Journal: Frontiers in Cell and Developmental Biology

Article Title: Regulation of the Transferrin Receptor Recycling in Hepatitis C Virus-Replicating Cells

doi: 10.3389/fcell.2020.00044

Figure Lengend Snippet: Reduced amounts of the TfR in α-taxilin knockdown (KD) and α-taxilin knockout (KO) cells. (A) (Left) Western blot analysis of cellular lysates derived from Huh7.5 cells transfected with an α-taxilin-specific siRNA (50 nM) or scrRNA (50 nM) (control). 24 h after siRNA transfection, the cells were transfected with pDest26-TXLNA (1 μg) or pUC18 (1 μg) (control) to rescue the α-taxilin KD. The cells were analyzed 72 h after siRNA transfection using α-taxilin- and TfR-specific antibodies. Total protein staining served as a loading control (lower panel). (Right) Densitometric quantification of α-taxilin and the TfR from two independent experiments. scrRNA transfected cells served as a control and were set as 1 ( n = 2; mean ± SEM). Two-tailed unpaired t -test, * p < 0.05, ** p < 0.01. (B) (Left) Western blot analysis of cellular lysates derived from Huh7.5 cells and stable α-taxilin KD Huh7.5 cells (Huh7.5 α-taxilin KD) using α-taxilin- and TfR-specific antibodies. ß-actin served as a loading control. (Right) Densitometric quantification of α-taxilin and the TfR from three independent experiments. Huh7.5 cells served as a control and were set as 1 ( n = 3; mean ± SEM). Two-tailed unpaired t -test, *** p < 0.001, **** p < 0.0001. (C) (Left) Western blot analysis of cellular lysates derived from HepaRG cells (HepaRG off target K8) and stable α-taxilin KO HepaRG cells (HepaRG α-taxilin KO T1K6) using α-taxilin- and TfR-specific antibodies. ß-actin served as a loading control. (Right) Densitometric quantification of α-taxilin and the TfR from one experiment. HepaRG cells (HepaRG off target K8) served as a control and were set as 1 ( n = 1). (D) CLSM analysis of HepaRG wt and stable α-taxilin KO HepaRG cells (HepaRG α-taxilin KO T1K6) using α-taxilin (green) and TfR (red)-specific antibodies. Nuclei were stained with DAPI (blue). Laser intensity and digital gain were kept constant for HepaRG wt and HepaRG α-taxilin KO (HepaRG α-taxilin KO T1K6) cells. Magnifications, x100. Scale bar represents 10 μm. (E) (Left) Western blot analysis of cellular lysates derived from Huh7.5.1 cells (Huh7.5.1 α-taxilin off target K8) and stable α-taxilin KO Huh7.5.1 cells (Huh7.5.1 α-taxilin KO K15/K21) using α-taxilin- and TfR-specific antibodies. ß-actin served as a loading control. (Right) Densitometric quantification of α-taxilin and the TfR from three independent experiments. Huh7.5.1 cells (Huh7.5.1 off target K8) served as a control and were set as 1 ( n = 3; mean ± SEM). Two-tailed unpaired t -test, * p < 0.05, **** p < 0.0001, ns = not significant. (F) (Top) The amount of TfR in Huh7.5 α-taxilin KD (stable) cells can be rescued by transient transfection with an α-taxilin mCherry plasmid (red). Huh7.5 α-taxilin KD (stable) cells were stained using a TfR-specific antibody (green). Nuclei were stained with DAPI (blue). Laser intensity and digital gain were kept constant. Magnifications, x100. (Bottom) Quantification of the fluorescence intensity of TfR (green) in Huh7.5 α-taxilin KD (stable) cells transfected with the α-taxilin mCherry plasmid and the pUC18 control compared to the transfected Huh7.5 cells. Total fluorescence per cell was calculated using ImageJ software and the following formula: corrected total cell fluorescence (CTCF) = integrated density - (area of selected cell × mean fluorescence of background readings). In total, a minimum of 10 cells were measured (mean ± SEM). Two-tailed unpaired t -test, **** p < 0.0001. (G) Viability assay for Huh7.5 and α-taxilin deficient cells. LDH release was measured by a microplate reader to determine cytotoxicity. Treatment with Triton X-100 (1%) served as a positive control.

Article Snippet: Hepatitis C virus-negative Huh7.5 cells were transfected 4 h after seeding either with 50 nM α-taxilin specific siRNA or scrRNA (sc-39644 and sc-37007, Santa Cruz Biotechnology) using N-TER TM Nanoparticle siRNA Transfection System (Sigma) according to the manufacturer’s protocol.

