climp63  (R&D Systems)

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were infected with WSN or VIC at a MOI of 5 PFU/cell for 8 h, or mock-infected. Fixed cells were stained for the viral NP and the cellular markers for ER sheets and ER tubules, CLIMP63 and RTN3, respectively. The RTN3 staining was used to delineate the cell edges. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10 µm. B. A549 cells treated as in A were analyzed with the Cell Profiler software in order to segment CLIMP63+ and RTN3+ areas based on the Otsu thresholding method. The ratios of the CLIMP63+ area to the RTN3+ area are shown. Each dot represents one cell, and the data from three independent experiments are shown (black, grey and white dots). The median and interquartile values are represented as box-plots (200-350 cells per condition). *** : p-value < 0.001, one-way ANOVA. C-D. A549 cells were infected or mock-infected as in A. At 8 hpi total cell lysates were prepared and analysed by western blot, using the indicated antibodies. (C) Cropped blots of one representative experiment out of three are shown. (D) The signals for CLIMP63 and RTN3 are normalised over the tubulin signal and expressed as percentages (100% : mock-infected cells). The data shown are the mean ± SD of three independent experiments. No significant difference is detected between infected and mock-infected cells (two-way ANOVA with Sidak’s multiple comparison test).

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Staining, Microscopy, Software, Western Blot, Comparison

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were infected with WSN at a MOI of 5 PFU/cell for 8 h. Fixed cells were stained for the viral HA and cellular CLIMP63 proteins. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10µm. B. Fluorescence intensity profile for HA (cyan) and CLIMP63 (magenta) along the white line drawn in panel A (merge inset), starting from the knob. C. A549 cells were infected with ZIKV PF13 at a MOI of 5 PFU/cell for 24 h, or mock-infected. Fixed cells were stained for the viral NP and the cellular RTN3 and CLIMP63 proteins. The RTN3 staining was used to delineate the cell edges. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. White arrows indicate viral factories surrounded with remodelled ER membranes. Scale bar: 10µm.

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Staining, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549cells were infected with the WSN-PB2-Strep virus at a MOI 5 PFU/cell for 8 h. Fixed cells were stained for NP, PB2 (StrepTactin-488) and RAB11. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10µm. B. Fluorescence intensity profile for NP (red), PB2-Strep-tag (cyan) and RAB11 (magenta) along the white line drawn in panel A (merge inset), starting from the knob. C. A549 cells were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 8 h, or mock infected. Fixed cells were stained for the viral NP and cellular RTN3 and CLIMP63 proteins. The difference in permeabilisation protocols (saponin versus Triton) most likely accounts for the difference in NP signal patterns in this experiment compared to the experiment shown in Figure 2A. Scale bar: 10µm.

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Infection, Virus, Staining, Microscopy, Fluorescence, Strep-tag, Control

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 4 h. Fixed cells were stained for the viral NP and the cellular RAB11 and CLIMP63 proteins. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. White stars: non-specific nuclear staining enhanced upon siRNA treatment. Scale bar: 10µm. B. Fluorescence intensity profiles for NP (red) and CLIMP63 (magenta) along the white lines drawn in panel A (merge insets), starting from the knob. C. U2OS cells stably expressing the ER translocon Sec61ß fused to mEmerald (U2OS-Sec61ß-mEmerald) were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 8 h. Fixed cells were stained for NP and Sec61ß-mEmerald (anti-GFP antibody) and images were acquired using STED microscopy. Scale bar: 10µm. D. U2OS-Sec61ß-mEmerald cells treated as in A were analyzed to assess colocalization of NP and Sec61ß, using a pixel-based method to determine the Pearson coefficient on a region of interest corresponding to the whole cell or to the perinuclear region, as defined in the Methods section. Each dot represents one cell, and the data from three independent experiments are shown (black, grey and white dots. The median and interquartile values are represented as box-plots (18 and 19 cells treated with the NT or RAB11A-specific siRNAs, respectively). ns: non-significant (unpaired t-test).

Article Snippet: Cells were then incubated overnight at 4°C with primary antibodies directed against RAB11 (Invitrogen 71-5300, 1:100), CLIMP63 (R&D systems AF7355, 1:500), ATG16L1 (MBL PM040, 1:200), the HA tag (Invitrogen 26183, 1:100), or the influenza NP (BioRad MCA 400 or Invitrogen PA5-32242, 1:1000).

