Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 1. Tumor-associated soluble uPAR (s-uPAR) enhances HUVEC invasion, migration and angiogenesis. (a) Conditioned medium (CM) was collected from tumor cells (parental and stably expressing empty vector (EV), uPAR-cDNA (UR) and uPAR siRNA (UR-Si)). Immunoblot analyses were performed for s-uPAR and DDK using specific antibodies. (b) s-uPAR levels in CM were quantified using uPAR Quantikine Immunoassay kit. Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental control. (c) Cells were labeled (tumor cells: Qtracker-525-Green and HUVECs: Qtracker- 655-Red) and seeded into separate chambers of culture inserts. After 16 h, the culture inserts were removed and cells were allowed to migrate for a further 24 h. Images were captured at 0 and 24 h of incubation and cell migration was quantified using ImageJ software (NIH). The levels of HUVEC migration were normalized to HUVEC migration in parental cells and are represented as arbitrary units. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental control. (d) HUVEC invasion experiments were performed using ThinCertTM inserts as described in Materials and methods. The levels of HUVEC invasion was quantified and normalized to HUVEC invasion in parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (e, f) In vitro angiogenesis assay was performed as described in Materials and methods. The degree of angiogenic induction by CM was quantified by ImageJ software (NIH) for the numerical value of the product of the relative capillary length per microscopic field. Serum-free medium (SFM) and recombinant human uPAR (rh-uPAR) in SFM were used as controls (insets). Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM. uPAR antibody, uPAR-Ab; isotype control, NSp.IgG. (g) Migration assay was performed using CM. In this case, both chambers of culture inserts were seeded with HUVECs. After 16 h, the culture inserts were removed, CM was added and cells were allowed to migrate for 24 h. Invasion assay was performed as described above. uPAR-Ab. or Nsp.IgG were added to UR-CM before adding onto cells. rh-uPAR was added to SFM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Labeling, Incubation, Software, In Vitro, Angiogenesis Assay, Recombinant, Invasion Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 2. s-uPAR recruits onto HUVEC membrane. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured on CM for 24 h, labeled with anti-uPAR antibody, followed by Alexa Fluor-488-conjugated secondary antibody and were analyzed by fluorescence-activated cell sorting (FACS) for uPAR expression. Serum-free medium (SFM) and rh-uPAR were used as controls. Isotype control (Neg.). (b) HUVECs were cultured in chamber slides on CM for 24 h and fixed in 4% paraformaldehyde and 0.2% glutaraldedyde in phosphate-buffered saline for 1 h. Immunocytochemical analysis was performed as described in Materials and methods. Isotype control (Neg.; inset). Slides were mounted and photographed. (c) Equal amounts of proteins were used for the extraction of HUVEC membrane fractions and were subjected to immunoblot analysis for uPAR expression using specific antibodies. The blot was re-probed for DDK-tag expression.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Membrane, Cell Culture, Labeling, FACS, Expressing, Control, Saline, Extraction, Western Blot

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 3. s-uPAR colocalizes in lipid rafts on HUVECs. Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. (a) HUVECs were cultured in chamber slides on CM for 24 h and incubated with anti-uPAR antibody followed by Alexa Fluor-488- conjugated secondary antibody at 4 1C. Cells were again labeled with Alexa Fluor-595-CTxB subunit. Slides were mounted and analyzed by confocal microscopy. Negative controls, using an isotype antibody, showed no staining (inset). Serum-free medium (SFM) and DDK-tag containing rh-uPAR were used as controls. To disrupt lipid rafts, HUVECs were pretreated with MBCD, as described in Materials and methods. (b) HUVECs lipid rafts were isolated as described in Materials and methods. Lipid raft-enriched fractions were analyzed for uPAR and DDK-tag levels using immunoblot analysis. Flotillin-1 and caveolin-1 served as controls. Protein band intensities were quantified by densitometric analysis using ImageJ software (NIH). The levels of uPAR protein were normalized to protein levels in HUVECs cultured on parental-CM. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM. (c) Invasion and migration assays were performed as described in Figure 1d In vitro angiogenesis assay was performed as described in Figure 1. To deplete cholesterol, HUVECs were pretreated with MBCD as described in Materials and methods (c and d). Columns: mean; bars: s.d.; n ¼ 3; *po0.01 vs parental-CM; **po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Culture, Incubation, Labeling, Confocal Microscopy, Staining, Isolation, Western Blot, Software, Migration, In Vitro, Angiogenesis Assay

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 4. s-uPAR induces ERK/Rac1-mediated migration and tube formation in HUVECs. Conditioned medium (CM) was collected from tumor cells, as described in Materials and methods. (a) HUVECs lysates were used to perform GST-Rac1 pull-down assay. The protein complexes were subjected to immunoblot analysis to detect active Rac1. Rac1 from total cell lysates was used as a control. (b) Total cell lysates were subjected to immunoblot analysis for phospho-ERK1/2 (pERK1/2) and total ERK1/2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. HUVECs grown on rh-uPAR were used as a control. (c) HUVECs were cultured on CM alone and/or supplemented with functional blocking anti-uPAR antibody (uPAR-Ab) or isotype control (Nsp.IgG.) or MEK inhibitor (U0126) for 24 h. Cell lysates or GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1, Rac1 pERK1/2 and ERK1/2. GAPDH served as a loading control. (d) HUVECs were transfected with dominant-negative mutant Rac1 (Dn-Rac1) for 24 h and cultured on UR-CM. Micrographs were captured for green fluorescent protein (GFP) expression (green) and phase contrast (gray) immediately after the addition of UR-CM (magnification 60). (e) HUVECs were transfected with Dn-Rac1 for 24 h, cultured on CM for another 24 h, collected and lysed. GST-Rac1 pull- down protein complexes were subjected to immunoblot analysis to detect active Rac1. GFP and Rac1 from total cell lysates were used as controls. (f) HUVECs were transfected with Dn-Rac1 for 24 h and cultured on CM alone and/or supplemented with uPAR-Ab., or Nsp.IgG or U0126 for another 24 h. Invasion and migration assays were performed as described in Figure 1. Columns: mean; bars: s.d.; n ¼ 3; *Po0.01 vs parental-CM; **Po0.01 vs UR-CM.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Migration, Pull Down Assay, Western Blot, Control, Cell Culture, Functional Assay, Blocking Assay, Transfection, Dominant Negative Mutation, Expressing

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 5. Diverse forms of tumor-associated s-uPAR in vitro and in vivo. (a) Conditioned medium (CM) was collected from tumor cells as described in Materials and methods. CM was subjected to deglycosylation using a deglycosylation kit and analyzed by immunoblot for uPAR using specific antibodies. (b) Equal amount of proteins containing HUVEC lysates were used for extraction of cell membrane fractions and were subjected to deglycosylation, and analyzed by immunoblot for uPAR using specific antibodies. (c) In vivo angiogenic assay was performed by using the dorsal air sac model. 4910EV (EV), 4910UR (UR), 4910UR-Si (UR-Si) cells or a recombinant human uPAR (rh-uPAR) containing chamber was implanted in the dorsal cavity of mice. The micrographs for the presence of tumor-induced neovasculature (microvessels with curved thin structures and many tiny bleeding spots) and pre-existing vasculature (straight) were captured. Representative micrographs are shown. (d, e) Blood was collected from mice orthotopically xenografted with stably expressing EV, UR and UR-Si cells. Total uPAR levels were estimated using a commercial human uPAR Quantikine Immunoassay kit according to the manufacturer’s instructions. The data quantification for a set I (n ¼ 4; d) and set II (n ¼ 6; e), on day 15 and 40, respectively, after cell implantation are shown. Columns: mean; bars: s.d.; *Po0.01 vs parental control. (f) Blood serum (from mice 1–6; on day 40) was subjected to deglycosylation and analyzed by immunoblot for uPAR using specific antibodies. D2-D3, D2-D3 domain containing truncated s-uPAR; D3, D3 domain containing truncated s-uPAR; FL, full-length s-uPAR; .

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Western Blot, Extraction, Membrane, Recombinant, Stable Transfection, Expressing, Control

Journal: Oncogenesis

Article Title: Tumor-associated soluble uPAR-directed endothelial cell motility and tumor angiogenesis.

doi: 10.1038/oncsis.2013.19

Figure Lengend Snippet: Figure 6. uPAR overexpression enhances tumor growth, vascularity and s-uPAR recruits onto endothelial cells in vivo. (a) Stably expressing EV, UR and UR-Si cells were injected intracerebrally into mice. Mice were euthanized and brains were collected and fixed as described in Materials and methods. Brain sections were stained with hematoxylin and eosin (H&E) solution, and representative micrographs are shown (upper panel). H&E-stained micrographs showing the tumor invasive front ( 20; lower panel). (b) Brain tumor areas were calculated using Image Pro Discovery Program software (Media Cybernetics, Inc., Rockville, MD, USA). Columns: mean; bars: s.d.; n ¼ 6; *Po0.01 vs parental controls. (c) Immunohistochemical analysis of brain sections using anti-uPAR and anti-vascular endothelial growth factor (VEGF). Blood vessels in tumor sections were visualized with biotin-labeled tomato lectin. Inset: isotype control. (d, e) Fluorescence microscopy for colocalization of an endothelial cell marker (von Willebrand factor (vWF)/anti-CD31) and DDK-tag in tumor sections from mice that were implanted with 4910 EV (EV) and 4910UR (UR) cells. Inset, isotype control.

Article Snippet: Enzyme-linked immunosorbent assay Blood plasma or CM was prepared as mentioned in appropriate sections, and s-uPAR levels were determined using a commercial human uPAR Quantikine Immunoassay kit (R&D Systems) according to the manufacturer’s instructions.

Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Injection, Staining, Software, Immunohistochemical staining, Labeling, Control, Fluorescence, Microscopy, Marker

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: VP treatment enhances apical AQP2 expression and its colocalization with ezrin. (A) VP treatment enhances apical AQP2 expression and its colocalization with ezrin in cultured renal epithelial cells. AQP2-MDCK cells were stained with antibodies against ezrin (green) and AQP2 (red) in the presence (VP) and absence (Control) of VP treatment (AVP 20 nM for 20 min). The larger panels represent confocal sections through the subapical regions of the cells above the nucleus. The smaller horizontal strips at the bottom of each panel are z-sections through the entire cell for direct comparison of the respective staining intensities of the apical and basolateral membranes, and the cytosol. Upper panels show that in the absence of VP stimulation, ezrin staining localized to the cytosol and basolateral region, while AQP2 staining was mainly detected in the subapical region. Lower panels show that after VP treatment, the ezrin signal was redistributed toward the apical and sub-apical regions and partially colocalized with the similarly apically redistributed AQP2. Scale bar: 10 μm. (B) Super-resolution Airyscan confocal microscopy imaging revealed that AQP2 and ezrin partially colocalize on the apical membrane in VP-treated MDCK cells. Left panels show no apparent colocalization of ezrin and AQP2, in the absence of VP stimulation. Right panels are cells treated with VP. Scale bar: 5 μm. (C) AQP2 and ezrin are co-expressed in principal cells of the Brattleboro rat collecting duct, and co-accumulate on the plasma membrane after vasopressin treatment. Without VP treatment (Control), ezrin was located in the cytosol and basal region, while AQP2 was detected mainly in the sub-apical region of the principal cells of the collecting ducts. After 7 days of VP treatment (VP), ezrin (red in the merge panel) colocalized with AQP2 (green in the merge panel) on the plasma membrane of the principal cells. Scale bar: 20 μm.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Expressing, Cell Culture, Staining, Confocal Microscopy, Imaging

