siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells

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.

1. Generation and handling of immature dendritic cells (iDCs) and mature dendritic cells (mDCs)

1. Isolate human periphera....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 (4x104 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 (0.1x106 U/mL)	Cell Genix GmbH (Freiburg, Germany)	1411-050
IL-4 (1x106 U/mL)	Miltenyi Biotec (Bergisch Gladbach, Germany)	130-093-924
IL-6 (1x106 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