Journal: Journal of Virology

Article Title: Antagonism of epidermal growth factor receptor signaling favors hepatitis E virus life cycle

doi: 10.1128/jvi.00580-24

Figure Lengend Snippet: HEV-replicating cells show increased internalization of EGFR into lysosomal structures. Uninfected and persistently HEV-infected A549 cells were treated with 200 µM leupeptin for 24 h. ( A ) Representative confocal microscopy images of uninfected (top) and persistently HEV-infected (bottom) A549 cells immunostained for pORF2 (green), EGFR (red), and LAMP2 (cyan) after EtOH fixation. Nuclei were counterstained with DAPI (blue). Cell shown in the zoom section is indicated within the merge image. Scale bar: 40 µm. ( B ) Quantification of co-localization between cyan LAMP2 signal and red EGFR signal in panel A, depicted as tMOC per cell; N = 2; n ≥ 10. Data are presented as mean with SEM. ( C ) CTCF quantification of EGFR intensity in confocal microscopy images described in panel A; n = 20. Statistical analysis was performed using unpaired t -tests with Holm-Sidak correction for multiple-group comparison: ns: not significant; * P < 0.05, *** P < 0.001, and **** P < 0.0001. ( D ) Profile plot of region indicated in panel A (white arrow) corroborates the localization of EGFR (red) and HEV pORF2 (green) in LAMP2-positive structures (cyan). ( E and F ) 3D reconstruction of cell depicted in panel A clipped within the z-axis ( E ) or y-axis ( F ). White arrows indicate EGFR (red) inside LAMP2-positive structures (cyan). ( G ) Determination of EGFR protein half-life via a CHX chase assay in uninfected (uninf.) and persistently HEV-infected A549 cells (pers. inf.). Representative western blot of HEV pORF2 (blue = unglycosylated, gray = glycosylated), EGFR, p62, β-tubulin, and GAPDH over a time of 3 h post CHX treatment. ( H and I ) Quantification of EGFR ( H ) or p62 ( I ) amounts in panel G, shown as relative change in protein amount referred to 0 min treatment (set to 100%); N ≥ 3. A one-phase decay model was applied with an intercept of y (0) = 100 and a plateau of y = 0. Microscopy was performed on Leica TCS SP8 system with 100× objective (numerical aperture 1.4).

Article Snippet: Primary antibodies against pORF2 (HCD3K129, polyclonal rabbit α-pORF2, raised against aa112-608 of recombinant pORF2 protein), pORF3 (HPO3.1A6, purified polyclonal rabbit α-pORF3, raised against recombinant pORF3 protein), LAMP2 (polyclonal goat α-LAMP-2, AF6228; R&D Systems, Minneapolis, USA), EGFR (monoclonal mouse α-EGFR, sc-120, Santa Cruz Biotechnology), and CD63 (monoclonal mouse α-CD63, ab59479, Abcam) were incubated as previously described ( ).

Techniques: Infection, Confocal Microscopy, Comparison, Western Blot, Microscopy

Journal: Journal of Virology

Article Title: Antagonism of epidermal growth factor receptor signaling favors hepatitis E virus life cycle

doi: 10.1128/jvi.00580-24

Figure Lengend Snippet: Erlotinib treatment interferes with intracellular cholesterol homeostasis. Uninfected and persistently HEV-infected A549 cells were treated with erlotinib (25 µM) for 48 h and fixed with FA. ( A ) Representative confocal microscopy images immunostained for HEV pORF2 (green) and LAMP2 (red). Intracellular cholesterol was counterstained with filipin (magenta) and acidic organelles with lysotracker (cyan). Cell shown within the zoomed merge section is indicated within the filipin image. Scale bar: 40 µm. ( B ) Quantification of filipin CTCF per cell in panel A; n ≥ 19. Microscopy was performed on Leica TCS SP8 system with 100× objective (numerical aperture 1.4). ( C ) Quantification of colocalization (tMOC) per cell for magenta filipin signal present in red LAMP2 areas in panel A; n ≥ 20. ( D ) Quantification of lysotracker intensity as CTCF per cell in panel A; n ≥ 15. ( E ) Quantification of HEV pORF2 intensity as CTCF per cell in panel A; n ≥ 23. ( F ) Representative western blot of HEV pORF2 (blue = unglycosylated, gray = glycosylated) in lysates of uninfected and persistently HEV-infected A549 cells as well as density-gradient fractions loaded with supernatant (SN) from persistently HEV-infected A549 cells. ( G ) Extracellular HEV RNA in fractions of density-gradients as determined by qRT-PCR; depicted as % of whole genomes in gradient; N = 5. All statistics refer to respective untreated control (DMSO) or uninfected A549 cells as indicated. Unpaired t -tests with Holm-Sidak correction for multiple-group comparison: ** P < 0.01 and **** P < 0.0001.