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Interaction of AQP2 with ezrin is detected in co-IP experiments. (A) List of ezrin peptides detected by mass spectrometry from the AQP2 co-IP complex. (B,C) By using an anti-ezrin antibody for co-IP, we were able to detect AQP2 in the co-IP complex from stable AQP2-expressing LLC-PK1 cell lysates and mouse kidney (B). Similarly, ezrin signal was detected in the co-IP complex using anti-AQP2 antibody (C). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Expressing, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 interacts with ezrin via the ezrin N-terminal FERM-containing domain. (A) Recombinant His-tagged ezrin full-length (FL, amino acids 1–586), N-terminus (NT, amino acids 1–308), and C-terminus (CT, amino acids 285–586) were expressed in E. coli and purified to homogeneity as revealed by SDS-PAGE together with purified recombinant AQP2 C-terminus (AQP2CT). Schematic representation of each recombinant protein is shown in B. (C) The purified His-tagged ezrin full-length protein (FL) and the N-terminal FERM-containing recombinant protein (but not the C-terminal protein) were able to pull down AQP2 from both LLC-AQP2 cell lysates and mouse kidney lysates. Lanes 1–3, beads alone pulled down with PBS (lane 1), LLC-AQP2 cell lysate (lane 2) and kidney lysate (lane 3); lanes 4–6, ezrin FL pulled down with PBS (lane 4), LLC-AQP2 cell lysate (lane 5) and kidney lysate (lane 6); lanes 7–9, ezrin NT pulled down with PBS (lane 7), LLC-AQP2 cell lysate (lane 8) and kidney lysate (lane 9); lanes 10–12, ezrin CT pulled down with PBS (lane 10), LLC-AQP2 cell lysate (lane 11) and kidney lysate (lane 12). WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Recombinant, Purification, SDS Page, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 C-terminus directly interacts with ezrin N-terminal FERM-containing domain. The direct interaction of AQP2 and ezrin is revealed by pulldown experiments using purified recombinant ezrin and AQP2 proteins. Only the purified ezrin full-length (FL) and the N-terminus FERM domain-containing (NT) recombinant protein were able to pull down the purified AQP2 C-terminus. The ezrin C-terminal domain (CT) did not pull down the AQP2 C-terminal domain. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Purification, Recombinant, Western Blot

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin causes membrane accumulation of AQP2. (A) Immunoblotting revealed a strong knockdown of endogenous ezrin in LLC-AQP2 cells by ezrin shRNA lentivirus. There was an ∼80% reduction of endogenous ezrin in ezrin shRNA knockdown LLC-AQP2 cells (graph shows the quantification of ezrin band intensity relative to actin). Results are mean±s.e.m. (n≥3). ***P<0.001 (one-way ANOVA). (B) Immunofluorescence staining of AQP2 in control LLC-AQP2 cells and cells infected with ezrin shRNA lentivirus. After knocking down ezrin in LLC-AQP2 cells, AQP2 was found to increasingly accumulate on the cell surface under baseline conditions (without any stimulation). VP-treated LLC-AQP2 cells were used for comparison. Scale bar: 10 μm. (C) Surface biotinylation experiment revealed a significantly increased accumulation of AQP2 signal on cell surface after knocking down ezrin expression in cells. Transferrin receptor 1 (TFR-1) was used as an internal control for stable membrane proteins. WB, western blotting.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Immunofluorescence, Staining, Infection, Expressing

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Phosphorylation of key residues in AQP2 was not altered in ezrin knockdown cells. (A) Representative western blot showing that there is no alteration of the levels of total AQP2 and AQP2 phosphorylated at residues 256 or 261 in ezrin shRNA lentivirus-infected LLC-AQP2 cells unlike in cells treated with VP. Immunoblot using anti-β-actin antibody was used as control. Quantification of western blotting results for AQP2 phosphorylated on S256 (p256), S261 (p261) and total AQP2. (B) Intracellular cAMP measurement in LLC-PK1 cells. The intracellular cAMP concentration was significantly increased by 30 min VP treatment (20 nM LVP) without clonal variation. After ezrin knockdown no significant difference in cAMP concentration was observed compared to control. Results in A and B are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Western Blot, shRNA, Infection, Concentration Assay

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: Downregulating ezrin reduces clathrin-mediated endocytosis and causes concomitant membrane accumulation of AQP2 without affecting the overall exocytosis. (A) Ezrin knockdown does not affect the overall exocytosis in LLC-AQP2-ssYFP cells. LLC-AQP2 cells were stably transfected with ssYFP. The fluorescence signal in the extracellular medium was measured in LLC-AQP2-ssYFP cells with and without treatment with VP or ezrin shRNA lentivirus, respectively. The fluorescence intensity in the medium reflected the exocytotic activity of LLC-AQP2-ssYFP cells, and therefore the rate of exocytosis. No significant increase was seen in the overall exocytosis in LLC-AQP2-ssYFP cells treated with ezrin shRNA lentivirus compared to control. In contrast, a significant increase in exocytosis was observed in VP-treated LLC-AQP2-ssYFP cells, which is consistent with our previous reports (Nunes et al., 2008). (B) Endocytosis assay using Rhodamine-conjugated transferrin showed that Alexa Fluor 568-labeled transferrin accumulated on the apical membrane following ezrin knockdown (upper panel), and a simultaneous acute membrane accumulation of AQP2 (lower panel). Scale bar: 10 μm. (C) A bar graph showing that ezrin knockdown affects clathrin-mediated endocytosis. A block of endocytosis with MβCD was used as a positive control. Results in A and C are mean±s.e.m. (n≥3). **P<0.01, ***P<0.001 (one-way ANOVA).

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Stable Transfection, Transfection, Fluorescence, shRNA, Activity Assay, Endocytosis Assay, Labeling, Blocking Assay, Positive Control

Journal: Journal of Cell Science

Article Title: Ezrin directly interacts with AQP2 and promotes its endocytosis

doi: 10.1242/jcs.204842

Figure Lengend Snippet: AQP2 internalization was reduced after ezrin knockdown in a cold block experiment. (A) Dynamic distribution of AQP2 after a 20°C cold block was revealed by immunofluorescence staining of LLC-AQP2 cells. Cells were incubated at 20°C for 30, 60 and 120 min to block AQP2 release from the trans-Golgi network. AQP2 formed a ‘perinuclear patch’ in cells at 20°C. In the absence of protein synthesis (blocked by cycloheximide), the speed of formation and immunostaining intensity of the perinuclear patch reflect the speed and extent of AQP2 internalization from the cell surface over time. Scale bar: 10 μm. (B) Quantification of the growth of the AQP2-positive perinuclear patch after 20°C cold block over time. The fluorescence intensity of AQP2 staining in the perinuclear patch was measured using Volocity software as described previously (Arthur et al., 2015). Results are mean±s.e.m. (n≥13 for each data point). Experiments were repeated at least three times.

Article Snippet: The commercial primary antibodies are from the following vendors: rabbit polyclonal anti-AQP2 antibody (#AQP2-002, Alomone Labs, Jerusalem, Israel), goat polyclonal anti-AQP2 antibody (sc9882, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-ezrin (3145s, Cell Signaling), rabbit anti-AQP2 p256 (PhosphoSolutions, Aurora, CO, USA), rabbit anti-AQP2 p261 (PhosphoSolutions), mouse monoclonal anti-GST antibody (sc138, Santa Cruz Biotechnology), mouse monoclonal anti-His antibody (70796, EMD Millipore, Billerica, MA), mouse anti-GAPDH antibody (AM4300, Ambion/Thermo Fisher Scientific), mouse anti β-actin antibody (A5441, Sigma-Aldrich), and rabbit anti-transferrin receptor 1 antibody (ab84036, Abcam, Cambridge, MA).

Techniques: Blocking Assay, Immunofluorescence, Staining, Incubation, Immunostaining, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: PCV2 infection led to translocation of HMGB1 from nuclei to cytoplasmic compartments. PK-15 cells and porcine monocytic cells (3D4/31) were infected for 36 h with PCV2 (MOI = 1) or mock infected as a control. (A) Confocal imaging of HMGB1 distribution in PCV2-infected cells immunostained with anti-HMGB1 (green) and anti-Cap (red) antibodies. Nuclei were labeled with DAPI (blue). Representative micrographic images are shown. (B) Immunoblotting of PCV2 Cap and HMGB1 in nuclear and cytoplasmic extracts from PCV2- or mock-infected PK-15 cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic fractions, respectively. (C) The intensity of protein bands was quantified densitometrically using Gel-Pro Analyzer. Ratios of nuclear or cytoplasmic HMGB1 to Histone H3 or GAPDH were quantified, respectively. (D and E) Quantification of hmgb1 mRNA by qPCR in PK-15 and 3D4/31 cells infected with PCV2 for different times using total RNA extracts from the cells. (F and G) Immunoblotting of HMGB1 and PCV2 Cap in the lysates of PK-15 and 3D4/31 cells infected with PCV2 for different times. β-Actin was used as a loading control. The data in panels A, B, F, and G are representative of three independent experiments. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Translocation Assay, Imaging, Labeling, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Overexpression of HMGB1 inhibited PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (A) Effect of HMGB1 overexpression on PCV2 Cap expression as shown by immunoblotting using protein samples from the whole-cell lysates. β-Actin was used as a loading control. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of HMGB1 overexpression on PCV2 orf2 (encoding Cap) transcription measured by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in cells overexpressing HMGB1 as assessed by indirect immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the HMGB1 overexpressing cells are shown with nontransfected but PCV2-infected cells set at 100%. (E) PCV2 genomic DNA copies in cells overexpressing HMGB1 quantified by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Over Expression, Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Downregulation of HMGB1 promoted PCV2 replication. PK-15 cells were transfected with hmgb1-specific RNA interference (RNAi) plasmid (sh-HMGB1) or control RNAi plasmid (sh-NC) for 24 h and then infected with PCV2 (MOI= 1) for 36 h. (A) Effect of hmgb1 knockdown on PCV2 Cap expression (β-actin used as a loading control) as shown by immunoblotting using protein samples from the whole-cell lysates. The gel shown is representative of three independent experiments. (B) The ratios of band intensity of HMGB1 or PCV2 Cap to β-actin (as shown in panel A). (C) Effect of hmgb1 knockdown on PCV2 orf2 (encoding Cap) transcription examined by qPCR using total RNA extracted from the whole-cell lysates. (D) PCV2 replication in hmgb1-silenced cells as assessed by indirect immunofluorescence. Representative fluorescence images are shown (top). Percentage of PCV2-infected cells was calculated by dividing the number of PCV2-infected cells by the total cell number in each group (n = 2 images for each experiment per group) that were counted using ImageJ software. Relative percentages of PCV2-infected cells in the hmgb1-silenced cells are shown with nontransfected but PCV2-infected cells set at 100% (bottom). (E) Effect of hmgb1 silencing on PCV2 genomic DNA copies measured by qPCR using total DNA extracts from whole-cell lysates. Bar charts in panels B, C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Plasmid Preparation, Infection, Expressing, Western Blot, Immunofluorescence, Fluorescence, Software

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Nuclear HMGB1 repressed PCV2 replication. PK-15 cells were transfected with recombinant plasmid expressing HMGB1 (pHMGB1) or control plasmid (pFlag) for 24 h and then infected with PCV2 (MOI = 1) for 36 h. Nuclear and cytoplasmic extracts were prepared for immunoblotting as described in the legend for Fig. 1 Immunoblotting of HMGB1 and PCV2 Cap in the nuclear (A) and cytoplasmic (B) fractions. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel A) in the nuclear fraction (C) or to GAPDH (as shown in panel B) in the cytoplasmic fraction (D). (E) Effect of HMGB1 overexpression on PCV2 orf2 transcription in the nuclei examined by qPCR using total RNA extracted from the nuclear fractions. Results were normalized to histone H3 mRNA in the same samples. (F) PCV2 genomic DNA replication in the nuclei of HMGB1-overexpressing cells quantified by qPCR using total DNA extracted from nuclear fractions. Bar charts in panels C to F show means ± SDs from three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Transfection, Recombinant, Plasmid Preparation, Expressing, Infection, Western Blot, Over Expression

