Fig 2: Impaired motility of LPS-stimulated macrophages following Pdpn-deletion. (A) Graph shows percentages of wound healing in WT and Pdpn-KO cells. Confluent cells were scratched and viewed under NanoEntek JuLi BRD04 live cell imager for 24 h. Area of wound was measured every 4 h using ImageJ software. Note that Pdpn-KO demonstrated impaired wound healing ability as compared to WT cells. Data were presented as mean ± SD and were representative of three independent experiments. See Supplemental Video 1 . (B) Comparison of cell proliferation rate among the non-stimulated wild type (WT) and Pdpn-knockout (KO) stable clones using MTT assay. Similar number of cells were seeded and cultured over a period of 4 days. Cell growth was measured each day by adding MTT solution for 4 h, replaced with DMSO, before measuring on a plate reader. All samples were run in triplicate. Data are presented as mean ± SD from duplicate readings and are representative of two independent experiments. (C) Representative images of migrated cells for wild-type, Pdpn-KD/TC, and Pdpn-KO cells in transwell migration assay. Cells seeded at upper chamber of 8-µm transwell insert were induced with 20% FBS, 100 ng/ml CCL2, and 1 µg/ml LPS from the lower chamber and processed with crystal violet staining after 24 h. Circles indicate the pores in the transwell. Scale bars indicate 100 µm. (D) Histogram shows number of migrated cells in transwell migration assay. Data were shown as mean ± SD from duplicates and were representative of three independent experiments. Statistical significance was analyzed with two-way ANOVA test (F value: 5.77; degree of freedom: 6; *p < 0.05; n.s., not significant). (E) Real-time migration rate of wild-type, Pdpn-KD/TC, and Pdpn-KO cells in RTCA analysis. Cells seeded at the upper chamber of CIM-16 plate were induced with 20% FBS, 100 ng/ml CCL2, and 1 µg/ml LPS, from the lower chamber. Data were normalized at the sixth hour time point and are representative of two independent experiments.

Fig 3: Endosomal transduction of micellized PTD-BMP-7 (mPTD-BMP-7) and exosomal secretion of bone morphogenetic protein-7 (BMP-7). (A) HEK 293 cells were treated with 200 ng of mPTD-BMP-7 for the indicated period, and the insoluble fraction of whole cell lysates were subjected to immunoblot analysis with anti-HA antibody (left). Arrow and arrowhead denote monomeric and aggregated PTD-BMP-7, respectively. The 293 cells were treated with 200 ng of mPTD-BMP-7, and intracellular transduction was monitored by confocal microscopy (right). Z-stack in blow. Scale bar, 5 µm. (B) The 293A cells were transiently transfected with mCherry-tagged Rab7 and treated with 200 ng of HA-tagged mPTD-BMP-7 for 3 h. Intracellular localization was observed with confocal microscopy and Nuc-blue served as nuclear staining of live cell imaging. (C) The 293 cells were treated with 200 ng mPTD-BMP-7, and the cells were subjected to transmission electron microscopy. Note a membrane-coated mPTD-BMP-7 in endosomes (middle) and Golgi (right). (D) The 293 cells were transfected with flag-tagged N-terminal of fibrillin-1 (FBN-1) and treated with 500 ng HA-tagged mPTD-BMP-7 for 16 h. The culture medium was harvested and subjected for immunoblot analysis (left) and immunoprecipitation with anti-flag (FBN-1). Arrows indicate active BMP-7. (E) Exosomal secretion of active BMP-7 (arrow) and mPTD-BMP-7 (arrow head) with FNB-1. CD63 serves as exosomal marker. (F) Cellular toxicity of mPTD-BMP-7. The 293A cells were incubated in various concentrations of mPTD-BMP-7, and cell growth was monitored by JuLi real-time live cell analyzer (NanoEntek).

Fig 4: Typical images taken from SH-5YSY cells after 72 h of exposure to 100 µM of tested compounds. Juli Fl apparatus (NanoEntek); magnification 4× g; scale bar 100 µm.

Fig 5: The effect of (+)-usnic acid on FaDu cells morphology (a), cell cycle distribution (b, c) and apoptosis (d, e). Representative images of FaDu cells were taken after 24 h incubation with ( +)-usnic acid or vehicle using JuLI FL microscope (NanoEntek, South Korea) (a). Cell cycle distribution was analyzed by flow cytometry after staining with propidium iodide (b). Topotecan (50 and 100 nM) was used as a positive control. Representative plots are presented (c). Apoptosis was evaluated by flow cytometry after staining with Annexin-V and 7-Aminoactinomycin D (d). Topotecan (200 nM) was used as a positive control. Representative plots are presented (e). Mean values ± SEM from three independent experiments are shown. Asterisk inside bars denotes statistically significant changes from DMSO treated (control) cells, *p < 0.05. Hash (#) above bars denotes statistically significant changes of the total number of apoptotic cells, p < 0.05