Fig 1.
Protease treatment decreases binding of LGTV to A549 cells.
To assess whether the cellular receptor for LGTV included a protein component, A549 cells were pre-treated with increasing amounts of the proteases Ficin and Proteinase K, prior to binding with LGTV. The bars indicate the percentage of cell-bound LGTV compared to untreated control. To demonstrate its effect, protease-treated cells were incubated with human adenovirus 37 (HAdV-37), shown to bind to proteins [21,22]. Results represent the mean of three independent experiments performed in duplicate. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test for multiple comparisons and t test for single comparisons (***, P≤0.001; ****, P≤0.0001).
Fig 2.
Heparan sulfate (HS) is not involved in LGTV binding to A549 cells.
A549 cells were pre-incubated with increasing amounts of Heparinse III to cleave HS (black bars). To demonstrate its effect, Heparinase III-treated cells were incubated with pre-formed complexes of human adenovirus 5 (HAdV-5) virion:coagulation factor X (FX) (grey bars). Data in the figure represents means of three independent experiments performed in duplicate. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test for multiple comparisons and t test for single comparisons (****, P ≤ 0.0001; ns, non-significant).
Fig 3.
LGTV cellular receptor does not include N-linked glycans.
(A) A549 cells were pre-incubated with 100 mU/mL of PNGase F (black bars). (B) The parental cell line Pro-5 and the N-glycan deficient cell line Lec1 were assessed for LGTV binding (black bars). (A and B) To demonstrate the reduction in N-linked glycan content, cells were incubated with the lectin Phaseolus vulgaris erythroagglutinin (E-PHA) (grey bars). Data in the figure represents means of three independent experiments performed in duplicate. Error bars represent standard deviation. Statistical analysis was performed using t test (**, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).
Fig 4.
LGTV binding to A549 cells does not depend on O-linked glycans.
A549 cells were treated with benzyl-α-GalNAc to inhibit O-glycosylation (black bars). As a control for this inhibition, HAdV-37 was used to bind to A549 cells (grey bars). Data represents means of three independent experiments performed in duplicate. Error bars represent standard deviation. Statistical analysis was performed using t test (**, P ≤ 0.01; ns, non-significant).
Fig 5.
LGTV binding to A549 cells does not depend on glycolipids.
A549 cells were pre-incubated with DL-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), in its active (1R,2R) or inactive (1S,2S) form to inhibit glycolipid synthesis (black bars). To confirm its effect, P4-treated cells were incubated with FITC-conjugated cholera toxin B (CT-B) (grey bars). Data represents means of three independent experiments performed in duplicate. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test for multiple comparisons and t test for single comparisons (****, P ≤ 0.0001; ns, non-significant).
Fig 6.
LGTV cellular receptor does not include sialic acid components.
(A) A549 cells were pre-incubated with 10 mU/mL of V. Choleare neuraminidase (black bars). (B) The parental cell line Pro-5 and the sialic-acid deficient cell line Lec2 were assessed for LGTV binding (black bars). (A and B) To demonstrate the reduction in sialic acid content, cells were incubated with HAdV-37, known to bind to sialic acids (grey bars). Data in the figure represents means of three independent experiments performed in duplicate. Error bars represent standard deviation. Statistical analysis was performed using t test (***, P ≤ 0.001; ****, P ≤ 0.0001; ns, non-significant).
Fig 7.
Binding of LGTV to 60 cell lines of the NCI60 panel.
Each cell line of the NCI60 panel was assessed for the binding of LGTV. The results are expressed in percentage as compared to the control cell line A549, which was normalized to 100% (represented by the dotted line). Data in the figure represents the means of at least three independent experiments performed in duplicate. Error bars represent standard deviation.
Fig 8.
Inhibition or overexpression of LGR6 does not influence LGTV binding to A549 or SW620 cell lines.
(A) A549 and (B) SW620 cells were pre-treated with 10-fold dilutions of two pAbs anti-LGR6: anti-LGR6 LL and HR. LGTV was pre-incubated with 50 or 5 μg/mL of rhLGR6, prior to binding with (C) A549 and (D) SW620 cells. (E) Hek293 cells were transfected with flag-LGR6 prior to binding to LGTV. Overexpression of flag-LGR6 was confirmed by flow cytometry (F) and western blotting (G) by using an anti-flag monoclonal antibody (mAb). The bars in the figures indicate the percentage of cell-bound LGTV compared to the untreated control. Data in the figure represents means of at least three independent experiments performed in duplicate, except for (C), for which two experiments were performed. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test for multiple comparisons and t test for single comparisons (ns, non-significant).
Fig 9.
SW620 trypsinized peptides inhibit binding of LGTV to SW620 but not A549.
(A) A549 cells and (B) SW620 cells were tested for the binding of LGTV after pre-incubation with 100 or 10 μg/mL of trypsinized membrane proteins from A, A549; S, SW620 and O, Ovcar5, a control cell line to which LGTV does not bind. The bars in the figures indicate the percentage of cell-bound LGTV compared to the control. Data in the figure represents means of three independent experiments performed in duplicate. Error bars represent standard deviation. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (*,P ≤ 0.05; **** P ≤ 0.0001; ns, non-significant).