Fig. 2: Laminin γ1 is involved in H-1PV cell attachment and entry.

a Silencing of LAMC1 decreases H-1PV cell binding. HeLa cells were transfected with control siRNA or two distinct siRNAs targeting two separate regions of LAMC1. At 46 h post transfection, cells were infected with H-1PV (MOI 1 pfu/cell) for 2 h at 4°C. The results are presented as a percentage of cell-associated virus genome normalised to control siRNA. The representative experiment shown in the figure is repeated twice; n = 3 biologically independent samples; control siRNA vs LAMC1 siRNA1 (P = 0.0165) and control siRNA vs LAMC1 siRNA2 (P = 0.0101). b Silencing of LAMC1 decreases H-1PV cell uptake. HeLa cells were transfected with the same siRNAs used for a. At 46 h post transfection, cells were infected with H-1PV (MOI 1 pfu/cell) for 4 h at 37°C. Cell processing and data representation were performed as per a. The independent experiment shown is repeated twice; n = 3; Control siRNA vs LAMC1 siRNA1 (P = 0.0231) and control siRNA vs LAMC1 siRNA2 (P = 0.0498). Western blot analysis ascertained the downregulation of LAMC1 in siRNA-transfected cells using β-tubulin as a loading control. c Silencing of LAMC1 decreases H-1PV transduction. HeLa cells were transfected with the same siRNAs used for a. At 46 h post transfection, cells were infected with recH-1PV-EGFP (1 TU/cell) and grown for an additional 24 h and analysed for EGFP-positive (+ve) cells. Numbers indicate the fraction of EGFP-positive cells (%) normalised to that obtained in cells transfected with control siRNA. The scale bar depicted in the figure corresponds to 100 μm. d Silencing of LAMC1 protects cells from H-1PV oncotoxicity. HeLa cells were transfected with control or LAMC1 siRNAs. At 72 h post transfection, cells were infected or not with H-1PV (MOI 0.25 pfu/cell) and grown for an additional 72 h before measuring cell viability by the CellTitre-Glo 2.0 assay. The data shown represent the percentage of cell viability normalised to uninfected cells. The independent experiment shown is repeated twice; n = 3 biologically independent samples; uninfected vs virus alone (P = 0.0027); Control siRNA vs LAMC1 siRNA (P = 0.0024). e Anti-laminin γ1 antibody impairs H-1PV cell uptake. HeLa cells were incubated with either IgG isotype (control) or anti-laminin-γ1 chain antibodies for 45 min on ice. An H-1PV cell membrane binding/entry assay was performed by treating the cells with H-1PV (MOI 0.1 pfu/cell) first for 30 min on ice and then for 60 min at 37°C in a serum-free medium. Cells were then processed as described in a. The independent experiment shown is repeated twice; n = 3 biologically independent samples; control IgG vs LAMC1 Ab (P = 0.031). f CRISPR-Cas9-mediated knockdown of LAMC1 impairs H-1PV uptake, which is rescued by re-introduction of exogenous LAMC1. Parental HeLa and LAMC1 KD (a LAMC1-knockdown HeLa cell line constructed using CRISPR-Cas9 technology) were transfected or not with either empty vector (+ vector alone) or vector expressing LAMC1 (+LAMC1). At 48 h post transfection, cells were infected with H-1PV (MOI 100 pfu/cell) for 4 h at 37°C for the virus uptake assay. Cells were then processed as described in a. The independent experiment shown is repeated twice; n = 2 biologically independent samples. g Overexpression of LAMC1 enhances H-1PV uptake. HeLa cells were transfected either with empty vector (+ vector alone) or vector carrying LAMC1. Cells were then processed as described in a. The independent experiment shown is repeated twice; n = 3 biologically independent samples; + vector alone vs + LAMC1 (P = 0.0329). h Overexpression of LAMC1 in HeLa enhances H-1PV transduction. HeLa cells were transfected with either vector alone or vector expressing LAMC1. At 46 h post transfection, cells were infected with recH-1PV-EGFP (0.25 TU/cell) and grown for an additional 24 h and processed as described above. Scale bar corresponds to 100 μm. Statistical significance was calculated using a paired two-tailed t test by GraphPad Prism 8; *P < 0.05; **P < 0.01. Error bars for all data indicate the mean values ±SD. Source data are provided as Source Data file.