Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) HDAC6- or DYNC1I1-depleted A549 cells were infected with IAV at a multiplicity of infection (MOI) of 0.1 for 4 hours (h). NS1 and NP mRNA levels were quantified by RT-PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expressed relative to siCtrl cells. ( B ) Cells in (A) were infected for 8 hours before WB with anti HDAC6, DYNC1I1, NP, and β-actin antibodies. NP levels were quantified relative to siCtrl cells. ( C ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 200 nM Baf, 1 μM Noc, or 10 μM CilioD before WB analysis using anti-NP and anti-GAPDH antibodies. ( D ) NP levels in (C), relative to untreated cells (Ctrl). ( E ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 10 μM CilioD, 1 μM Noc, 0.1 μM LatA, or 1 μM Noc and 0.1 μM LatA simultaneously before WB analysis with anti-NP and anti-GAPDH antibodies. ( F ) NP levels in (E), relative to untreated cells (Ctrl). ( G ) The cytoplasm entry assay was carried out in siCtrl-, siHDAC6-, or siDYNC1I1-treated sHeLa cells, which were then processed for IF with anti-M1 and anti-LAMP1 antibodies. Baf (200 nM) was used under the siCtrl condition to block IAV cytoplasm entry. Insets highlight M1-positive LAMP1 puncta. Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( H to J ) Quantification of the cell percentage with dispersed M1 (H), the number of M1 puncta per cell (I), and the percentage of M1-positive LAMP1 puncta (J) in (G). Error bars represent SDs [ n = 3 in (A), (B), (D), and (F); n = 10 in (H) to (J), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Microscopy

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) sHeLa cells treated as in in the presence or absence of Baf were processed for IF with anti-M1 and anti-LC3 antibodies. Insets highlight the colocalization between LC3 and M1, and white arrowheads point to colocalization (in untreated cells) and noncolocalization (in Baf-treated cells). Images were collected using a DeltaVision microscope. Scale bars, 5 μm. ( B ) Quantification of the LC3-positive M1 puncta in (A). Error bars represent SDs. ( C ) IAV cytoplasm entry in sHeLa cells was carried out as in , except that IAV was at an MOI of 30. Baf (200 nM) was used to block IAV fusion at LEs. Cell extracts were subjected to IP with LC3 antibodies before separating the coisolated proteins and WB analysis with anti-LC3, NP, and immunoglobulin G (ΙgG) (control) antibodies. ( D ) Quantification of the NP bound to LC3 in (C), expressed relative to infected cells not treated with Baf. ( E ) Cell extracts from sHeLa cells treated and processed as in (C). WB membranes were probed with anti-LC3, HDAC6, DYNC1I1, KIF5B, and ΙgG antibodies (control). ( F ) Quantification of the DYNC1I1 bound to LC3s in (E), expressed relative to mock-treated cells. Error bars represent SDs [ n = 3 in (D) and (F); n = 3 in (B), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Microscopy, Blocking Assay, Control, Infection

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) Working flow that led to the identification of PCNT as a factor in IAV cytoplasm entry. ( B ) DYNC1I1-, CDK1-, PCNT-, or PPP1CC-depleted sHeLa cells were infected with IAV at an MOI of 10 and processed for IF with the anti-M1 antibody at 3 hpi as in . Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( C and D ) Quantification of both the percentage of cells with dispersed M1 (C) and the amount of M1 puncta per cell (D) in (B). ( E ) sHeLa cells were transfected with siCtrl, siPCNT, or siDYNC1I1 for 48 hours, infected, and processed for ExM as in . Baf (200 nM) was used to block IAV fusion at LEs in sHeLa cells. Scale bars, ~4.5 μm (maximum projection images) and ~0.2 μm (inset images). ( F ) Quantification of the distance of luminal M1 puncta from the LAMP1-positive LE membrane in single slices of expanded cells shown in (E). ( G ) DYNC1I1- or PCNT-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 8 hours. Cell extracts were examined by WB with anti-NP, M2, PCNT, DYNC1 I1, and β-actin antibodies. ( H ) NP and M2 level quantification in (G). Bars represent average amounts relative to infected cells treated with siCtrl. ( I ) PCNT-depleted sHeLa cells were infected with luc-HSV-1 or luc-VaV at an MOI of 1 for 6 hours. Luciferase activity in cell extracts was then measured. Data represent the average luciferase activities expressed relative to the siCtrl for each virus. Error bars represent SDs [ n = 10 in (C) and (D), 50 cells counted per repeat; n = 3 in (F), (H), and (I)]. Asterisks indicate significant differences. h, hours.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Microscopy, Transfection, Blocking Assay, Membrane, Luciferase, Activity Assay, Virus