Techniques: Control, Infection, Staining, Microscopy, Fluorescence, Stable Transfection, Expressing

Journal: Journal of Virology

Article Title: The Japanese encephalitis virus NS1 protein concentrates ER membranes in a cytoskeleton-independent manner to facilitate viral replication

doi: 10.1128/jvi.02113-24

Figure Lengend Snippet: Vimentin regulates ER morphology. ( A ) The strategy for quantifying the ER membrane distribution (left). A demonstration of the distribution of the ER within a cell (right). PN, perinuclear; PP, cell periphery. ( B ) WT and Vim-KO HeLa cells were transfected with the RR and KDEL plasmids. Immunofluorescence assays were performed to observe the ER. Scale bars, 10 µm. ( C ) The ER of WT and Vim-KO HeLa cells stained with the dye was observed using confocal microscopy. Scale bars, 10 µm. ( D ) The quantification of the ER signal distribution in PN and PP regions. Each point represents a single cell from two independent experiments, two-tailed t -test, *** P < 0.001. ( E ) The immunofluorescence images of CLIMP63 and VAP-A in WT and Vim-KO HeLa cells. Scale bars, 10 µm. ( F ) The expression of CLIMP63 and VAP-A in WT and Vim-KO HeLa cells was detected by immunoblotting.

Article Snippet: Commercial reagents used in this study are as follows: dsRNA (J2) mouse monoclonal antibody (10010200, Nordic MUbio), JEV NS3 protein rabbit polyclonal antibody (GTX125868, GeneTex), JEV NS4B protein rabbit polyclonal antibody (GTX125865, GeneTex), JEV NS5 protein rabbit polyclonal antibody (GTX131359, GeneTex), RRBP1 rabbit polyclonal antibody (22015–1-AP, Proteintech), vimentin rabbit monoclonal antibody (ab92547, Abcam), vimentin mouse monoclonal antibody (BF8006, Affinity), CLIMP63 mouse monoclonal antibody (sc-393544, Santa cruz), VAP-A mouse monoclonal antibody (sc-293278, Santa cruz), HA tag rabbit polyclonal antibody (51064–2-AP, Proteintech), FLAG (DYKDDDDK) tag mouse monoclonal antibody (66008–4-Ig, Proteintech), GAPDH mouse monoclonal antibody (AC033, ABclonal), goat anti-rat IgG Alexa Fluor 555 (A-21434, Invitrogen), goat anti-rabbit IgG Alexa Fluor 488 (A-11008, Invitrogen), donkey anti-rabbit IgG Alexa Fluor 647 (ab150075, Abcam), goat anti-mouse IgG Alexa Fluor 647 (ab150115, Abcam), goat anti-rabbit IgG Alexa Fluor 594 (ab150080, Abcam), goat anti-mouse IgG Alexa Fluor 488 (ab150113, Abcam), goat anti-rabbit IgG-horseradish peroxidase (HRP; 31460, Invitrogen), goat anti-mouse IgG-HRP (31430, Invitrogen), ER Staining kit-Red Fluorescence-Cytopainter (ab139482, Abcam), DMSO (M3850, AbMole), acrylamide (M11469, AbMole), withaferin A (M7490, AbMole), latrunculin A (ab144290, Abcam), nocodazole (M3194, AbMole), MβCD (M11339, AbMole), and TVB-2640 (M10746, AbMole).

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

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were infected with WSN or VIC at a MOI of 5 PFU/cell for 8 h, or mock-infected. Fixed cells were stained for the viral NP and the cellular markers for ER sheets and ER tubules, CLIMP63 and RTN3, respectively. The RTN3 staining was used to delineate the cell edges. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10 µm. B. A549 cells treated as in A were analyzed with the Cell Profiler software in order to segment CLIMP63+ and RTN3+ areas based on the Otsu thresholding method. The ratios of the CLIMP63+ area to the RTN3+ area are shown. Each dot represents one cell, and the data from three independent experiments are shown (black, grey and white dots). The median and interquartile values are represented as box-plots (200-350 cells per condition). *** : p-value < 0.001, one-way ANOVA. C-D. A549 cells were infected or mock-infected as in A. At 8 hpi total cell lysates were prepared and analysed by western blot, using the indicated antibodies. (C) Cropped blots of one representative experiment out of three are shown. (D) The signals for CLIMP63 and RTN3 are normalised over the tubulin signal and expressed as percentages (100% : mock-infected cells). The data shown are the mean ± SD of three independent experiments. No significant difference is detected between infected and mock-infected cells (two-way ANOVA with Sidak’s multiple comparison test).

Article Snippet: In the case of dual stainings of ER markers CLIMP63 and RNT3 (Santa Cruz sc-374599, 1:150), together with the influenza NP or Zika NS3 antigen (anti-NS3 antibody [ ] kindly provided by Andres Merits, 1:1000), cells were incubated in a saponin-containing permeabilization and blocking solution (PBS supplemented with 0.1% saponin, 1% Bovine Serum Albumin) for 30 min at room temperature.