Article Snippet: Primary antibodies against pORF2 (HCD3K129, polyclonal rabbit α-pORF2, raised against aa112-608 of recombinant pORF2 protein), pORF3 (HPO3.1A6, purified polyclonal rabbit α-pORF3, raised against recombinant pORF3 protein), LAMP2 (polyclonal goat α-LAMP-2, AF6228; R&D Systems, Minneapolis, USA), EGFR (monoclonal mouse α-EGFR, sc-120, Santa Cruz Biotechnology), and CD63 (monoclonal mouse α-CD63, ab59479, Abcam) were incubated as previously described ( ).

Techniques: Infection, Confocal Microscopy, Microscopy, Western Blot, Quantitative RT-PCR, Control, Comparison

Journal: Journal of Virology

Article Title: Antagonism of epidermal growth factor receptor signaling favors hepatitis E virus life cycle

doi: 10.1128/jvi.00580-24

Figure Lengend Snippet: EGFR inhibition promotes the endosomal system used by HEV for viral release. Uninfected and persistently HEV-infected A549 cells were treated with erlotinib (25 µM) for 48 h and fixed with EtOH. ( A ) Representative confocal microscopy images immunostained for HEV pORF2 (green), CD63 (red), and LAMP2 (cyan). Nuclei were counterstained with DAPI (blue). Cell shown in the zoom section is indicated within the merge image. Scale bar: 40 µm. Microscopy was performed on Leica TCS SP8 system with 100× objective (numerical aperture 1.4). ( B ) Quantification of CD63 CTCF per cell in panel A; n ≥ 17. ( C ) Quantification of colocalization (tMOC) per cell for green HEV pORF2 signal present in red CD63 areas in panel A; n ≥ 14. ( D ) Quantification of colocalization (tMOC) per cell for red CD63 signal present in cyan LAMP2 areas in panel A for uninfected and persistently HEV-infected cells; n ≥ 10. ( E ) Relative change in amount of intracellular syntaxin 17 (STX17) mRNA after erlotinib treatment (12.5 µM or 25 µM) for 48 h in uninfected and persistently HEV-infected A549 cells. Values refer to untreated control and were normalized toward the respective reference gene hRPL27; N = 5. All statistical analyses were performed using unpaired t -test related to respective untreated A549 cells (DMSO): * P < 0.05, ** P < 0.01, and **** P < 0.0001. ( F–H ) Volcano plot for predicted kinase activity. Shown are significantly up- (red dots) or downregulated (blue dots) kinases either upon 48 h erlotinib treatment (25 µM) in uninfected ( F ) and persistently HEV-infected ( G ) A549 cells compared to the untreated control (DMSO) or upon HEV infection comparing untreated persistently HEV-infected A549 cells with respective uninfected control ( H ). A mean final score above 1.3 was considered to be significant. Several autophagy-related kinases that were significantly changed in their activity were labeled by name. ( I ) Schematic representation of HEV- and erlotinib-based induction of the autophagy pathway. HEV impairment of lysosomal acidification changes the fate of autophagosomal structures.