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate inhibited nucleocytoplasmic translocation of HMGB1 in PCV2-infected cells. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Confocal microscopic images show inhibition of nuclear HMGB1 migration into the cytosol by EP. Cells were immunostained for HMGB1 (green) and PCV2 Cap (red), with nuclei stained with DAPI (blue). Bars, 10 μm. (B) Immunoblotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic fractions of PCV2-infected and EP-treated cells. Histone H3 and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. Representative images from three independent experiments are shown. The ratios of band intensities of HMGB1 or PCV2 Cap to those of histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of EP on PCV2 genomic DNA replication by qPCR using DNA extracted from nuclei of PCV2-infected cells treated with 7.5 mM EP. Bar charts in panels C, D, and E show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Inhibition, Migration, Staining, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: N-Acetylcysteine inhibited PCV2-induced HMGB1 translocation from nuclei to cytosol and repressed PCV2 replication. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) for 12 h and then treated with 10 mM N-acetylcysteine (NAC). The cell samples were harvested at 36 hpi. (A) Confocal imaging of HMGB1 distribution in PCV2-infected and NAC-treated cells after the cells were fixed and immunostained for HMGB1 (green) and Cap (red). Nuclei were stained with DAPI (blue). Bars, 10 μm. (B) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without NAC treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. The figure is representative of three independent experiments. The ratios of band intensities of HMGB1 or PCV2 Cap to histone H3 (as shown in panel B, left) in the nuclear fraction (C) or to GAPDH (as shown in panel B, right) in the cytoplasmic fraction (D). (E) Effect of NAC on PCV2 genomic DNA replication by qPCR using DNA extracted from lysates of PCV2-infected cells treated with NAC. (F) Cytosolic ROS levels in PCV2-infected cells with or without treatment by NAC or ethyl pyruvate (EP) as measured by flow cytometry after probing with DCFH-DA. Bar charts in panels C, D, E, and F show means ± SDs from three independent experiments. *, P < 0.05; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Infection, Imaging, Staining, Flow Cytometry

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Ethyl pyruvate was inhibitory to PCV2 infection. PK-15 cells were mock infected or infected with PCV2 (MOI = 1) with or without ethyl pyruvate (EP; 7.5 mM) treatment. The cell samples were harvested at 36 hpi. (A) Effect of EP on PCV2 replication in PK-15 cells by immunofluorescence. Percentages of PCV2-infected cells were calculated as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the EP-treated cells are shown with untreated but PCV2-infected cells set at 100%. (B) Immunoblotting of HMGB1 and PCV2 Cap in whole-cell lysates with β-actin used as a loading control. (C) The ratios of band intensities of HMGB1 or PCV2 Cap to β-actin (as shown in panel B). Bar charts in panels A and C show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: The B box domain of HMGB1 was involved in inhibition of PCV2 replication. (A) Schematic illustration of the full-length and truncated forms of porcine HMGB1 according to its human homolog. All truncated versions, A box, AB box, and B box plus C terminus (B boxCT), were flag tagged. The numbers indicate positions of amino acids. Arrows with C followed by numbers represent key cysteine residues. NLS, nuclear localization signal. PK-15 cells were transfected with recombinant plasmids expressing flag-tagged or full-length HMGB1 for 24 h and then infected with PCV2 (MOI = 1) for 36 h. (B) Numbers of PCV2-infected cells examined by immunofluorescence using anti-Cap monoclonal antibody as the probe (top). Expression of PCV2 Cap and different forms of HMGB1 as assessed by immunoblotting using the whole-cell lysates harvested at 36 hpi and antibodies against Flag, Cap, and HMGB1 (bottom). β-Actin was used as a loading control. The panel B images are representative of three individual experiments. (C) The ratios of band intensities of PCV2 Cap to those of β-actin (as shown at the bottom panel of B). (D) Effect of different HMGB1 truncations on PCV2 DNA replication estimated by qPCR using total DNA extracted from the whole-cell lysate. Bar charts in panels C and D show means ± SDs from three independent experiments. ns, not significant; **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Inhibition, Transfection, Recombinant, Expressing, Infection, Immunofluorescence, Western Blot

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: HMGB1 bound to the Ori region of the PCV2 genome. (A) Binding of porcine HMGB1 to PCV2 DNA using the gel shift assay. PCV2 DNA (500 ng) and various concentrations (0 to 5 μg) of purified His-tagged recombinant HMGB1 were mixed in binding buffer. The DNA-protein mixtures were subjected to 0.8% agarose gel electrophoresis to visualize changes of the DNA motility. (B) Binding of HMGB1 to a specific region of PCV2 DNA: full-length and different fragments of PCV2 genome (orf1, orf2, and Ori) were incubated with recombinant HMGB1 protein to identify the region of PCV2 genome involved in HMGB1 binding. (C) To confirm the Ori region is required for HMGB1 binding, the Ori fragment was combined with orf1 or orf2 (Ori-orf1 or Ori-orf2) that were then compared with orf1 or orf2 alone by the gel shift assay. (D) Immunoprecipitation of purified HMGB1 protein (500 μg) and PCV2 DNA (500 ng) mixture by anti-HMGB1 antibody (rabbit IgG as control) and protein A/G agarose. The precipitates were probed with anti-His and anti-HMGB1 antibodies by immunoblotting. (E) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from PCV2 DNA-HMGB1 precipitates (shown in panel D) after DNase pretreatment. (F) Blotting of HMGB1 in immunoprecipitates of whole-cell lysates (WCL) of the PK-15 cells infected with PCV2 (36 h) by anti-HMGB1 (rabbit IgG as control) and protein A/G agarose. (G) Quantification of PCV2 genomic Ori copies by qPCR in DNA extracts from immunoprecipitates of whole-cell lysates (shown in panel F) after DNase pretreatment. (H) Blotting of HMGB1 in immunoprecipitates of nuclear extracts of the PK-15 cells infected with PCV2. (I) Quantification of PCV2 genomic Ori copies in the precipitates shown in panel H. Bar charts in panels E, G, and I show means ± SDs from three independent experiments.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Agarose Gel Electrophoresis, Incubation, Immunoprecipitation, Western Blot, Infection

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Effect of hydrogen peroxide treatment on subcellular localization of HMGB1 and PCV2 replication. (A) H2O2 treatment promoted nucleocytoplasmic translocation of HMGB1. PK-15 cells were treated with or without N-acetylcysteine (NAC; 10 mM) before adding 50 μM H2O2. Cells were fixed and immunostained with anti-HMGB1 (green) for confocal microscopy. Nuclei were labeled with DAPI (blue). (B) Immunoblotting of HMGB1 in the nuclear and cytoplasmic fractions of PK-15 cells treated with H2O2 and NAC. (C) Confocal imaging of PK-15 cells infected by PCV2 with or without 50 μM H2O2 treatment after immunostaining with anti-HMGB1 (green) and anti-Cap (red) antibodies. (D) Blotting of HMGB1 and PCV2 Cap in the nuclear and cytoplasmic extracts of PCV2-infected cells with or without H2O2 treatment. Histone H3 and GAPDH were used as internal controls for the nuclear and cytoplasmic fractions, respectively. (E) Percentages of PCV2-infected cells were calculated from immunofluorescence images as described in the legend for Fig. 2. Relative percentages of PCV2-infected cells in the H2O2-treated cells are shown with nontreated but PCV2-infected cells set at 100%. Bar chart in panel E shows means ± SDs from three independent experiments. **, P < 0.01.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Translocation Assay, Confocal Microscopy, Labeling, Western Blot, Imaging, Infection, Immunostaining, Immunofluorescence

Journal: Journal of Virology

Article Title: PCV2 Induces Reactive Oxygen Species To Promote Nucleocytoplasmic Translocation of the Viral DNA Binding Protein HMGB1 To Enhance Its Replication

doi: 10.1128/JVI.00238-20

Figure Lengend Snippet: Schematic illustration of the interaction between PCV2 and HMGB1 in infected cells. HMGB1 in the nucleus restricts PCV2 replication by binding to the Ori region of the PCV2 genome. PCV2 infection causes increased generation of cellular ROS. Increased ROS promotes nucleocytoplasmic translocation of HMGB1 and lessens sequestration of the viral DNA by HMGB1 in the nucleus, thus enhancing PCV2 replication. N-Acetylcysteine (and probably ethyl pyruvate as well) scavenges PCV2-induced ROS and thus increases retention of HMGB1 in the nucleus, leading to sequestration of viral DNA and reduced PCV2 replication.

Article Snippet: Approximately 1 mg of total cellular proteins or nuclear proteins was transferred to a 1.5-ml microcentrifuge tube and incubated with 4 μg of rabbit anti-HMGB1 polyclonal antibody (ChIP grade, ab18256) (Abcam) or normal rabbit IgG (Beyotime) for 2 h at 4°C.

Techniques: Infection, Binding Assay, Translocation Assay

Journal: Frontiers in Immunology

Article Title: Next-generation cell lines for profiling different proteasome forms and their implications in cancer

doi: 10.3389/fimmu.2025.1672000

Figure Lengend Snippet: Generation of SW620B8-mCherryB5-GFP and TZM-blB8-mCherryB10-CFP cell lines. (A) Schematic representation of genome modifications in SW620B8-mCherryB5-GFP (left) and TZM-blB8-mCherryB10-CFP (right) cell lines. (B) Analysis of SW620- and TZM-bl-derived cells by flow cytometry. Populations of control cells and cells stimulated for 72 h with pro-inflammatory cytokines (1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF) are shown. Control cell populations are shown in red; cells expressing one chimeric subunit – in blue, and cells with two chimeric subunits – in orange. For each cell population, FSC vs SSC gating was used to exclude dead cells and cell debris; FSC area vs height gating was done to exclude cell doublets. 10.000 events are shown in each case. (C) Mean fluorescence intensity of mCherry, EGFP and PS-CFP2 in SW620 (left) and TZM-bl (right) -derived cells and the same cell lines treated with 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF. Tests were performed in triplicate. **p<0.01; ***p<0.001; ****p<0.0001; t-test.

Article Snippet: Preservation of endogenous regulatory mechanisms that control PSMB10-PS-CFP2 and PSMB8-mCherry expression in TZM-blB8-mCherryB10-PS-CFP2 was tested using qPCR after treatment of wt TZM-bl and modified cells with recombinant human IFN-γ (1000 mg/mL) and recombinant human TNF (500 mg/mL) (both R&D Systems, Minneapolis, MN, USA) for 72 h. Two sets of primers “O”,”P” ( PSMB10-PS-CFP2 ) and “W”,”X”( PSMB8-mCherry ), directed at chimeric genes, were used ( ; ).

Techniques: Derivative Assay, Flow Cytometry, Control, Recombinant, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: Next-generation cell lines for profiling different proteasome forms and their implications in cancer

doi: 10.3389/fimmu.2025.1672000

Figure Lengend Snippet: Immune and intermediate proteasomes accumulate in the nuclei of TZM-blB8mCherryB10-CFP cells following stimulation with pro-inflammatory cytokines. (A) Confocal microscopy of unstimulated TZM-bl, TZM-blB8-mCherry, TZM-blB8-mCherryB10-CFP cells, and the same cells treated with pro-inflammatory cytokines. Twenty four hours after seeding, 1000 U/mL of recombinant human IFN-γ and 500 U/mL of recombinant human TNF were added to the culture medium and the cells were incubated for an additional 72 h. After that, the cells were fixed in a 4% PFA solution. The photoconversion of PS-CFP2 was induced by intense 400 nm light irradiation. The fluorescence of the photoconverted protein was detected at 511 nm following excitation with a 488 nm light. Images are given in order from left to right: merged image, PS-CFP2 (blue channel), EGFP (green channel), mCherry (red channel). The fluorescence of PS-CFP2 can be seen in blue before photoconversion and in green after photoconversion. (B) (Left) The TZM-blB8-mCherryB10-CFP cells after photoconversion at a higher magnification. (Right) The Coloc 2 plugin FIJI (ImageJ) software 4 was used to perform pixel co-localization analysis, measuring the mean intensity value of pixels (0–255 for 8-bit images) within cell nuclei following photoconversion. The scale bar is 10 µm.