Techniques: Knockdown, Knock-Out, Western Blot, Derivative Assay, Transfection, Control, Staining, Two Tailed Test, Plasmid Preparation, Fluorescence, Software, Viability Assay, Positive Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Regulation of the Transferrin Receptor Recycling in Hepatitis C Virus-Replicating Cells

doi: 10.3389/fcell.2020.00044

Figure Lengend Snippet: Reduced amounts of the TfR in α-taxilin knockdown (KD) and α-taxilin knockout (KO) cells. (A) (Left) Western blot analysis of cellular lysates derived from Huh7.5 cells transfected with an α-taxilin-specific siRNA (50 nM) or scrRNA (50 nM) (control). 24 h after siRNA transfection, the cells were transfected with pDest26-TXLNA (1 μg) or pUC18 (1 μg) (control) to rescue the α-taxilin KD. The cells were analyzed 72 h after siRNA transfection using α-taxilin- and TfR-specific antibodies. Total protein staining served as a loading control (lower panel). (Right) Densitometric quantification of α-taxilin and the TfR from two independent experiments. scrRNA transfected cells served as a control and were set as 1 ( n = 2; mean ± SEM). Two-tailed unpaired t -test, * p < 0.05, ** p < 0.01. (B) (Left) Western blot analysis of cellular lysates derived from Huh7.5 cells and stable α-taxilin KD Huh7.5 cells (Huh7.5 α-taxilin KD) using α-taxilin- and TfR-specific antibodies. ß-actin served as a loading control. (Right) Densitometric quantification of α-taxilin and the TfR from three independent experiments. Huh7.5 cells served as a control and were set as 1 ( n = 3; mean ± SEM). Two-tailed unpaired t -test, *** p < 0.001, **** p < 0.0001. (C) (Left) Western blot analysis of cellular lysates derived from HepaRG cells (HepaRG off target K8) and stable α-taxilin KO HepaRG cells (HepaRG α-taxilin KO T1K6) using α-taxilin- and TfR-specific antibodies. ß-actin served as a loading control. (Right) Densitometric quantification of α-taxilin and the TfR from one experiment. HepaRG cells (HepaRG off target K8) served as a control and were set as 1 ( n = 1). (D) CLSM analysis of HepaRG wt and stable α-taxilin KO HepaRG cells (HepaRG α-taxilin KO T1K6) using α-taxilin (green) and TfR (red)-specific antibodies. Nuclei were stained with DAPI (blue). Laser intensity and digital gain were kept constant for HepaRG wt and HepaRG α-taxilin KO (HepaRG α-taxilin KO T1K6) cells. Magnifications, x100. Scale bar represents 10 μm. (E) (Left) Western blot analysis of cellular lysates derived from Huh7.5.1 cells (Huh7.5.1 α-taxilin off target K8) and stable α-taxilin KO Huh7.5.1 cells (Huh7.5.1 α-taxilin KO K15/K21) using α-taxilin- and TfR-specific antibodies. ß-actin served as a loading control. (Right) Densitometric quantification of α-taxilin and the TfR from three independent experiments. Huh7.5.1 cells (Huh7.5.1 off target K8) served as a control and were set as 1 ( n = 3; mean ± SEM). Two-tailed unpaired t -test, * p < 0.05, **** p < 0.0001, ns = not significant. (F) (Top) The amount of TfR in Huh7.5 α-taxilin KD (stable) cells can be rescued by transient transfection with an α-taxilin mCherry plasmid (red). Huh7.5 α-taxilin KD (stable) cells were stained using a TfR-specific antibody (green). Nuclei were stained with DAPI (blue). Laser intensity and digital gain were kept constant. Magnifications, x100. (Bottom) Quantification of the fluorescence intensity of TfR (green) in Huh7.5 α-taxilin KD (stable) cells transfected with the α-taxilin mCherry plasmid and the pUC18 control compared to the transfected Huh7.5 cells. Total fluorescence per cell was calculated using ImageJ software and the following formula: corrected total cell fluorescence (CTCF) = integrated density - (area of selected cell × mean fluorescence of background readings). In total, a minimum of 10 cells were measured (mean ± SEM). Two-tailed unpaired t -test, **** p < 0.0001. (G) Viability assay for Huh7.5 and α-taxilin deficient cells. LDH release was measured by a microplate reader to determine cytotoxicity. Treatment with Triton X-100 (1%) served as a positive control.

Article Snippet: Hepatitis C virus-negative Huh7.5 cells were transfected 4 h after seeding either with 50 nM α-taxilin specific siRNA or scrRNA (sc-39644 and sc-37007, Santa Cruz Biotechnology) using N-TER TM Nanoparticle siRNA Transfection System (Sigma) according to the manufacturer’s protocol.

Techniques: Knockdown, Knock-Out, Western Blot, Derivative Assay, Transfection, Control, Staining, Two Tailed Test, Plasmid Preparation, Fluorescence, Software, Viability Assay, Positive Control