Article Snippet: Primary antibodies against pORF2 (HCD3K129, polyclonal rabbit α-pORF2, raised against aa112-608 of recombinant pORF2 protein), pORF3 (HPO3.1A6, purified polyclonal rabbit α-pORF3, raised against recombinant pORF3 protein), LAMP2 (polyclonal goat α-LAMP-2, AF6228; R&D Systems, Minneapolis, USA), EGFR (monoclonal mouse α-EGFR, sc-120, Santa Cruz Biotechnology), and CD63 (monoclonal mouse α-CD63, ab59479, Abcam) were incubated as previously described ( ).

Techniques: Inhibition, Infection, Confocal Microscopy, Microscopy, Control, Activity Assay, Labeling

Journal: Autophagy

Article Title: Direct homophilic interaction of LAMP2A with the two-domain architecture revealed by site-directed photo-crosslinks and steric hindrances in mammalian cells.

doi: 10.1080/15548627.2021.1911017

Figure Lengend Snippet: Figure 1. The N-domain truncation of mouse LAMP2A impairs the CMA activity. (A) Domain architectures and orientations of five LAMP family proteins. The core domain common to LAMPs 1–5 has four conserved cysteine residues that can form two disulfide bonds (pink). Both LAMP1 and LAMP2 have two core domains, whereas LAMP3 and LAMP4 have a mucin-like domain (gray) in addition to the core domain [50,51] and LAMP5 is composed of the single core domain [52]. (B) Schematic representations of the full- length and N-domain truncated LAMP2A proteins used in this study. (C, D) rbc1cc1−/- MEFs either with or without LAMP2 (generated by guide RNA #2) were transfected with GAPDH-HT. The full-length or truncated FLAG-LAMP2A (the N-terminally FLAG-tagged) was expressed together with GAPDH-HT in LAMP2-deficient rbc1cc1−/- MEFs. The fluorescence images after labeling with TMR-HT ligand (red) for 18–20 h are shown. Nuclei were stained with Hoechst (blue). (D) Quantitative analysis of GAPDH-HT puncta. n = 50, 42, 51, 47. ***P < 0.001. Essentially the same results were obtained in three independent experiments, including one using guide RNA #1. (E) Quantitative analysis of GAPDH-HT puncta upon expression of the full-length or truncated LAMP2A, which is not tagged (left panel, n = 51, 59), or the C-terminally FLAG-tagged LAMP2A (right panel, n = 64, 53), in LAMP2-deficient rbc1cc1−/- MEFs. (F) GAPDH-HT was transiently expressed in rbc1cc1−/- MEFs (lane 2) or in the clones of LAMP2-deficient rbc1cc1−/- MEFs stably expressing the full-length (FL) or truncated (TR) N-terminally FLAG-tagged LAMP2A (lanes 3, 4 and 5, 6, respectively). As a control, rbc1cc1−/- MEFs were used without transfection of GAPDH-HT (lane 1). Twenty-four hours after the transfection of GPADH-HT, biotin-HT ligand was added to the medium and the cells were cultured for an additional 24 h (lanes 3–6). The proteins in the lysates and retrieved by NeutrAvidin beads (Pulldown) were analyzed by immunoblotting using an anti-HaloTag protein antibody, and reprobed with an anti-tubulin antibody. Closed and open triangles show GAPDH-HT (70 kDa) and likely the HT protein (30 kDa), respectively. Essentially the same results were obtained in an independent experiment. See also Fig. S2.

Article Snippet: The mouse Lamp1 and Lamp2a cDNA clones in pCMV6AC-GFP and the human LAMP2, transcript variant A, cDNA clone in pCMV6 were obtained from ORIGENE (MG225631, MG222878, and RC221216, respectively).

Techniques: Activity Assay, Generated, Transfection, Fluorescence, Labeling, Staining, Expressing, Clone Assay, Stable Transfection, Control, Cell Culture, Western Blot

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 1. Effect of LAMP2A expression on known CMA substrates. Proliferating T47D cells were either transfected with LAMP2A or empty pcDNA3 vector for 48 h (A) and with LAMP2A-siRNA or control siRNA for 72 h (B). The cells were then harvested, and protein lysates were fractionated on a 4–20% Tris Glycine SDS PAGE and immunoblotted with indicated antibodies. Densitometric analyses with respect to ACTB from three independent experiments are shown on the right of each panel. Error bars are SEM and asterisk represents a statistically significant difference (*p < 0.05, ANOVA; Tukey test). LAMP2A downregulated T47D cells were either treated with proteasome inhibitor, MG132 (C) or incubated with lysosomal inhibitor, bafilomycin A1 (BAFA1) (D) as described under methods and followed by SDS PAGE and immunoblotting with indicated antibodies. DMF and DMSO were the solvents for the inhibitors and thus serve as controls.

