DOI: 10.5281/zenodo.10784727
title: Genome-wide analysis of the effects of siRNA-mediated removal of components of ESCRT-I or HOPS complexes on gene expression in HEK293 cells

description: To identify genes regulated by ESCRT-I or HOPS complexes, whole-genome expression profiling (Illumina Human HT-12 BeadChip arrays) of cells lacking TSG101 or VPS28 (ESCRT-I components) or VPS11, VPS39 or VPS41 (HOPS components) was performed. We found that loss of ESCRT-I activates the expression of inflammatory genes and genes encoding glycolytic enzymes and leads to reduced expression of genes encoding enzymes of oxidative metabolism of amino acids and fatty acids. In contrast to ESCRT-I dysfunction, inactivation of HOPS has no major impact on the cellular transcriptional program, as it leads to up-regulated expression of a relatively small number of genes, which encode mainly plasma membrane or secreted proteins.
description: The microarray experiment was performed in cells transfected with Ambion Silencer Select siRNAs (ThermoFisher Scientific) to remove TSG101, VPS28, VPS11, VPS39 or VPS41 proteins using two single siRNAs against each target. Cells transfected with two single non-targeting siRNAs served as controls. At 3 days post transfection cells were lysed for RNA isolation. Three biological replicates of each experimental condition were prepared. In order to identify ESCRT-I- or HOPS-regulated genes, we used the Illumina Human HT-12 BeadChip arrays.
description: Sample name
title
source name
organism
characteristics: Time
characteristics: siRNA
characteristics: sample type
molecule
label


siCtrl#1_rep.1
siCtrl#1_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
Negative Control No. 1, 4390843
Control
RNA
 


siCtrl#1_rep.2
siCtrl#1_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
Negative Control No. 1, 4390843
Control
RNA
 


siCtrl#1_rep.3
siCtrl#1_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
Negative Control No. 1, 4390843
Control
RNA
 


siCtrl#2_rep.1
siCtrl#2_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
Negative Control No. 2, 4390846
Control
RNA
 


siCtrl#2_rep.2
siCtrl#2_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
Negative Control No. 2, 4390846
Control
RNA
 


siCtrl#2_rep.3
siCtrl#2_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
Negative Control No. 2, 4390846
Control
RNA
 


siTSG101#1_rep.1
siTSG101#1_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against TSG101 (s14439)
ESCRT-I depletion
RNA
 


siTSG101#1_rep.2
siTSG101#1_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against TSG101 (s14439)
ESCRT-I depletion
RNA
 


siTSG101#1_rep.3
siTSG101#1_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against TSG101 (s14439)
ESCRT-I depletion
RNA
 


siTSG101#2_rep.1
siTSG101#2_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against TSG101 (s14440)
ESCRT-I depletion
RNA
 


siTSG101#2_rep.2
siTSG101#2_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against TSG101 (s14440)
ESCRT-I depletion
RNA
 


siTSG101#2_rep.3
siTSG101#2_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against TSG101 (s14440)
ESCRT-I depletion
RNA
 


siVPS28#1_rep.1
siVPS28#1_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS28 (s27577)
ESCRT-I depletion
RNA
 


siVPS28#1_rep.2
siVPS28#1_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS28 (s27577)
ESCRT-I depletion
RNA
 


siVPS28#1_rep.3
siVPS28#1_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS28 (s27577)
ESCRT-I depletion
RNA
 


siVPS28#2_rep.1
siVPS28#2_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS28 (s27579)
ESCRT-I depletion
RNA
 


siVPS28#2_rep.2
siVPS28#2_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS28 (s27579)
ESCRT-I depletion
RNA
 


siVPS28#2_rep.3
siVPS28#2_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS28 (s27579)
ESCRT-I depletion
RNA
 


siVPS11#1_rep.1
siVPS11#1_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS11 (s31595)
HOPS depletion
RNA
 


siVPS11#1_rep.2
siVPS11#1_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS11 (s31595)
HOPS depletion
RNA
 


siVPS11#1_rep.3
siVPS11#1_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS11 (s31595)
HOPS depletion
RNA
 


siVPS11#2_rep.1
siVPS11#2_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS11 (s31596)
HOPS depletion
RNA
 


siVPS11#2_rep.2
siVPS11#2_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS11 (s31596)
HOPS depletion
RNA
 


siVPS11#2_rep.3
siVPS11#2_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS11 (s31596)
HOPS depletion
RNA
 


siVPS39#1_rep.1
siVPS39#1_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS39 (s23599)
HOPS depletion
RNA
 


siVPS39#1_rep.2
siVPS39#1_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS39 (s23599)
HOPS depletion
RNA
 


siVPS39#1_rep.3
siVPS39#1_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS39 (s23599)
HOPS depletion
RNA
 


siVPS39#2_rep.1
siVPS39#2_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS39 (s23601)
HOPS depletion
RNA
 


siVPS39#2_rep.2
siVPS39#2_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS39 (s23601)
HOPS depletion
RNA
 


siVPS39#2_rep.3
siVPS39#2_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS39 (s23601)
HOPS depletion
RNA
 


siVPS41#1_rep.1
siVPS41#1_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS41 (s25768)
HOPS depletion
RNA
 


siVPS41#1_rep.2
siVPS41#1_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS41 (s25768)
HOPS depletion
RNA
 


siVPS41#1_rep.3
siVPS41#1_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS41 (s25768)
HOPS depletion
RNA
 


siVPS41#2_rep.1
siVPS41#2_rep.1
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS41 (s25770)
HOPS depletion
RNA
 


siVPS41#2_rep.2
siVPS41#2_rep.2
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS41 (s25770)
HOPS depletion
RNA
 


siVPS41#2_rep.3
siVPS41#2_rep.3
transfected HEK293 cells
human cell line
3 days post transfection
siRNA against VPS41 (s25770)
HOPS depletion
RNA
 
description: extract protocol
RNA was extracted with Roche High Pure RNA Isolation Kit followed by clean-up and DNase I treatment in accordance with the prescribed protocol provided with the kit. Quality control was performed with Agilent Bioanalyser.


label protocol
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays.


hyb protocol
Standard Illumina hybridization protocol.


scan protocol
Standard Illumina scanning protocol.


data processing
The data were preprocessed using beadarray R package and normalized using quantile normalization.


value definition
quantile normalized signal intensity