Article Snippet: Preservation of endogenous regulatory mechanisms that control PSMB10-PS-CFP2 and PSMB8-mCherry expression in TZM-blB8-mCherryB10-PS-CFP2 was tested using qPCR after treatment of wt TZM-bl and modified cells with recombinant human IFN-γ (1000 mg/mL) and recombinant human TNF (500 mg/mL) (both R&D Systems, Minneapolis, MN, USA) for 72 h. Two sets of primers “O”,”P” ( PSMB10-PS-CFP2 ) and “W”,”X”( PSMB8-mCherry ), directed at chimeric genes, were used ( ; ).

Techniques: Confocal Microscopy, Recombinant, Incubation, Irradiation, Fluorescence, Software

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Schematic of the protocol for generation of tdTomato-CD63 + BV-2 cells and lentivirus-induced expression of tGFP-shRNA targeting Sepp1 , created with BioRender. (B) Transduced tdTomato-CD63 + tGFP-shRNA + BV-2 cells were sorted by FACS. (C) Representative images of double-positive (tdTomato + /tGFP + ) BV-2 cells transduced with tGFP-shRNA for downregulation of Sepp1 . Scale bar, 50 μm. (D) Workflow for purification of EVs from the conditioned medium (CM) of tdTomato-CD63 + BV-2 microglia upon stimulation with LPS and ATP; created with BioRender . (E–G) Size distribution and concentration of EVs secreted on CM of BV-2 cells were measured by nanoflow cytometry using a flow nanoanalyzer (NanoFCM); ** p < 0.01 and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). The assessment of tdTomato-CD63 + molecule loading on EVs was performed using the Nanoimager system (ONI). (H) Representative image of single tdTomato-CD63 + EVs. Scale bar, 2 μm. (I and J) Single-molecule fluorescence analysis of tdTomato-CD63 loading on EVs was performed at single-EV level, and the number of tdTomato-CD63 molecules present in single EVs was quantified using the CODI platform from ONI; one-way ANOVA with Holm-Šidák post hoc analysis ( n = 3 replicates per group). * p < 0.05, ** p < 0.01, and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 per group). Scale bar, 50 nm. Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Expressing, shRNA, Transduction, Purification, Concentration Assay, Cytometry, Fluorescence

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Principal-component analysis of transcriptomic profile of tdTomato-CD63 + BV-2 cells transduced with tGFP-shRNA targeting scramble and Sepp1 (three different clones) ( n = 6 replicates per group). (B) Heatmap displaying the Pearson correlation of scramble and Sepp1-shRNA replicates from RNA sequencing (RNA-seq). (C–E) Volcano plots comparing the DEGs of three Sepp1 -shRNA clones versus scramble. Dashed lines indicate a significant threshold using q < 0.05 (−log10 q- value > 1.3) on the y axis and log2 fold change > +0.5 or < −0.5 on the x axis. (F) Venn diagram comparison of up- and downregulated DEGs across different Sepp1 -shRNAs clones using the same criteria as in (D) and (E) ( q < 0.05 and log2 fold change > +0.5 or < −0.5). (G) Common Sepp1 -shRNA DEGs were subjected to Gene Ontology classification, and the top 5 significant ( q < 0.05) for each cellular component (CC), biological process (BP), and molecular function (MF) are displayed as a bar graph based on gene count enrichment.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Transduction, shRNA, Clone Assay, RNA Sequencing, Comparison

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Top 10 enriched cellular processes identified by KEGG pathway analysis of all common regulated DEGs by Sepp1 -shRNA in BV-2 microglia. (B) Heatmap showing the consistent regulation of the components of endocytosis and lysosome pathways from (A) by different Sepp1 -shRNAs in BV-2 microglia. Identified components related to endosome/exosome ESCRT machinery, the retromer complex, and lysosomal activity are highlighted in bold. (C–E) Bar graphs depicting normalized expression levels of highlighted genes from (B) among scramble and Sepp1 -shRNA groups in RNA-seq data. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). Bar graphs indicate mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: shRNA, Activity Assay, Expressing, RNA Sequencing

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Heatmap displaying the top common DEGs ( q < 0.05) regulated in response to Sepp1 downregulation. (B) Enriched pathways associated with the common DEGs in Sepp1 -shRNA were obtained using Metascape software. (C and D) Ingenuity Pathway Analysis (IPA) using all common DEGs regulated in different Sepp1 -shRNA groups predicted LXR/RXR pathway downregulation and identified a network interaction composed mainly of Ptger4 , Npc1 , Trem2 , and Cop1 in response to Sepp1 downregulation (D). (E) Analysis of normalized expression of Ptger4 , Npc1 , Trem2 , and Cop1 in RNA-seq data. * p < 0.05, ** p < 0.01, and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). Bar graphs indicate mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: shRNA, Software, Expressing, RNA Sequencing

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Schematic of the experimental design for Sepp1 silencing using siRNA in primary neurons and primary astrocytes. (B) Representative MAP2 and GFAP immunostaining images in primary neurons and astrocytes. Scale bar, 100 μm. (C) Bar graphs showing relative expression of Sepp1 mRNA in primary neurons and astrocytes treated with siRNA targeting the scramble control sequence or Sepp1 ( n = 6 replicates per group from 2 independent experiments). **** p < 0.0001 by two-tailed student t test. (D and E) Size distribution and concentration of EVs secreted in CM of primary neurons (D) and astrocytes (E) measured by nanoflow cytometry (NanoFCM) ( n = 6–8 replicates per group from 2 independent experiments). * p < 0.05 by two-tailed student t-test; ns, no significance. Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Immunostaining, Expressing, Control, Sequencing, Two Tailed Test, Concentration Assay, Cytometry

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Experimental design for microglia-specific EV labeling and Sepp1 silencing in APP NL-G-F mice. Six-month-old mice received injections into the hippocampus with a lentivirus for microglia-specific expression of mEm-CD9, followed by a lentivirus injection for scramble-shRNA:mCherry or Sepp1 -shRNA:mCherry expression 2 weeks later. (B) Representative images of mEm-CD9 + (green) microglia cells and cells expressing mCherry:shRNA (red) in the APP NL-G-F mouse dentate gyrus in the hippocampus brain region. Scale bar, 100 μm. (C) Confocal microscopy and Imaris 3D rendering of mouse hippocampal tissue. Shown are mEm-CD9 + (green) and P2ry12 + (red). Scale bar, 100 μm. (D) Percentage of the P2ry12 microglial marker overlapped with mEm-CD9 + cells in the hippocampus of APP NL–G-F mice injected with shRNA. (E) Super-resolution confocal z stack images of single microglia co-transduced with mEm-CD9 and shRNA:mCherry in the hippocampus of APP NL-G-F mice. 3D images of shRNA:mCherry (red) and Mac2 (magenta) surfaces overlapped with mEm-CD9 + (green) microglia as well as mEm-CD9 + particles surrounding microglia were generated using Imaris software. Scale bar, 4 μm. (F) Measured Mac2 fluorescence intensity in scramble- and Sepp1-shRNA-transduced microglia in APP NL-G-F mice ( n = 3–4 microglia per mouse, 3–4 mice per group). Two-tailed student t test; ns, no significance. (G) Representative high-magnification images of mEm-CD9 + voxels obtained in areas surrounding Mac2 + MGnD microglia as in (E). Scale bars: total EVs, 1 μm; small EVs, 0.2 μm. (H–K) In situ analysis of mEm-CD9 + voxels surrounding mEm-CD9 + /Mac2 + MGnD microglia. Shown are (H) the size frequency distribution of mEm-CD9 + particles and quantification of the number of mEm-CD9 + voxels per microglia in total EVs (I, total EVs <1,000 nm), small EVs (J, <150 nm), and large EVs (K, >150 nm) among scramble- and Sepp1 -shRNA:mCherry-transduced mEm-CD9 + /Mac2 + MGnD microglia. Two-tailed student t test, * p < 0.05 ( n = 3–4 microglia per mouse, 3–4 mice per group). Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Labeling, Expressing, Injection, shRNA, Confocal Microscopy, Marker, Transduction, Generated, Software, Fluorescence, Two Tailed Test, In Situ

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Virus, shRNA, Control, Recombinant, Staining, RNA Sequencing, Software, Saline, Imaging

Journal: Molecular Carcinogenesis

Article Title: Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

doi: 10.1002/mc.22312

Figure Lengend Snippet: Cathepsin B silencing in human CRC cells inhibits tumorigenicity and metastasis in immunodeficient mice. (A) Representative digital images of mouse lungs 28 d after tail vein injection of 10 6 HT29 cells expressing either shScrambled (shSCR) or shCathepsin B (shCTSB). (B) HT29 cells expressing either shSCR or shCTSB were injected subcutaneously in immunodeficient mice and the growth of tumors (mm 3 ) over time was measured. The results represent the mean tumor volume obtained from two independent experiments in which at least six mice were injected for each cell line. *, significantly different from shCTSB tumors at P < 0.05 (Student's t ‐test). (C) Equal amounts of whole cell lysates from tumors were analyzed by Western blotting for the expression of cathepsin B, cyclin B1, phosphorylated ERK1/2 (pERK1/2), total ERK2, phosphorylated Akt (pAkt), p27 Kip1 , p21, p57 Kip2 , and β‐actin. (D) Densitometric analysis of cathepsin B, p27 Kip1 , and cyclin B1 was determined in each tumor (cells expressing shSCR and cells expressing shCTSB were injected in different flanks of the same mouse) using ImageJ software. *, significantly different from shSCR tumors at P < 0.05 (Wilcoxon matched‐pairs signed‐ranks t ‐test).

Article Snippet: For double p27 Kip1 ‐cathepsin B immunofluorescence, cells were incubated overnight at 4°C with anti‐p27 Kip1 (C‐19, Santa Cruz) and anti‐cathepsin B. Cathepsin B and p27 Kip1 were visualized using donkey anti‐goat AlexaFluor 594 and chicken anti‐rabbit AlexaFluor®488 (Invitrogen), respectively.

Techniques: Injection, Expressing, Western Blot, Software

Journal: Molecular Carcinogenesis

Article Title: Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

doi: 10.1002/mc.22312

Figure Lengend Snippet: Cathepsin B exhibits endo‐ and exopeptidase activity against p27 Kip1 . The pcDNA3 vectors without or with the HA‐tagged wild‐type p27 Kip1 cDNA were transiently co‐transfected with 0, 150, 300, or 600 ng of the cDNA encoding cathepsin B in 293T cells. After 48 h, lysates were analyzed by Western blotting for the expression of HA‐27 Kip1 , cathepsin B, and β‐actin. (B and C) The cDNA encoding HA‐tagged p27 Kip1 was transfected transiently in 293T cells. After 48 h, cells were lysed and 15 μg of protein lysate was incubated during 30 min (B) or during 1, 5, 10, 20, and 30 min (C) with purified human cathepsin B (300 ng) at 37°C (pH 6.0). Proteins were then solubilized in Laemmli buffer and analyzed by Western blotting for the expression of HA‐27 Kip1 and β‐actin. (D) Amino acid sequence of human p27 Kip1 ; putative cleavage sites of cathepsin B are indicated in bold and underlined. The sequence of amino acids deleted in each p27 Kip1 deletion mutant generated is highlighted in grey. (E and F) The cDNA encoding HA‐tagged p27 Kip1 and indicated mutants were transfected transiently in 293T cells. After 48 h, cells were lysed and 7.5 μg of protein lysates were incubated during 30 min with purified human cathepsin B (300 ng) at 37°C (pH 6.0). Proteins were then solubilized in Laemmli buffer and analyzed by Western blotting for the expression of HA‐27 Kip1 , cathepsin B, and β‐actin.

