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. 2022 Nov 14;44(5):1066–1082. doi: 10.1038/s41401-022-01021-2

Fig. 5. Effects derived from autophagy inhibition on NRP1 protein expression and NRP1-associated antitumor actions of lenvatinib.

Fig. 5

All the assays were performed 48 h post-silencing with the last 24 h of treatment with 2.5 µM lenvatinib (Lvt) and/or 100 nM bafilomycin A1 (Baf). Protein levels of NRP1 were analyzed by (a) Western blot and (b) ICC. Cell proliferation was assessed by (c) CellTiter-Glo® assay, (d) colony formation assay, and (e) Ki67 proliferation index determination. Data from (a), (b), (d) and (e) are represented as mean values of arbitrary units (a.u.) ± SD (n = 3), showing for (a) a representative immunoblot. Data from (c) are represented as % of mean values relative to control ±SD (n = 7). Bar graphs from (b) and (d) represent the NRP1 CTCF ratio and the nuclear CTCF ratio of Ki67, respectively. Magnification 63×, scale bar 10 µm. f Cell migration ability was evaluated by wound-healing assay, representing the % of the wound closure area after 24 h from the scratch. Magnification 10× and scale bar 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001 vs non-treated cells in each siR group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs siR Control; σP < 0.05, σσP < 0.01, σσσP < 0.001 Lvt+Baf vs Lvt treatment.