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, SDS Page, Incubation, Western Blot

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 2. Effect of LAMP2A expression on the half-life of GAPDH. Proliferating MCF-7 cells were transfected with LAMP2A or pcDNA3 for 24 h (A), and similarly proliferating T47D cells were transfected with LAMP2A siRNA or control siRNA (C). Representative autoradiograms of 35[S]-Methionine labeled GAPDH at the indicated time points from MCF7 (A) and T47D (C) cells are shown following immunoprecipitation with anti-GAPDH antibody in a pulse-chase experiment (see methods). Lanes are labeled according to treatments and the duration of hours after removal of 35[S]-Methionine (chase period). ‘0’ represents initial labeling. Quantifications of the labeled bands from three independent experiments were performed using Image J software and are shown in (B) (MCF-7) and (D) (T47D) with error bars (SD). Linear trend lines were drawn through the time points, and the equations of the lines are shown on the graph.

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Expressing, Transfection, Control, Labeling, Immunoprecipitation, Pulse Chase, Software

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 3. Breast tumors express higher levels of LAMP2A. The breast cancer OncoPair INSTA-BlotTM that contains 14 lanes of paired breast tumor, and matched normal adjacent tissue lysates from seven different patient donors was used. (A) Amido black staining of the blot. (B) Table portraying the type, grades, stages of breast tumors, sex and age of the breast cancer patients. (C) Patient tissue blot was immunoblotted with indicated antibodies. (D–I) Protein expression bands (as indicated) were quantified by densitometry, and the levels were normalized to ACTB (arbitrary units). The values are means ± SEMs of at least three independent experiments, and asterisk represents a statistically significant difference between samples (*p < 0.05).

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Staining, Expressing

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 4. LAMP2A overexpression stimulates breast cancer cell survival during oxidative stress. Proliferating MCF-7 (A and B) and T47D (C and D) cells were either transfected with LAMP2A, or empty pcDNA3 vector for 48 h (A and C), as well as transfected with LAMP2A siRNA or control siRNA for 72 h (B and D). (E) demonstrates T47D cells overexpressed with LAMP1 and LAMP2B. Treatment with H2O2 was performed in the final 24 h of incubation and cell survival assay was performed as described under methods. Cell viability values are means + SEM of three independent experiments and are normalized to the control (vehicle only) treatment. Significant induction in survival due to LAMP2A and reduction due to LAMP2A siRNA are indicated by an asterisk (*p < 0.05, ANOVA; Tukey test).

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Incubation, Clonogenic Cell Survival Assay

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 5. Ectopic expression of LAMP2A inhibits protein carbonyl content (PCC) formation. Proliferating T47D cells were transiently transfected with LAMP2A or empty pcDNA3 vector for 48 h (A and C) as well as transfected with LAMP2A siRNA or control siRNA for 72 h (B and D). Cytosolic extracts were prepared; and 50 μg aliquots were tested for their PCC. (C and D) represent the quantitative estimation of PCC, expressed as means ± SEMs of three independent experiments. Significant changes in the PCC contents are indicated by an asterisk (*p < 0.05 ANOVA; Tukey test). Western blots showing the protein levels of LAMP2A and ACTB in different transfection conditions are provided in (E and F).

