Fig. 4: Determination of the mechanism underlying the lenvatinib-derived downregulation of NRP1.

a Protein expression of NRP1 was analyzed by Western blot after 24 h treatment with 2.5 µM lenvatinib (Lvt) and/or 300 µM cycloheximide (CHX) or 30 µM MG132 (MG). b NRP1 protein levels were also determined by Western blot after 3, 6, 12 and 24 h treatment with 2.5 µM lenvatinib (Lvt) alone and combined with 100 nM bafilomycin A1 (Baf). *P < 0.05, **P < 0.01, ***P < 0.001 vs control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 combined treatment vs Lvt treatment; ^σP < 0.05^{, σσ}P < 0.01, ^σσσP < 0.001 combined treatment vs inhibitor treatment. c Analysis of autolysosome cell content by acridine orange staining and fluorescence microscopy. Magnification 40×, scale bar 25 µm. Bar graphs represent the quantification of red/green CTCF ratio (n = 5). d Protein levels of p62/SQSTM1 and LC3 (LC3-I and LC3-II) were analyzed by Western blot. LC3 turnover assay was performed to determine the autophagic flux index. Data from (a), (b) and (d) are represented as mean values of arbitrary units (a.u.) ± SD (n = 3), showing a representative immunoblot. *P < 0.05, **P < 0.01, ***P < 0.001 vs control for each time point; ^##P < 0.01, ^###P < 0.001 combined treatment vs Lvt treatment.