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 200 nM Baf or 100 nM Ctn before WB with the indicated antibodies. ( B ) NP levels in (A) relative to siCtrl. ( C ) PCNT-depleted sHeLa cells transfected with the GFP-PCNTB or GFP-PCNTS plasmid were infected with IAV at an MOI of 0.1 for 8 hours and examined by WB with the indicated antibodies. NP levels are relative to siCtrl. The GFP antibody was used for GFP-PCNTS. The PCNT antibody only detects PCNTB. ( D ) Cells as in (C) were infected with WSN-luc IAV for 16 hours, and luciferase activity was measured relative to infected siCtrl cells. ( E ) PCNT-depleted sHeLa cells were transfected with the GFP-PCNTS or GFP-PCNTS ΔPACT plasmid before IAV infection at an MOI of 0.1 for 8 hours and examined by WB analysis with the indicated antibodies. NP levels are relative to siCtrl. ( F ) Cells as in (E) were infected with WSN-luc IAV for 16 hours, and luciferase activity was measured relative to siCtrl. ( G ) DYNC1I1-, PCNT-, or LC3/PCNT-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 4 hours, and NP and NS1 mRNA levels were quantified by RT-PCR, normalized to GAPDH, and expressed relative to siCtrl. ( H ) Cells from (G) were infected with IAV for 8 hours before WB with the indicated antibodies. NP levels are relative to siCtrl. ( I ) DYNC1I1-, PCNT-, HDAC6-, or PCNT/HDAC6-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 4 hours, and NP and NS1 mRNA levels were quantified as in (G). ( J ) Cells as in (I) were infected for 8 hours before WB with the indicated antibodies. NP levels are relative to siCtrl. Error bars represent the SDs [ n = 3 in (B) to (J)]. Asterisks indicate significant differences. h, hours.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) PCNT-depleted atg7 −/− cells were infected with IAV at MOI 30 for 3 hours, and cell extracts were subjected to IP with an anti-LC3 antibody before examining the input and the coisolated proteins by WB with anti-LC3, NP, PCNT, DYNC1I1, and ΙgG (control) antibodies. ( B ) DYNC1I1, NP, and PCNT bound to LC3s in (A) relative to the infected siCtrl cells. ( C ) sHeLa APEX2KI and LC3 APEX2KI cells were infected with IAV as in , but 500 μM biotin phenol (BP) and 1 mM H 2 O 2 were added 30 and 1 min, respectively, before isolating biotinylated proteins. sHeLa APEX2KI cells without BP incubation were used as a negative control. The input and the affinity-purified proteins were analyzed by WB with antibodies against biotin, NP, PCNT, DYNC1I1, or β-actin. ( D and E ) Biotinylated DYNC1I1 (D) and NP (E) in (C) relative to the noninfected sHeLa APEX2KI cells. ( F ) PCNT-depleted atg7 −/− cells were processed for IF as in with antibodies against M1 and LC3. Insets highlight colocalization between M1 and LC3. Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( G ) Percentage of the LC3-positive M1 puncta in (F). ( H ) Model for IAV host cytoplasm entry. The lower pH of LEs triggers the fusion between endocytoses IAV VPs at LEs. Uncoating and cytoplasmic vRNP release is mediated by two dynein-dependent systems that take advantage of the pulling force of MT-based motors. vRNPs are linked to dynein motors via the LC3-PCNT adaptor complex or HDAC6, which binds ubiquitin. It is unknown which vRNP components interact with LC3s and HDAC6. Error bars represent SDs [ n = 3 in (B), (D), and (E); n = 5 in (C), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Control, Incubation, Negative Control, Affinity Purification, Microscopy, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

