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. 2019 Jan 17;11(1):107. doi: 10.3390/cancers11010107

Figure 6.

Figure 6

Elevated expression of PCDHB13 inhibits cell invasion and triggers cell death. (A,C) The underside of Matrigel-coated polycarbonate membranes used for cell invasion assays on EGFP-, PCDHB13-, and FOSB-transfected cells (A) or cells incubated with 1.68 and 3.35 μM TP4 (C). Cells were stained by Hoechst33342 shown in blue (A) or white (C). Bar: 200 μm. (B,D) Quantification of the cells that migrated across the membrane. Data were calculated by normalizing GFP/tGFP-positive to the total cell counts (n = 463 in EGFP transfected groups, n = 300 in PCDHB13-tGFP transfected groups, and n = 297 in FOSB-tGFP transfected group) in (B) or by counting Hoechst-dye stained cells in (D). (E) Schematic of the A549 cell xenotransplantation procedure. Cells were used for RO injection at 24 h post-transfection. Cell-transplanted zebrafish were cultured for 4 d. (F) Transmitted light and fluorescent images of A549 cells without or with transfection. Bar: 100 μm. (G) Quantification of fluorescent signal in dissected organs (n = 5 in the nontransfected/NT group and n = 9 in the transfected groups). au: arbitrary unit. (H) Tissue extracts from tGFP-, PCDHB13-, and FOSB-transfected A549-transplanted zebrafish were analyzed by Western blot using antibodies against N-Cadherin, α/β-Tubulin, and tGFP. P: Lysates from tGFP/PCDHB13/FOSB-transfected A549 cells. N: Lysate from nontransfected A549 cell. (I) Quantification of the EGFP/tGFP levels from Western blots (n = 9). (J) Cell viability was determined by the ATP assay for A549 and NCI-H1975 cells after transfection with PCDHB13. Ten replicate wells were analyzed for each dose (n = 3). (K) LDH release in A549 and NCI-H1975 cultures were determined 24 h after FOSB and PCDHB13 transfection. Triton-X was used as a positive control. Each independent replicate was measured at least in triplicate (n = 3). (L) Total lysates from A549 cells transfected with control (Neg.) or PCDHB13 siRNAs were analyzed by Western blot using antibodies against GAPDH and PCDHB13. Bottom, PCDHB13 levels were quantified and normalized to GAPDH. (M) Cell viability was determined by the ATP assay for A549 cells transfected with siRNAs targeting PCDHB13. Eight replicate wells were analyzed for each condition (n = 3). Quantitative results represent the mean ± SD (One-way ANOVA test in (K); two tailed t-test in (M): ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001).