shown in the right panel. Scale bar = 50 μm. All data are presented as the means ± SD, n = 6 per group. Statistical differences were determined using Student’s t test or ANOVA. ns not statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001
Supplier Page from Abcam for Anti-Galectin 3 antibody [EPR19244]