Article Snippet: For double p27 Kip1 ‐cathepsin B immunofluorescence, cells were incubated overnight at 4°C with anti‐p27 Kip1 (C‐19, Santa Cruz) and anti‐cathepsin B. Cathepsin B and p27 Kip1 were visualized using donkey anti‐goat AlexaFluor 594 and chicken anti‐rabbit AlexaFluor®488 (Invitrogen), respectively.

Techniques: Activity Assay, Transfection, Western Blot, Expressing, Incubation, Purification, Sequencing, Mutagenesis, Generated

Journal: Molecular Carcinogenesis

Article Title: Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

doi: 10.1002/mc.22312

Figure Lengend Snippet: Cathepsin B reduces p27 Kip1 stability. (A) The cDNA encoding HA‐tagged wild‐type p27 Kip1 and R152A/△96–100 mutant were transiently transfected in 293T cells. Twenty‐four hours following transfection, cells were treated with cycloheximide (CHX, 30 μg/mL) and harvested after 0, 2, 6, 8, or 10 h. Cells were processed and lysed at the same time. Expression of HA‐27 Kip1 proteins and β‐actin was analyzed by Western blotting. Representative Western blot analysis is shown in upper panel. The graph illustrates the densitometric analysis of data from three independent experiments. HA‐p27 Kip1 expression at 0 h of cycloheximide was set at 100%. Relative HA‐p27 Kip1 protein levels were calculated using β‐actin as reference. (B) The pcDNA3 vectors without or with the cDNA encoding HA‐tagged wild‐type p27 Kip1 or HA‐tagged R152A/△96–100 mutant were transiently co‐transfected with 0, 150, 300, or 600 ng of the cDNA encoding cathepsin B in 293T cells. After 48 h, protein lysates were analyzed by Western blotting for the expression of HA‐27 Kip1 , cathepsin B, and β‐actin.

Article Snippet: For double p27 Kip1 ‐cathepsin B immunofluorescence, cells were incubated overnight at 4°C with anti‐p27 Kip1 (C‐19, Santa Cruz) and anti‐cathepsin B. Cathepsin B and p27 Kip1 were visualized using donkey anti‐goat AlexaFluor 594 and chicken anti‐rabbit AlexaFluor®488 (Invitrogen), respectively.

Techniques: Mutagenesis, Transfection, Expressing, Western Blot

Journal: Molecular Carcinogenesis

Article Title: Cathepsin B promotes colorectal tumorigenesis, cell invasion, and metastasis

doi: 10.1002/mc.22312

Figure Lengend Snippet: Colocalization of p27 with cathepsin B in lysosomes. (A) Representative confocal microscopy images of Caco‐2/15 CRC cells showing cathepsin B (in green), LysoTracker staining (in red), and DAPI (in blue). (B) Representative confocal microscopy images of Caco‐2/15 CRC cells showing cellular distribution of endogenous p27 Kip1 (in green) and cathepsin B (in red) in a double immunofluorescence experiment. Boxed region in the low‐magnification image is enlarged on the right. The graph represents fluorescence profiles along the dashed white line showing areas of colocalization (white asterisks). (C) Caco‐2/15 cell lysates were fractionated by differential centrifugation (see Material and Methods), and equal amounts of proteins from each fraction were analyzed by Western blotting for the expression of p27 Kip1 and cathepsin B. Expression of lysosomal (LAMP1), nuclear (lamin B), cytosolic (calpain 2), and nuclear/cytoplasm shuttling markers (cyclin E and CDK2) was analyzed to assess purity of subcellular fractions. (D) SW480 cells stably expressing either shSCR or shCTSB were harvested and lysates were fractionated by differential centrifugation (see Material and Methods). Equal amounts of proteins from each fraction were analyzed by Western blotting for the expression of p27 Kip1 and cathepsin B. Expression of lysosomal (LAMP1), nuclear (lamin B), and cytosolic (calpain 2) markers was analyzed to assess purity of subcellular fractions.

Article Snippet: For double p27 Kip1 ‐cathepsin B immunofluorescence, cells were incubated overnight at 4°C with anti‐p27 Kip1 (C‐19, Santa Cruz) and anti‐cathepsin B. Cathepsin B and p27 Kip1 were visualized using donkey anti‐goat AlexaFluor 594 and chicken anti‐rabbit AlexaFluor®488 (Invitrogen), respectively.

Techniques: Confocal Microscopy, Staining, Immunofluorescence, Fluorescence, Centrifugation, Western Blot, Expressing, Stable Transfection

Journal: Nature communications

Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

doi: 10.1038/ncomms13889

Figure Lengend Snippet: Figure 2 | HS1BP3 regulates autophagy in zebrafish. (a) Representative confocal images of GFP-LC3 puncta (autophagosomes) in the trunk area of GFP-LC3 transgenic zebrafish embryos injected with control morpholino (C), Hs1bp3 translational-blocking morpholino (K), and the human Hs1bp3 mRNA coinjected with the morpholino (R) and imaged at 2 dpf with or without pre-treatment with chloroquine (10 mM) for 6 h. Scale bars, 10 mm. (b) GFP-LC3 puncta were counted in the trunk region (marked in d) of the transgenic zebrafish embryos at 2 dpf (mean±s.e.m., n ¼ 3). Total of 7–13 embryos were used for each condition per experiment. *Po0.05, **Po0.01, ***Po0.001, by Student’s t-test. (c) Representative immunoblotting of Hs1bp3 and Tubulin in whole lysates of zebrafish embryos at 2 dpf, treated with or without chloroquine for 6 h before harvest. (d) Representative light fluorescent microscopy images of whole embryos at 2 dpf. Scale bars, 300 mm.

Article Snippet: For immunoprecipitation from lysates, GFP, GFP-HS1BP3 or GFP-PLD1 were immunoprecipitated by GFP trap (Chromotek) following the manufacturer’s protocol.

Techniques: Transgenic Assay, Injection, Control, Blocking Assay, Western Blot, Microscopy

Journal: Nature communications

Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

doi: 10.1038/ncomms13889

Figure Lengend Snippet: Figure 3 | HS1BP3 localizes to ATG16L1- and ATG9-positive vesicles. HEK293 and U2OS cells expressing the indicated proteins were starved for 2 h before fixation and immunostaining with the indicated antibodies. Confocal micrographs show: (a) HEK cells expressing mCherry-HS1BP3 stained for endogenous ATG16L1 and WIPI2. Yellow arrows mark HS1BP3- and ATG16L1-positive structures. White arrow marks HS1BP3-, ATG16L1- and WIPI2-positive structure. (b) Co-localization of GFP-HS1BP3 with endogenous ATG9 and TfR (white arrows show triple co-localization) in U2OS cells. (c) Co-localization of endogenous HS1BP3 with endogenous ATG9 in HEK cells. (d) Control or HS1BP3-depleted U2OS cells expressing GFP-ATG16L1 stained for endogenous HS1BP3. Yellow arrows indicate ATG16L1-positive structures that are positive for HS1PB3, while white arrow heads indicate ATG16L1-positive structures that are not positive for HS1BP3. Note that in addition to the specific staining (co-localization with ATG16L1), the HS1BP3 antibody also recognizes other proteins non-specifically both on immunofluorescence and western blotting (Fig. 1d). (e) HEK cells expressing GFP-HS1BP3 stained for endogenous LC3. Scale bars, 10 mm.

Article Snippet: For immunoprecipitation from lysates, GFP, GFP-HS1BP3 or GFP-PLD1 were immunoprecipitated by GFP trap (Chromotek) following the manufacturer’s protocol.

Techniques: Expressing, Immunostaining, Staining, Control, Western Blot

Journal: Nature communications

Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

doi: 10.1038/ncomms13889

Figure Lengend Snippet: Figure 6 | PLD1 co-localization with ATG16L1 is affected by HS1BP3. (a) HEK cells were transfected with GFP-tagged PLD1 or PLD2. After starvation and fixation the cells were immunostained for ATG16L1 and analysed by confocal microscopy. (b) HEK cells were transfected with GFP-PLD1, starved, fixed and immunostained for ATG16L1 and TfR. (c) HEK cells were first treated with non-targeting or HS1BP3 siRNA, then transfected to express GFP-PLD1, starved, fixed and immunostained for ATG16L1. Yellow arrows indicate ATG16L1 vesicles positive for PLD1 and white arrow heads indicate ATG16L1 vesicles negative for PLD1. Co-localization of GFP-PLD1 to ATG16L1 vesicles was quantified in transfected cells using the ImageJ plugin Squassh, using 10 pictures of each condition from three independent experiments (mean±s.e.m., n ¼ 3). (d) HEK cells were transfected with HA-PLD1 together with GFP, GFP-HS1BP3 full-length, -PX or DPX constructs, starved and stained for endogenous ATG16L1. Arrows indicate co-localization between ATG16L1 and HA-PLD1. (e) Co-localization of HA-PLD1 with endogenous ATG16L1 vesicles was quantified in transfected cells in d with the Zen software (Zeiss) using 10 pictures of each condition from three independent experiments (mean±s.e.m., n ¼ 3). Scale bars, 10 mm. *Po0.05, by Student’s t-test.

Article Snippet: For immunoprecipitation from lysates, GFP, GFP-HS1BP3 or GFP-PLD1 were immunoprecipitated by GFP trap (Chromotek) following the manufacturer’s protocol.

Techniques: Transfection, Confocal Microscopy, Construct, Staining, Software

Journal: Nature communications

Article Title: HS1BP3 negatively regulates autophagy by modulation of phosphatidic acid levels.

doi: 10.1038/ncomms13889

Figure Lengend Snippet: Figure 7 | HS1BP3 regulates autophagy through PLD1. (a) HEK cells were first treated with the indicated siRNA and then transfected with the indicated GFP-tagged construct. Cells were starved and fixed before immunostaining for endogenous LC3. LC3 spots were counted only in transfected cells, minimum 200 transfected cells per condition in three independent experiments (mean±s.e.m., n ¼ 3). *Po0.05, by Student’s t-test. (b) Model for the role of HS1BP3 in autophagy. PLD1 generates PA on ATG16L1-positive autophagosome precursor membranes. HS1BP3 is recruited to these membranes by the generated PA, inhibiting PLD1 activity and displacing it from the ATG16L1 vesicles. HS1BP3 thus provides a negative feedback on PA generation on these vesicles. If HS1BP3 is depleted from the cells, this negative feedback is lost, causing the PA concentrations of these membranes to increase and thereby drive increased autophagosome formation.

Article Snippet: For immunoprecipitation from lysates, GFP, GFP-HS1BP3 or GFP-PLD1 were immunoprecipitated by GFP trap (Chromotek) following the manufacturer’s protocol.