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 6. Knockdown of LAMP2A triggers AKT1 phosphorylation at Ser473. Proliferating T47D cells were either transfected with LAMP2A, or empty pcDNA3 vector for 48 h (A, C and E) and with LAMP2A siRNA or control siRNA for 72 h (B, D and F). (A and B) showing western blots analysis of total AKT1 and phospho AKT1 (Ser 473). (C and D) demonstrate AKT1 kinase activity using AKT1 substrate GSK3 fusion protein as a measure of AKT1 activation. (E and F) demonstrate the AKT1 substrate assay under oxidative stress condition rendered by 150 nM H2O2. Densitometric analyses from (E and F) are given under each panel. Data are mean ± SEMs, and significant changes are indicated by an asterisk (*p < 0.05 ANOVA; Tukey test). “#” represents the sample lanes that were treated with 1 μM Wortmannin. (G and H) Starvation does not promote AKT1 phosphorylation. Both MCF-7 and T47D cells were starved as described under methods and the cells were harvested after 48 h followed by SDS PAGE, and immunoblotted with indicated antibodies.

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Knockdown, Phospho-proteomics, Transfection, Plasmid Preparation, Control, Western Blot, Activity Assay, Activation Assay, SDS Page

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 7. LAMP2A modulates ROS generation in breast cancer cells. Proliferating T47D cells were either transfected with LAMP2A or empty pcDNA3 vector for 48 h (A and C) and with LAMP2A siRNA or control siRNA for 72 h (B and D) in the presence of 150 nm H2O2 for the last 24 h. (A and B) Cells were assayed for carboxy-DCF fluorescence by flow cytometry, and the total geometric means of the carboxy-DCF tagged cells were plotted in each case. (C and D) Cellular uptakes of carboxy-DCF by the indicated cells were imaged live using confocal microscopy (Fig. S9). The green fluorescence due to carboxy DCF among different samples was analyzed using Metamorph imaging software and represented as bar diagram. Error bars are SEM and asterisk represents a statistically significant difference (*p < 0.05, ANOVA; Tukey test).

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Transfection, Plasmid Preparation, Control, Fluorescence, Flow Cytometry, Confocal Microscopy, Imaging, Software

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 8. Downregulation of LAMP2A triggers apoptosis in breast cancer cells. Proliferating T47D cells were either transfected with LAMP2A or empty pcDNA3 vector for 48 h or transfected with LAMP2A siRNA or control siRNA for 72 h followed by incubation with 150 nm H2O2 for the last 24 h. The cells were then assayed using Apo-Direct apoptosis kit. Percentage of FITC positive cells from both LAMP2A overexpressed (A) and underexpressed (B) samples were represented as a bar diagram. (C and D) demonstrate immunoblotting with CASP3 and cleaved CASP3 fragments in the indicated samples. Protein expression levels of BAX and BCL2 in LAMP2A overexpressed cells, as well as LAMP2A knock down cells, are shown in (E and F). The densitometric analyses of the indicated proteins with respect to ACTB from three independent experiments are shown as bar diagram in (G and H). Values are means ± SEMs from three independent experiments. Significant changes are indicated by an asterisk (*p < 0.05 ANOVA; Tukey test).

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Transfection, Plasmid Preparation, Control, Incubation, Western Blot, Expressing, Knockdown

Journal: Autophagy

Article Title: LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy

doi: 10.4161/auto.21654

Figure Lengend Snippet: Figure 9. Inhibition of LAMP2A sensitizes breast tumor cells to DNA damaging drug. Proliferating MCF-7 cells (A and B) and T47D cells (C and D) were transfected with LAMP2A siRNA or control siRNA for 72 h and treated/untreated with 2.5 μM doxorubicin for the last 24 h of transfections. The cells were then subjected to cell survivability assay as described under methods. – DOX indicates no doxorubicin treatments where as + DOX indicates cells treated with doxorubicin. The DOX treatments were compared with no DOX treatment in (B) for MCF-7 cells and (D) for T47D cells. Error bars representing SEM and significant changes is indicated by an asterisk (*p < 0.05 ANOVA; Tukey test).

Article Snippet: Expression vectors and reagents LAMP2A expression plasmid was purchased from ORIGENE, (SC118738) that harbors a full length open reading frame (ORF) and driven by the cytomegalovirus (CMV) promoter.

Techniques: Inhibition, Transfection, Control