siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells
Published: May 31, 2024
Abstract
Source: Düthorn, A., et al. siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells. J. Vis. Exp. (2019).
This video demonstrates a small interfering RNA (siRNA)-based technique to interfere with autophagic flux. A siRNA targeting FIP200 mRNA is introduced into monocyte-derived immature dendritic cells (iDCs) using electroporation, causing the knockdown of FIP200 expression and leading to the inhibition of nuclear lamin disruption via autophagy. Upon adding herpes simplex virus (HSV), the nuclear egress of viral progeny is inhibited due to the absence of lamin disruption via autophagic flux.
Protocol
Monocyte-derived DCs were generated from leukapheresis products of healthy donors. For this, a positive vote from the local ethics committee has been obtained (reference number 4556). The experiments of the present study were performed in accordance with the recommendations of the ethics committee of the "Friedrich-Alexander-Universität Erlangen-Nürnberg" (reference number 4556). All donors approved a written informed consent, including the accordance with the Declaration of Helsinki. <p class="jove…
Disclosures
The authors have nothing to disclose.
Materials
| 4D-Nucleofector Core Unit (electroporation apparatus II) | Lonza (Basel, Switzerland) | AAF-1002B | |
| AB-Serum | Sigma Aldrich Chemie GmbH (Steinheim, Germany) | H4522 | Dendritic cell cultivation |
| ACD-A | Sigma-Aldrich Chemie GmbH (Steinheim, Germany) | 9007281 | |
| Amaxa P3 Primary Cell 4D-Nucleofector X Kit L (electroporation kit apparatus II) | Lonza (Basel, Switzerland) | V4XP-3024 | |
| Amersham ECL Prime Western Blotting Detection Reagent | GE Healthcare (Solingen, Germany) | RPN2232 | Western Blot Detection |
| Ammonium persulfate (APS) | Sigma Aldrich Chemie GmbH (Steinheim, Germany) | A3678 | |
| anti-mouse-IgG (mouse, polyclonal, HRP) | Cell Signaling (Leiden, Netherlands) | 7076 | Western Blot detection |
| anti-rabbit-IgG (goat, polyclonal, HRP) | Cell Signaling (Leiden, Netherlands) | 7074 | Western Blot detection |
| BD FACS Canto II Flow Cytometer | BD Biosciences (Heidelberg, Germany) | 338962 | |
| Blotting Chamber Fastblot B44 | Biometra (Göttingen, Germany) | 846-015-100 | |
| CCR7 (mouse, Pe-Cy7) | BioLegend (Fell, Germany) | 557648 | Flow cytometry Dilution: 1:100 Clone: G043H7 |
| CD11c (mouse, Pe-Cy5) | BD Biosciences (Heidelberg, Germany) | 561692 | Flow cytometry Dilution: 1:100 Clone: B-ly6 |
| CD14 (mouse, PE) | BD Biosciences (Heidelberg, Germany) | 555398 | Flow cytometry Dilution: 1:100 Clone: M5E2 |
| CD3 (mouse, FITC) | BD Biosciences (Heidelberg, Germany) | 555332 | Flow cytometry Dilution: 1:100 Clone: UCHT1 |
| CD80 (mouse, V450) | BD Biosciences (Heidelberg, Germany) | 560442 | Flow cytometry Dilution: 1:100 Clone: L307.4 |
| CD83 (mouse, APC) | eBioscience Thermo Fisher Scientific (Langenselbold, Germany) | 17-0839-41 | Flow cytometry Dilution: 1:200 Clone: HB15e |