Techniques: Transfection, Construct, Immunostaining, Generated, Activity Assay

Journal: Research

Article Title: Polyarginine Peptide R11–Actin Interaction Induces a Domino Effect on Cytoskeleton Remodeling to Suppress Bladder Cancer Metastasis

doi: 10.34133/research.1109

Figure Lengend Snippet: The disruption of actin dynamics by R11. (A) MD stimulation analysis of the actin–R11 interplay. The full-length G-actin monomer crystal structure was obtained from the AlphaFold database, the crystal structure of plectin was downloaded from the PDB database, R11 was constructed based on PyMOL 2.5.5, and the simulation was conducted using AMBER 18 software. (B) R11–actin binding mode at the late phase of their interaction. Yellow dashed lines represent hydrogen bonding, and magenta dashed lines represent salt bridge interaction. (C) Spatial relationship of G-actin tetramer and R11 during the actin–R11 interplay (200 ns), illustrated by the MD analysis. (D) Changes in root mean square deviation (RMSD) during the actin–R11 interplay. (E) Binding affinity of G-actin/G-actin, G-actin/R11, and G-actin (with R11)/G-actin, illustrated by the MST analysis; the K d value is calculated by curve fitting; R11 (5 μM). Error bars represented ± SEM ( n = 3 independent experiments). (F) Intracellular colocalization of R11 and F-actin (left), illustrated by the CLSM analysis; R11 was observed via TAMRA fluorescence channel; F-actin was stained with the fluorescein isothiocyanate (FITC)–phalloidin and observed via FITC fluorescence channel; the TAMRA fluorescence intensity and the FITC fluorescence intensity along the white line in the right inset (right); scale bar, 10 μm; R11 (5 μM), 24-h incubation. (G) Ultra-high-resolution confocal microscopy imaging of F-actin in T24 and 5637 cells; nucleus was observed via DAPI fluorescence channel; F-actin was stained with the TRITC–phalloidin and observed via TRITC fluorescence channel; R11 (5 μM), 24-h incubation; scale bar: 10 μm. (H) Ultra-high-resolution CLSM imaging of G-actin and F-actin. G-actin was labeled with FITC-DBP and observed via FITC fluorescence channel; F-actin was stained with the TRITC–phalloidin and observed via TRITC fluorescence channel; R11 (5 μM), 24-h incubation; scale bar: 2 μm. (I) F-actin/G-actin ratio analyzed by ultra-high-resolution CLSM; n = 3 independent experiments. (J) F-actin/G-actin ratio analyzed by the differential sedimentation assay. * P < 0.05; ** P < 0.01.

Article Snippet: F-actin was stained with SF488–phalloidin (CA1640, Solarbio) and tetramethyl rhodamine isothiocyanate (TRITC)–phalloidin (CA1610, Solarbio).

Techniques: Disruption, Construct, Software, Binding Assay, Fluorescence, Staining, Incubation, Confocal Microscopy, Imaging, Labeling, Sedimentation

Journal: Research

Article Title: Polyarginine Peptide R11–Actin Interaction Induces a Domino Effect on Cytoskeleton Remodeling to Suppress Bladder Cancer Metastasis

doi: 10.34133/research.1109

Figure Lengend Snippet: The disruption of the interaction between actin and plectin by R11. (A) MD stimulation analysis of the R11-participated G-actin–plectin interplay. (B) R11-participated G-actin – plectin binding mode at the late phase of their interaction. Yellow dashed lines represent hydrogen bonding, and magenta dashed lines represent salt bridge interaction. (C) Spatial relationship of G-actin tetramer and plectin during the R11-participated actin–plectin interplay (200 ns), illustrated by the MD analysis. (D) Changes in RMSD during the R11-participated actin–plectin interplay. (E) Binding affinity of F-actin/R11, F-actin (with R11)/plectin, and F-actin (with R11)/plectin, illustrated by the MST analysis; the K d value is calculated by curve fitting; R11 (5 μM). Error bars represented ± SEM ( n = 3 independent experiments). More details on the 65-400 binding domain of plectin with actin can be found in UniProt Tools and related literature . (F) Intracellular colocalization of F-actin and plectin (left), illustrated by the CLSM analysis; plectin was observed via Alexa 488 fluorescence channel; F-actin was stained with the TRITC–phalloidin and observed via TRITC fluorescence channel; scale bar, 10 μm; the TRITC fluorescence intensity and the Alexa 488 fluorescence intensity along the white line in the right inset (right); R11 (5 μM), 24-h incubation.

Article Snippet: F-actin was stained with SF488–phalloidin (CA1640, Solarbio) and tetramethyl rhodamine isothiocyanate (TRITC)–phalloidin (CA1610, Solarbio).

Techniques: Disruption, Binding Assay, Fluorescence, Staining, Incubation

Journal: Research

Article Title: Polyarginine Peptide R11–Actin Interaction Induces a Domino Effect on Cytoskeleton Remodeling to Suppress Bladder Cancer Metastasis

doi: 10.34133/research.1109

Figure Lengend Snippet: The cascade reaction down the R11–actin–plectin interplay. (A) Intracellular distribution of F-actin and vimentin in T24 and 5637 cells, illustrated by the ultra-high-resolution CLSM analysis; nucleus was observed via DAPI fluorescence channel; F-actin was stained with the TRITC–phalloidin and observed via TRITC fluorescence channel; vimentin was stained with the Alexa 488 fluorescence and observed via Alexa-488 fluorescence channel; R11 (5 μM), 24-h incubation; scale bar: 10 μm. (B) Interactions between plectin and vimentin/ITGβ4, revealed by the immunoprecipitation assay. (C) Intracellular colocalization of vimentin-EGFP and plectin-mCherry (left), plectin-EGFP, and ITGβ4-mCherry (right), revealed by the fluorescence intensity change along the white line drawn in the merged view. Scale bar: 10 μm. (D) Intracellular colocalization and E-FRET of vimentin-EGFP and plectin-mCherry (left), plectin-EGFP, and ITGβ4-mCherry (right), measured by the quantitative FRET analysis in live cells. E d values and E d – R c plots representing the efficiency of FRET. Scale bar: 10 μm.

Article Snippet: F-actin was stained with SF488–phalloidin (CA1640, Solarbio) and tetramethyl rhodamine isothiocyanate (TRITC)–phalloidin (CA1610, Solarbio).

Techniques: Fluorescence, Staining, Incubation, Immunoprecipitation

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Intracellular P. gingivalis ( P. g ) Significantly Induces and Co-Localizes with LC3C, an Isomer of LC3, and this Specific Event is Highly Dependent on HSp27 for Successful Autophagic Survival. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated for 6 or 24 h. ( A ) GECs were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Following HSp27 depletion, P. g appeared to readily start to degrade. Representative transmission electron microscopy images of P. g -infected GECs were also taken at 80 kV and 100000x magnification. Scale bar is 800 nm. ( B ) 6 h and 24h P. g-infected GECs were also stained for P. g (rabbit anti-P . g ; Alexa 488; green) and LC3C (mouse anti-LC3C; Alexa 568; red) to examine whether LC3C characterizes P. g -specific autophagosomes. These cells were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. The range of z-stacks was kept consistent and representative images were selected from the mid-ranged sections. ( Bi ) The Imaris software was utilized to obtain a xoomed 63x Orthogonal Image of 24 h P. g infection and found heightened co-localization between P. g and LC3C. LC3C was found to readily colocalize with P. g, having an average Pearson correlation coefficient of 0.96 via the Imaris post-processing software. ( C ) Lysates of infected and HSp27-depleted GECs were also analyzed via western blotting. Non-target controls were performed and not shown. (Ci) Quantitative ImageJ analysis was performed for the western blot results. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. **p<.005.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Labeling, Transmission Assay, Electron Microscopy, Infection, Staining, Confocal Microscopy, Software, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: The Autophagic Lifestyle of P. gingivalis (P. g) is Highly Characterized by Only the LC3C Isoform of LC3, Which is not Increased During Starvation-Induced Autophagy in GECs. (A ) The LC3 A/B lipidation results of the same assay provided in . (B ) GECs were separately treated with LC3B siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6 h. Intracellular P. g survival after LC3B siRNA depletion was determined using a standard antibiotic protection assay using P. g- specific 16S rRNA primers. ( C ) GECs were subjected to starvation conditions in HBSS for 24 h. GECs were then collected and fixed so that immunofluorescence could be performed. GECs were stained for LC3C (rabbit anti-LC3C;Alexa 568; red). GECs were then imaged via confocal microscopy (Super Resolution Zeiss Airyscan LSM 880) at 63x. Western blotting (Not Shown) was utilized to confirm the lack of induction of LC3C I and LC3C II. Data is represented as Mean±SD; n=3; p<0.05 is considered statistically significant (Student two-tailed T-test).

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 Presence Permits the Prolonged Existence of LC3C-characterized, P. gingivalis Specific Autophagosomes by Hampering Canonical Autolysosomal Fusion in Primary GECs. ( A ) Human primary GECs were transfected with mCherry-eGFP-LC3C for 48 h. Select GECs were also treated with 1 uM of the autophagolysosomal fusion inhibitor Bafilomycin A1, 1 uM Pepstatin A, or 5 mM 3-MA. Others were treated with Hsp27 siRNA (100nM) for 24 h. P. g was added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs were then stained for P. g (mouse anti- P.g; Alexa 405; blue) and were mounted. GECs were then imaged via confocal microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 63x. ( Ai ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the high co-localization levels between P. g and the LC3C Reporter System. P. g localized readily to the LC3C construct, with a Pearson correlation coefficient of 0.82. (B) Separately, GECs were additionally stained for P. g (rabbit anti-P . gingivalis ; Alexa 488; green) and LAMP-1 (mouse anti-LAMP-1; Alexa 568; red) and were imaged. The range of all z-stacks was kept consistent and representative images were selected from the mid-ranged sections. Scale bar is 40 µm for 63x Magnification. ( Bi ) Imaris was used to obtain a zoomed 63x orthogonal image of 24 h P. g infection and measure the co-localization levels between P. g and LAMP-1 in infected and treated GECs. While P. g infected GECs did not exhibit high co-localization with LAMP-1 (Pearson correlation coefficient of .25), their HSp27-depleted counterparts did, with an average Pearson correlation coefficient of 0.83. The Scale bar is 20 µm for 63x Magnification. ( C ) Finally, GECs treated with autophagic inhibitors were targeted for P. g (rabbit anti- P. g ; goat anti-rabbit Ultra Small Gold Antibody) and labeled P. g was found to be readily ensconced within double-membraned autophagosomes in GECs. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Following HSp27 depletion, P. g appeared to readily start to degrade, however treatment with late-stage autophagic inhibitors Bafilomycin A1 or Pepstatin A appeared to rescue P. g from degradation. Representative transmission electron microscopy images of P. g -infected GECs were taken at 80 kV and 30000x or 100000x magnification. Scale bar is 800 nm.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Transfection, Incubation, Staining, Confocal Microscopy, Infection, Construct, Labeling, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Depletion of LC3C via siRNA Collapses P. gingivalis (P. g) -Induced Non-Canonical Autophagosomal Integrity. Human primary GECs were treated with LC3C siRNA (100nM) for 48h. P. g was added at MOI 100 to GECs for 6, 12, or 24 h. ( A ) Intracellular P. g survival after LC3C siRNA depletion was determined using a standard antibiotic protection assay. In brief, any extracellular bacteria were killed via 1h gentamicin (300 μg/mL) and metronidazole (200 μg/mL) treatment. cDNAs were synthesized for qPCR using P. g -specific 16S rRNA primers to quantify intracellular levels of live P. g . Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005. ( B ) P. g -specific autophagosomes were also selectively isolated. Autophagosomes were stained for P. g (rabbit anti- P. g ; Alexa 488; green) and reduced GSH (ThiolTracker Violet; blue). Confocal images of P. g -specific autophagosomes at 6 h post-infection (63x) were taken utilizing the Super Resolution Zeiss Airyscan LSM 880.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Bacteria, Synthesized, Two Tailed Test, Isolation, Staining, Infection

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Causes the Nucleation of Hsp27-Mediated LC3C Accumulation and Lipidation; this Specific Assembly is Highly Dependent on Host Cells’ Redox Potential Determined by eATP Treatments. Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h. P. g was added at MOI 100 to GECs, which were incubated 6 and 24 h. Non-Depleted and HSp27-depleted GECs also were treated with the physiologically-relevant oxidative stress inducer eATP (3mM) treatment for 30 min prior to infection, and were analyzed by western blot.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Infection, Western Blot