doi: 10.1371/journal.pone.0159646

Figure Lengend Snippet: A) Confocal immunofluorescence images of Hela cells depicting the localization of LIC1 (red) and LIC2 (green), shown by arrows. B) Fluorescence images of Hela cells showing the presence of LIC2 (green, using the ThermoScientific antibody) at kinetochores (CREST, red) in prometaphase. C) Fluorescence imaging after treatment of cells with anti-LIC2 siRNA, showing loss of LIC2 signal from spindle poles and kinetochores (arrow in inset). DAPI was used to visualize chromosomes. Scalebar is 5 μm in all images that are not zoomed.

Article Snippet: The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam.

Techniques: Immunofluorescence, Fluorescence, Imaging

Journal: PLoS ONE

Article Title: Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

doi: 10.1371/journal.pone.0159646

Figure Lengend Snippet: A) Metaphase index (% of total cells present in metaphase +/- SD) in Hela cells treated with respective siRNAs as indicated. (3 experiments, n = approximately 500 cells per experiment). B) & C) Western blots showing siRNA mediated LIC1 and LIC2 specific knockdown. LIC2a and LIC2b represent two different siRNA sequences against human LIC2 . The band marked by the arrow represents human LIC2 that gets reduced upon siRNA treatment, the upper bands are non-specifically recognized by the LIC2 antibody (ThermoScientific). Actin = loading control. D) Rescue of the LIC2-depletion induced metaphase arrest by transgenic expression of rat LIC2 (3 experiments, n = at least 500 cells per experiment). E) Western blots showing specific depletion of LIC2 upon treatment with LIC2-specific siRNAs, using a different LIC2 antibody (Abcam). LIC2a and LIC2b represent two different siRNA sequences against human LIC2 . The band marked by the arrow represents human LIC2 that appears at the same molecular weight as in C.

Article Snippet: The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam.

Techniques: Western Blot, Knockdown, Control, Transgenic Assay, Expressing, Molecular Weight

Journal: PLoS ONE

Article Title: Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

doi: 10.1371/journal.pone.0159646

Figure Lengend Snippet: Representative images showing accumulation of SAC proteins A) Mad1, C) Zw10 and F) BubR1 at metaphase kinetochores upon prolonged metaphase arrest following indicated siRNA treatments. B, D, E, G) Left panels: integrated fluorescence intensities of SAC proteins normalized to the respective kinetochore (CREST) intensities for B) Mad1, D) Zw10, E) Mad2 and G) BubR1 at metaphase kinetochores. Y-axis = mean fluorescence intensity (+/- SEM) from 3 independent experiments, n = at least 18 metaphase cells per experiment for LIC1 and LIC2 depletion. Right panels: fold increase in normalized fluorescence intensity over control (GFP siRNA) for the various SAC proteins. Scalebar is 5 μm in all images.

Article Snippet: The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam.

Techniques: Fluorescence, Control

Journal: PLoS ONE

Article Title: Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

doi: 10.1371/journal.pone.0159646

Figure Lengend Snippet: Novel functions of mitotic LIC2 uncovered from this study in silencing the spindle assembly checkpoint (SAC) are shown. LIC2-dynein strips attachment sensing SAC proteins (Mad1, Mad2, Zw10) from metaphase kinetochores, like LIC1-dynein. LIC2-dynein has the additional capability of causing removal of tension-sensing SAC protein BubR1, which is lacking in LIC1-dynein. The model offers a possible mechanistic explanation for the differential effects of the two LICs in mediating metaphase to anaphase progression.

Article Snippet: The following primary antibodies against respective antigens were used: LIC1, Mad1, Mad2, Zw10 (Pierce/ Thermo-scientific); LIC2 (Pierce/ Thermo-Scientific PA5-25392 for immunoblotting and immunofluorescence staining, Abcam ab178702 for immunoblotting), BubR1 (Bethyl laboratories); dynein heavy chain from Abcam and ProteinTech; α-tubulin (Dm1α), β–actin, monoclonal antibodies from Sigma; IC-74 monoclonal antibody from Abcam.

Techniques: