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. 2019 Nov 5;8(11):1395. doi: 10.3390/cells8111395

Figure 5.

Figure 5

HCV promotes invadopodium precursor formation via down-regulation of T-cell protein tyrosine phosphatase (TC-PTP) and activation of epidermal growth factor receptor (EGFR). (A) Huh7.5 cells transfected with plasmids containing FLAG-tagged HCV-Core, FLAG-tagged HCV-NS3/4A, FLAG-tagged TC-PTP, or empty vector (pCMV) as control were immunostained with anti-core (for the core) positive serum from HCV-infected patient (for NS3) or anti-TC-PTP. Bar, 5 μm. (B) Transfected cells were plated on unlabeled gelatin, fixed, and labeled for actin and cortactin. Co-localization of actin and cortactin dots as markers of invadopodium precursors was counted. Presented is the quantification of invadopodium precursors per cell. n = 20 cells per group from three independent experiments. (C) Huh7.5 cells were transfected as above, plated on Alexa 488 gelatin, and allowed to degrade for 72 h. Presented is the quantification of matrix degradation by control and transfected cells. n = 10 fields per group from three independent experiments. (D) Huh7.5 cells were plated on unlabeled gelatin for 24 h and then transfected with plasmids containing FLAG-tagged HCV-NS3/4A or empty vector (pCMV) as a control. Erlotinib (1 μM) was added six hours following transfection for additional 24 h. Cells were then fixed and labeled for actin and cortactin as invadopodia markers. Invadopodium precursor formation (n = 20 cells per group from three independent experiments) was quantified. (E) Matrix degradation of Huh7.5 cells transfected and treated with Erlotinib (n = 10 fields per group from three independent experiments) was quantified. (F) Cell lysates from non-infected, HCV-infected, and NS3-transfected cells were analyzed by western blot using antibodies for TC-PTP (Abcam) and β actin (Sigma). (G) Cell lysates from non-infected, HCV-infected, and NS3-transfected cells, all transfected with FLAG-tagged TC-PTP, were analyzed by western blot using antibodies for TC-PTP (Abcam) and β actin (Sigma). (H) HCV-infected or non-infected Huh7.5 cells were transfected with a plasmid containing FLAG-tagged TC-PTP or with empty vector (pCMV) as control, plated on unlabeled gelatin for 24 h, fixed and labeled for actin and cortactin as invadopodia markers. Invadopodium precursor formation in cells that showed a positive signal in anti-FLAG staining was quantified. n = 15 cells per group from two independent experiments. (I) Quantification of matrix degradation of Huh7.5 cells transfected as above is presented. n = 10 fields per group from three independent experiments. * p < 0.05; *** p < 0.001, Student’s t-test.