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis ( P. g ) Induces and Prolongs the Autophagosomal LC3C/Beclin 1/ATG14 Nucleation Complex in a Manner Dependent upon HSp27 and the Reduced Redox State of Infected GECs as Determined by Isolated P. g- Specific Autophagosomes. GECs were treated with HSP27siRNA (100nM) for 48 h. Select GECs were also treated with N-acetyl Cysteine (NAC)(50 uM) for 1 h and/or eATP (3mM) for 30 min. P. g was added at MOI 100 to GECs, which were incubated 6 and 12 h. Autophagosomes were then isolated and prepared for analysis. ( A ) The glutathione (GSH) levels of primary GECs were also measured using chemiluminescence detection. ( B) Isolated autophagosomes were analyzed via western blot. ( Bi ), ( Bii ), ( Biii ) Quantitative ImageJ analysis was performed of each of the western blot results. Data is represented as Mean±SD, where n=3 for results. p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Infection, Isolation, Incubation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 and LC3C Recruit Beclin 1 and ATG14 to Form a Temporal Pro-bacterial Autophagic Complex, which Can Be Disrupted by Increased Oxidative Stress. Human Primary GECs were treated with HSp27siRNA (100nM) for 48 h. Select GECs were also treated with N-acetyl Cysteine (NAC) (50 uM) for 1 h and/or eATP (3mM) for 30 min. P. gingivalis (P. g) was added at MOI 100 to GECs, which were incubated 6 and 12 h. GECs were then lysed and the extracts were incubated in rabbit anti-LC3C antibody over-night. Samples underwent co-immunoprecipitation. ( A ) The eluted protein complexes were then analyzed by western blot. ( Ai ), ( Aii ), and ( Aiii ) Quantitative ImageJ analysis of western blot results was performed for each of the proteins in question. ( B ) GECs also underwent staining for HSp27 (goat anti-HSp27; Alexa 405; blue), LC3C (rabbit anti-LC3C; Alexa 488; green), and Beclin 1 (sheep anti-Beclin 1; Alexa 568; red), and ATG14 (mouse anti-ATG14; Alexa 647; magenta) to examine the formation of the pro-bacterial autophagic initiation complex. GECs were then imaged via Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. The Imaris software was used to obtain zoomed orthogonal views of ( Bi ) An infected GECs and ( Bii ) a theoretical autophagosome with HSp27, LC3C, ATG14, and Beclin 1 highly co-localized about it. The scale bar is 20 µm for all Magnification. All of the markers were found to have a Pearson correlation coefficient greater than .9 with each other via Imaris, denoting their close theorized interactions. Data is represented as Mean±SD, where n=3 and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Immunoprecipitation, Western Blot, Staining, Software, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 and LC3C Selectively and Specifically Partner with One Another to Promote P. gingivalis ( P. g )-Induced Autophagy. P. g was added at MOI 100 to Human Primary GECs, which were incubated 6 and 24 h. ( A ) GECs were stained for LC3C (rabbit anti-LC3C; Alexa 488; green) and HSp27 (mouse anti-HSp37; Alexa 568; red) following infection. HSp27 was found to readily and temporally colocalize with LC3C, having a Pearsons correlation coefficient of .85 at 24 h post infection via the Imaris post-processing software. ( B ) To assess if full length HSp27 is truly capable of binding to full-length LC3C, a far western approach was implemented by probing 5 µg of recombinant LC3C with 10 µg of recombinant HSp27 for one hour. Antibody specificity was accounted for via probing the LC3C blot with monoclonal mouse anti-HSp27 antibody (Not shown), which showed no cross-reactivity.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Incubation, Staining, Infection, Software, Binding Assay, Western Blot, Recombinant

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 does not Interact with either LC3A or LC3B isoforms in the way that it interacts with LC3C. A Far Western approach was implemented. rLC3A or rLC3B were loaded and incubated with 10 ug of rHSp27. Interactions for ( Bi ) LC3A and ( Bii ) LC3B were then detected by probing the rLC3A or rLC3B blot with mouse Anti-Hsp27 antibody.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Western Blot, Incubation

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Phosphorylated HSp27 (P-HSp27) Preferentially Binds to the C-terminal Tail of LC3C, Inhibiting the Canonical Cleavage of LC3C and Halting the Canonical Maturation of LC3C-Specific Autophagosomes. The structural models of monomeric full-length wild-type ( A ) HSp27 (Uniprot: P04792) and ( B ) LC3C (Uniprot: Q9BXW4) were acquired from the AlphaFold database. Optimized complex configurations between ( C ) LC3C and unmodified HSp27 and ( D ) LC3C and P-HSp27 were then obtained, and the modifications in the theorized interaction sites in their N-terminal regions were highlighted. ( E ) Surface electrostatic potentials of the complexes were additionally mapped, contrasting the varied potentials between the two complexes. ( F ) Finally, the buried surface areas and interaction areas between HSp27 or P-HSp27 and LC3C proteins were also assessed and contact maps were generated.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Generated

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: P. gingivalis (P. g) Secretes its Ndk Effector Molecule to Activate HSp27 and Induce Temporal HSp27-LC3C Partnering to Inhibit Canonical LC3C Cleavage by ATG4B and Halt Autolyosomal Fusion in GECs. A) Human primary GECs were treated with HSp27 siRNA (100nM) for 48 h or were transfected with 1 µg of the constitutively activated pFLAG-CMV2-HSP27-S78D/S82D construct for 48h. Select GECs were then jointly treated with the late stage autophagy inhibitors 1 µM Pepstatin A, or 1 µM lactostatin for 24h. Wild-type P. g or ΔNDK P. g was then added at MOI 100 to GECs, which were incubated for 24 h. ( A ) GECs also underwent staining for HSp27 (goat anti-HSp27; Alexa 405; blue), LC3C (rabbit anti-LC3C; Alexa 488; green), and Beclin 1 (sheep anti-Beclin 1; Alexa 568; red), and ATG14 (mouse anti-ATG14; Alexa 647; magenta) to examine the formation of the pro-bacterial autophagic initiation complex. GECs were then imaged via Leica DM6 CS Stellaris 5 Confocal/Multiphoton System at 63x. ( B ) A diagram was created detailing how LC3C preferentially partners to P-HSp27 over its non-phosphorylated counterpart, causing a confirmational shift to the C-terminal tail of LC3C. This shift results in the inhibition of the final lipidated LC3C tail cleavage by the ATG4B protease, lending to LC3C not disassociating from the autophagosome and halting fusion with the lysosome. ( C ) A diagram was also created to highlight the temporal relationship between HSp27 and LC3C, where LC3C can initially bind to HSp27 but via the actions of Ndk, it preferentially binds to P-HSp27, resulting in a limiting of mature, cleaved LC3C.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Transfection, Construct, Incubation, Staining, Inhibition

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Cross-Sectional Human in Situ Sample and Expression Analyses Support High Levels and Increased Co-localization of P. gingivalis (P. g) , HSp27, and LC3C in Periodontitis-Afflicted Oral Tissues. Publicly available mRNA expression data (GEO accession: GSE79705) was obtained from previously collected and examined periodontitis-afflicted and healthy gingival tissues. This microarray expression data was then analyzed via GEO2R and the relative levels of ( A ) HSp27 and ( B ) LC3C were obtained and compared. Data is represented as Mean±SD, where n=12 and p<0.05 was considered as statistically significant via One-Way Anova. *p<0.05. Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis-afflicted patients were also taken and examined. DAPI staining was utilized to visualize cellular DNA. ( C ) P. g (mouse anti-P . gingivalis ; Alexa 488; green) and LC3C (rabbit anti-LC3C; Alexa 594; red) were detected via dual staining. ( D ) HSp27 (mouse anti-HSp27; Alexa 488; green) and LC3C detection (rabbit anti-LC3C; Alexa 594; red) were also detected. Images were then captured using super resolution confocal laser scanning microscopy (Leica DM6 CS Stellaris 5 Confocal/Multiphoton System) at 10x and 63x magnification with oil immersion. Zoomed (2x) 3D versions of the 63x magnifications were obtained via the Imaris software. The range of z-stacks was kept consistent. SC: Stratum corneum, SL: Stratum lucidum, SG: Stratum granulosum, SB: Stratum basale, LT: Lamina propria. Scale bars = 200µm for 10x and 20 µm for 63x. Quantification of mean fluorescence intensity provided in Supplement. LC3C and HSp27 both were found to exhibit high levels of co-localization with each other and with P. g, as the Pearson coefficient was calculated to be .93 for LC3C and P. g and .85 for LC3C and HSp27 via the Imaris Software at the most severe state of disease.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: In Situ, Expressing, Microarray, Staining, Confocal Laser Scanning Microscopy, Software, Fluorescence

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: Quantifications of Cross-Sectional Human Ex-Vivo Samples Support High Levels of P. gingivalis (P. g) , HSp27, and LC3C in Chronically Diseased Oral Tissues (i.e. Periodontitis). Representative confocal images of gingival biopsy specimens from healthy individuals and periodontitis patients were obtained using the Leica DM6 CS Stellaris 5 Confocal/Multiphoton System so that the mean fluorescence intensity of ( A ) HSp27 ( B ) P. g and ( C ) LC3C could be calculated using ImageJ with JACoP Plugin. Data are presented as mean ± SD. Representative images from at least 5 different patients per group were used for quantitative analysis and p<0.05 was considered as statistically significant via Student two-tailed T-test. *p<.05 **p<.005

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Ex Vivo, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Porphyromonas gingivalis activates Heat-Shock-Protein 27 to drive a LC3C-specific probacterial form of select autophagy that is redox sensitive for intracellular bacterial survival in human gingival mucosa

doi: 10.1101/2024.07.01.601539

Figure Lengend Snippet: HSp27 is a Critical Regulator in Pro-bacterial LC3C-Characterized Autophagy, Facilitating the Intracellular Autophagic Survival of P. gingivalis (P. g) and Influencing the Bacterial Symbiosis of the Oral Mucosa. The proposed diagram of the identified mechanisms of P. gingivalis persistence in GECs. ( A ) HSp27 is largely induced and spatially recruited by P. g invasion of the host cells. After the initial periods of cellular infection, the gradually growing secretion of the bacterial Nucleoside-diphosphate-kinase (Ndk) into the cytoplasmic space causes heightened activation of HSp27 (P-HSp27) via direct phosphorylation. P-HSp27 abrogates extracellular ATP (eATP)-induced antimicrobial Reactive-Oxygen-Species (ROS) production via increasing glutathione (GSH) levels. In parallel, P. g -mediated induction of HSp27 promotes the specific recruitment and lipidation of LC3C, an isomer of the LC3 autophagosomal structural molecule, which is strictly dependent upon the large presence and the strong antioxidant activity of HSp27. LC3C and HSp27 partner in a stepwise manner, 1) their coupling drives the formation of Beclin1/ATG14 induction, 2) where the temporally increased phosphorylation of HSp27 by P. g Ndk both strengthens the P-HSP27 and LC3C partnering and shifts the confirmation of the LC3C tail so that LC3C cannot be successfully further cleaved by ATG4. ( B ) P-HSp27 and LC3C become increasingly assembled to the ATG14-Incorperated Nucleation Complex, where they prolong the complex’s formation and result in the accumulation of the complex on forming autophagic membranes. Thus, the strengthened partnering between P-HSp27 and LC3C is the proposed mechanism for inhibiting the autolysosomal fusion of P. g- specific autophagosomes, which is also controlled by the host cell redox homeostasis ( C ) Autophagic P. g does not undergo lysosomal degradation and is instead able to survive, multiply and subsequently intercellularly spread to neighboring cells to propagate. ( D ) Thus, the non-canonical, pro-bacterial autophagic events create a favorable and protected cellular environment for P. g , thereby establishing long-term intracellular bacterial persistence. The chronic colonization of P. g in the epithelia can lead to host-microbial dysbiosis in oral mucosa and systemic disorders.