| CD86 (mouse, PE) | BD Biosciences (Heidelberg, Germany) | 553692 | Flow cytometry Dilution: 1:100 |
| EVOS FL Cell Imaging | System AMG/Life Technologies (Carlsbad, USA) | AMF4300 | |
| FIP200 (rabbit) | Cell Signaling (Leiden, Netherlands) | 12436 | Western Blot detection Dilution: 1:1000 Clone: D10D11 |
| Gene Pulser II apparatus (electroporation apparatus I) | BioRad Laboratories GmbH (München, Germany) | 165-2112 | |
| GM-CSF (4×104 U/mL) | Miltenyi Biotec (Bergisch Gladbach, Germany) | 130-093-868 | |
| HLA-DR (mouse, APC-Cy7) | BioLegend (Fell, Germany) | 307618 | Flow cytometry Dilution: 1:200 Clone: L243 |
| HSV-1/17+/CMV-EGFP/UL43 | BioVex | DC infection | |
| IL-1β (0.1×106 U/mL) | Cell Genix GmbH (Freiburg, Germany) | 1411-050 | |
| IL-4 (1×106 U/mL) | Miltenyi Biotec (Bergisch Gladbach, Germany) | 130-093-924 | |
| IL-6 (1×106 U/mL) | Cell Genix GmbH (Freiburg, Germany) | 1404-050 | |
| ImageQuant LAS 4000 | GE Healthcare (Solingen, Germany) | 28955810 | |
| L-glutamine | Lonza (Basel, Switzerland) | 17-605E | |
| LIVE/DEAD Fixable Violet dead cell stain kit | Life Technologies (Carlsbad, CA, USA) | L34964 | L/D staining in Flow cytometry |
| Lymphoprep | Alere Technologies AS (Oslo, Norway) | 04-03-9391/01 | |
| Magnesium chloride | Carl Roth GmbH (Karlsruhe, Germany) | A537.1 | |
| Megafuge 2.0 RS | Heraeus (Hanau, Germany) | 75015505 | |
| Neubauer counting chamber | Brand (Wertheim, Germany) | 717805 | |
| Nunc Cell culture flasks (175.0 cm2) | Thermo Scientific (Rockford, USA) | 159910 | |
| Paraformaldehyde, 16 % | Alfa Aesar, Haverhill, USA | 43368.9M | |
| PerfectSpin 24 Plus | Peqlab (Erlangen, Germany) | C2500-R-PL | |
| PGE2 (1 mg/mL) | Pfizer (Berlin, Germany) | BE130681 | |
| Phosphate buffered saline (PBS) | Lonza (Basel, Switzerland) | 17-512F | |
| RestoreTM Western Blot Stripping Buffer | Thermo Scientific, Rockford, USA | 21059 | |
| Rocking Platform wt 15 | Biometra (Göttingen, Germany) | 042-590 | |
| RotiBlock | Carl Roth GmbH (Karlsruhe, Germany) | A151.4 | |
| Roti-Load 1 (4x) | Carl Roth GmbH (Karlsruhe, Germany) | K929.3 | |
| Rotiphorese Gel 30 (37.5:1) | Carl Roth GmbH (Karlsruhe, Germany) | 3029.1 | |
| RPMI 1640 | Lonza (Basel, Switzerland) | 12-167F | |
| Sodium dodecyl Sulfate (SDS) | Carl Roth GmbH (Karlsruhe, Germany) | 2326.2 | |
| Thermomixer comfort | Eppendorf (Hamburg, Germany) | 5355 000.011 | |
| TNF-α (10 μg/mL) | Peprotech (Hamburg, Germany) | 300-01A | |
| Tris | Carl Roth GmbH (Karlsruhe, Germany) | 4855.3 | |
| Trypan blue solution (0.4 %) | Sigma-Aldrich Chemie GmbH (Steinheim, Germany) | T8154 | |
| Tween 20 | Carl Roth GmbH (Karlsruhe, Germany) | 9127.1 | |
| Whatman 0.2 μm nitrocellulose membrane | GE Healthcare (Solingen, Germany) | 10600001 | |
| WhatmanTM Chromatography Paper 3 mm Chr | Fisher Scientific GmbH (Schwerte, Germany) | 3030917 |
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Cite This Article
siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells. J. Vis. Exp. (Pending Publication), e22233, doi: (2024).