Techniques: Infection, Staining, Microscopy, Software, Western Blot, Comparison

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were infected with WSN at a MOI of 5 PFU/cell for 8 h. Fixed cells were stained for the viral HA and cellular CLIMP63 proteins. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10µm. B. Fluorescence intensity profile for HA (cyan) and CLIMP63 (magenta) along the white line drawn in panel A (merge inset), starting from the knob. C. A549 cells were infected with ZIKV PF13 at a MOI of 5 PFU/cell for 24 h, or mock-infected. Fixed cells were stained for the viral NP and the cellular RTN3 and CLIMP63 proteins. The RTN3 staining was used to delineate the cell edges. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. White arrows indicate viral factories surrounded with remodelled ER membranes. Scale bar: 10µm.

Article Snippet: In the case of dual stainings of ER markers CLIMP63 and RNT3 (Santa Cruz sc-374599, 1:150), together with the influenza NP or Zika NS3 antigen (anti-NS3 antibody [ ] kindly provided by Andres Merits, 1:1000), cells were incubated in a saponin-containing permeabilization and blocking solution (PBS supplemented with 0.1% saponin, 1% Bovine Serum Albumin) for 30 min at room temperature.

Techniques: Infection, Staining, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549cells were infected with the WSN-PB2-Strep virus at a MOI 5 PFU/cell for 8 h. Fixed cells were stained for NP, PB2 (StrepTactin-488) and RAB11. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. Scale bar: 10µm. B. Fluorescence intensity profile for NP (red), PB2-Strep-tag (cyan) and RAB11 (magenta) along the white line drawn in panel A (merge inset), starting from the knob. C. A549 cells were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 8 h, or mock infected. Fixed cells were stained for the viral NP and cellular RTN3 and CLIMP63 proteins. The difference in permeabilisation protocols (saponin versus Triton) most likely accounts for the difference in NP signal patterns in this experiment compared to the experiment shown in Figure 2A. Scale bar: 10µm.

Article Snippet: In the case of dual stainings of ER markers CLIMP63 and RNT3 (Santa Cruz sc-374599, 1:150), together with the influenza NP or Zika NS3 antigen (anti-NS3 antibody [ ] kindly provided by Andres Merits, 1:1000), cells were incubated in a saponin-containing permeabilization and blocking solution (PBS supplemented with 0.1% saponin, 1% Bovine Serum Albumin) for 30 min at room temperature.

Techniques: Infection, Virus, Staining, Microscopy, Fluorescence, Strep-tag, Control

Journal: bioRxiv

Article Title: Influenza A virus-induced PI4P production at the endoplasmic reticulum involves ATG16L1 and promotes the egress of viral ribonucleoproteins

doi: 10.1101/2024.11.29.625996

Figure Lengend Snippet: A. A549 cells were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 4 h. Fixed cells were stained for the viral NP and the cellular RAB11 and CLIMP63 proteins. Nuclei were stained with DAPI and cells were imaged with a confocal microscope. White stars: non-specific nuclear staining enhanced upon siRNA treatment. Scale bar: 10µm. B. Fluorescence intensity profiles for NP (red) and CLIMP63 (magenta) along the white lines drawn in panel A (merge insets), starting from the knob. C. U2OS cells stably expressing the ER translocon Sec61ß fused to mEmerald (U2OS-Sec61ß-mEmerald) were treated with RAB11A-specific or control Non-Target (NT) siRNAs for 48 h, and subsequently infected with WSN at a MOI of 5 PFU/cell for 8 h. Fixed cells were stained for NP and Sec61ß-mEmerald (anti-GFP antibody) and images were acquired using STED microscopy. Scale bar: 10µm. D. U2OS-Sec61ß-mEmerald cells treated as in A were analyzed to assess colocalization of NP and Sec61ß, using a pixel-based method to determine the Pearson coefficient on a region of interest corresponding to the whole cell or to the perinuclear region, as defined in the Methods section. Each dot represents one cell, and the data from three independent experiments are shown (black, grey and white dots. The median and interquartile values are represented as box-plots (18 and 19 cells treated with the NT or RAB11A-specific siRNAs, respectively). ns: non-significant (unpaired t-test).

Article Snippet: In the case of dual stainings of ER markers CLIMP63 and RNT3 (Santa Cruz sc-374599, 1:150), together with the influenza NP or Zika NS3 antigen (anti-NS3 antibody [ ] kindly provided by Andres Merits, 1:1000), cells were incubated in a saponin-containing permeabilization and blocking solution (PBS supplemented with 0.1% saponin, 1% Bovine Serum Albumin) for 30 min at room temperature.

Techniques: Control, Infection, Staining, Microscopy, Fluorescence, Stable Transfection, Expressing