Article Snippet: Antibody cross-reactivity was accounted for via probing non-incubated LC3C blots with mouse anti-HSp27 antibody (Santa Cruz Biotechnology, sc-13132; 1:1000).

Techniques: Infection, Activation Assay, Phospho-proteomics, Antioxidant Activity Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: A microscopic and molecular assay to measure parasite load. (A) Confocal microscopy images of THP-1 cell-derived macrophage-like cells infected or not with L. donovani promastigotes at the MOI shown at 100× magnification. The infection was carried out for 24 h before fixing and staining. DAPI stain was used to stain both the nuclear and parasite DNA (the pseudocolor red is shown instead of the blue color of DAPI for better visualization; laser wavelength: 405.0, power: 4.3). The arrow indicates an amastigote inside a cell. Images were obtained under the DAPI channel using the NIS-Elements Imaging software (version 5.20.00). (B) The images obtained in A were manually scanned to count the number of cells infected and the number of amastigotes/cell and plotted. Then, 100–200 cells were counted per condition. (C) Parasite count in THP-1-derived cells infected with L. donovani (L. d) or not (C, control). The Ct values obtained from qPCR carried out on DNA isolated from infected cells were compared with the same represented as a standard curve obtained from qPCR carried out on promastigote DNA that was isolated from serially diluted parasite cultures in M199 medium; from this standard curve, the parasite numbers were deduced and shown as mean and SD from two experiments. (D) A new method to estimate parasite load after normalizing for host DNA concentration. The kDNA was measured from DNA isolated from infected cells by qPCR and normalized to copies of GAPDH DNA. The mean is shown from three separate experiments along with SD. NC, no infection control. (E) RAW264.7 cells infected with L. donovani were stained with Geimsa stain for LD body counting. Images were obtained in a bright field using Cell D software at 100× magnification. The arrow indicates an amastigote inside a cell. The number of amastigotes in 100 macrophage cells was counted using oil immersion lenses. (F) Data obtained from counting cells in E are plotted, showing the mean and SD. (G) Parasite load was estimated as kDNA normalized to gapdh from two experiments (shown as mean and SD) that were performed simultaneously with those shown in E. L. d, L. donovani; NC, no infection control.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Confocal Microscopy, Derivative Assay, Infection, Staining, Imaging, Software, Control, Isolation, Concentration Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 inhibits L. donovani growth in cells. (A) Human (left) or mouse(right) IFN-λ3 at the shown concentration was added along with parasites during infection, and kDNA was measured after 24 h. The data are from two experiments in THP-1-derived cells and four experiments from RAW264.7 cells, showing the mean and SD. *P < 0.05; **P < 0.01; ***P < 0.001. (B) Human (left) or mouse (right) IFN-λ3 at 100 ng/mL was added at different time points after L. donovani infection, and kDNA was measured after 24 h of infection. The data are from two experiments in both cell lines, shown as the mean and SD. *P < 0.05. (C) IFN-λ4 at 6 µg/mL was added, and the experiment was as in A showing data from two experiments with mean and SD (**P < 0.01). A high concentration of IFN-λ4 was required as the specific activity of the recombinant protein preparation is low, and 6 µg/mL would give a comparable activity to 100 ng/mL of IFN-λ3, as detailed in our previous work (16). (D) Effect of IFN-λ3 and IFN-λ4 on cytokine secretion in L. donovani infected M2-macrophage-like cells. M2 macrophages were differentiated for 2 days from THP-1 cells as described in our previous work (16), and IFN-λ3 at 1 µg/mL or IFN-λ4 at 6 µg/mL were added during parasite infection. After 24 h, the supernatants and cells were collected, and qPCR and ELISA were carried out. The data are from two experiments, showing the mean and SD. *P < 0.05; **P < 0.01. L. d, L. donovani; NT, no treatment control.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Concentration Assay, Infection, Derivative Assay, Activity Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 inhibits L. donovani by increasing ROS production in cells. (A) (Top) RAW264.7 cells infected with L. donovani for different time points as shown were subject to qPCR for kDNA (normalized to Gapdh) and Ifnl3 (normalized to Rps29). (Bottom) Immunoneutralization of secreted mIFN-λ3 increases parasite load. The experiment was similar to the one described in the top, except that a rat anti-mouse IFN-λ3 antibody (shown as α-mIFN-λ3) was added or not at 24 h pi, at increasing concentrations as shown (1.5, 3.0, and 4.5 µg/mL) in the medium, and kDNA copies in the cells were measured after an additional 12 h (i.e., 36 h pi) of incubation. The data from both the top and bottom are from two experiments, showing the mean and SD. *P < 0.05i, post-infection. (B) ROS levels are induced by IFN-λ3, as measured by the NBT assay. (Top) The indicated treatment (IFN-λ3, LPS, or L. d ± IFN-λ3) was for 2 h; IFN-λ3 was at 100 ng/mL and LPS was at 1 µg/mL. (Bottom) L. d infection was for indicated time points. The means from three experiments (top) and two experiments (bottom) are shown; error bars denote SD. NT, no treatment. *P < 0.05; **P < 0.001; L.D, L. donovani. (C) mIFN-λ3 increases ROS production in RAW264.7 cells in a dose-dependent manner, as measured by a fluorescence-based assay. RAW264.7 cells were incubated with the shown concentrations of mIFN-λ3 or LPS (1 µg/mL) or NAC (100 mM) + mIFN-λ3 (0.5 µg/mL) for 2 h before staining with DCFDA. The fluorescence intensity quantified is shown at the top (data from two experiments with mean and SD are depicted; *P < 0.05), and the bottom shows representative images taken at 20× magnification of the cells under the conditions shown. (D) IFN-λ3 can inhibit parasite load even when ROS production is inhibited. (top) L. donovani (L. d) infection was given in THP-1-derived cells as before for 24 h in the absence (NT, no treatment) or presence of IFN-λ3 and in the presence or absence of an increasing concentration of NAC added to the medium. The parasite load was estimated after 24 h pi. (Bottom) As in top, but the kDNA copies are shown after normalizing to NT and NAC at 50 mM; data are from two experiments showing the mean and SD. **P < 0.01. (E) (Top) THP-1 cells were pretreated with 50 ng/mL of IFN-λ3 for 48 h (incubated along with PMA), and then the media was removed and L. donovani (L. d) or mock infection (1:10 MOI) was given for 2 h. ROS levels were measured by the NBT assay. Data from two experiments with mean and SD are shown. *P < 0.05. NT, no treatment. (Bottom) kDNA levels shown in THP-1 cells that were pretreated with 100 ng/mL IFN-λ3 or not (incubated along with PMA for 48 h) were washed off the media and infected or not with L. donovani for 24 h, and kDNA was measured by qPCR as before. The data are from two experiments, showing the mean and SD. Similar experiments with IFN-λ4 are shown in Fig. S2B.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Incubation, Fluorescence, Staining, Derivative Assay, Concentration Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 targets an early event in parasite uptake. (A) IFN-λ3 at the indicated doses (the color key for the doses shown is the same for A, B, and C) were incubated along with the promastigotes, and slides were stained with DAPI, and the amastigotes presence and numbers were manually counted. The data are from three (for no treatment) and four (for IFN-λ3 treatment) independent experiments, each with 100–200 individual cells shown as the mean and SD. *P < 0.05. (B) (Left) Fluorescence microscope image of THP-1-derived cells incubated with CFSE-stained heat-killed L. donovani showing different stains. The arrow indicates a parasite taken up by the cell by phagocytosis. (Right) Manual counting of the parasite inside the cells and numbers was done as above and plotted. The data are from four independent experiments, each involving 100 to 200 individual cells, showing the mean and SD. For B and C, live parasites inside the cells were observed with the DAPI channel, while heat-killed parasites were observed under the GFP channel. Images were obtained under DAPI (imparts red pseudocolor), GFP (green), and Texas Red (imparts gray pseudocolor). (C) (Top) Fluorescence microscopic image of THP-1-derived macrophage-like cells that have taken up zymosan in a phagocytosis assay as described in the Materials and Methods; images were obtained under DAPI (imparts red pseudocolor), GFP (green), and Brightfield. The arrow indicates zymosan inside a cell. (Bottom) Manual counting of the cells with zymosan was performed as in B and plotted. The data are from two separate experiments with ~250 cells in each, showing the mean and SD. For B and C, image analysis was done using Leica Application Suite X software (version 3.7.2.22383) at 100× magnification. (D) Schematic representation of an experimental design with five (I-V) strategies. L donovani (L. d) infection (downward arrow) was given at 1:20 MOI for only 6 h, after which the uninfected promastigotes were removed after rigorous washing with PBS. IFN-λ3 treatment (inverted dark triangle) was given at the depicted time points, and after the indicated incubation periods, cells were collected and quantified for kDNA copies by qPCR (downward arrow with round head). (E and F). IFN-λ3 targets an early event during parasite uptake. Experiments as designed in the schematic representation shown in D were carried out, and results are presented in E (strategies I-IV) for 24 (E, top), 48 (E, middle), or 72 h pi (E, bottom) and F (strategies I for 48 h pi and V). 1:10 MOI and continuous infection conditions as described for previous experiments were also carried out, but the incubation periods included 48 and 72 h pi along with 24 h pi (shown in E middle, bottom, and top, respectively). qPCR was performed to estimate kDNA copies and ISG expression. The kDNA copies were normalized to NT (no IFN-λ3 treatment) and shown in E, while the actual fold changes are shown in F. The data in both E and F are from two experiments showing the mean and SD. *P < 0.05; **P < 0.01. The data shown in E are also shown with actual fold changes and without normalization to NT control samples in Fig. S2E and F.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Incubation, Staining, Fluorescence, Microscopy, Derivative Assay, Phagocytosis Assay, Software, Infection, Expressing, Control

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 before infection fails to inhibit parasite load in the mouse model of VL. (A) and (B) Gene expression changes measured by qPCR from mice spleen (A) or liver (B) pretreated with 4 µg/mouse of mIFN-λ3 or PBS 18 h before infection with L. donovani. The infection was allowed for 6 weeks, and gene expression was quantified using primers listed in Table S1 and SYBR green (Applied Biosystems) protocol. kDNA was also measured and normalized using the 2−∆∆Ct method. Six female mice were used in each group, and 18 data points are shown for the six samples carried out in technical triplicate. The statistical significance, however, was calculated with n = 6 by considering the average of the technical triplicates for each animal as the actual value for that animal. **P < 0.01. No significant differences were observed in the body weight and organ weights for the two groups.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Gene Expression, SYBR Green Assay

Journal: Infection and Immunity

Article Title: IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans

doi: 10.1128/iai.00504-23

Figure Lengend Snippet: IFN-λ3 treatment during infection fails to inhibit parasite load in the mouse model of VL. (A) Liver and spleens of the mice treated with PBS or 4 µg/mouse of mIFN-λ3 (in two doses of 2 µg/mouse in each dose given 24 h apart) at 12 weeks of infection were collected and weighed before subjecting them to qPCR for kDNA (liver and spleen) and other genes (spleen only). Each group had six animals (n = 6); 18 data points are shown for the six samples carried out in technical triplicates for qPCR data. The statistical significance, however, was calculated with n = 6 by considering the average of the technical triplicates for each animal as the actual value for that animal; for the organ weights, single measurements were taken for each organ belonging to each animal. *P < 0.01. (B) The sera from mice were subjected to ELISA for the three mouse cytokines shown.

Article Snippet: For quantifying parasite load, DNA was isolated from infected cells, and parasite kinetoplast DNA (kDNA) and human GAPDH or mouse Gapdh regions were amplified in real time using SYBR green qPCR master mix (Applied Biosystems) in a Step One Plus Real-Time PCR system (Applied Biosystems).

Techniques: Infection, Enzyme-linked Immunosorbent Assay