EMBO/soda-nel huggingface dataset:
EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions (2020)
Figure 7

Figure 7: Y244F mSTING mutant translocates to autophagosomes but not late endosomes.

(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with LC3 (autophagosome marker, Red) antibody. Co-localization (Col) is indicated by white dots. Scale bars represent 10 µ m.

(B) The cells with the same treatment as A and labeled with CD63 (late endosome marker).

(C) A model for STING's trafficking routes in stimulated cells in the presence or the absence of EGFR.

Expanded View Figure Legends
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f1

(a) Hippocampal tissues obtained from Nrf2
(−/−) (6 months old, one male and two female) or
wild-type (WT) (11 months old, two male and one female) mice were
immunoblotted with a Nrf2
antibody, 12E8, PHF1, a
monoclonal to total tau
(Tau5) or a
polyclonal to total tau
(Tau) antibody. The
asterisk to the left of the Nrf2 blot designates the position at which a nonspecific
band migrated. The relative molecular masses (kDa) are indicated to the left
of each blot. (b) Bar graphs represent the relative optical density
of phosphorylated or total tau in the hippocampal tissues. n=6 (These data
are based on the mouse hippocampi immunoblotted in Supplementary Fig. 1). Data shown are
mean±s.e. and were analysed using Student’s
t-test. (**P0.01; ***P0.001)
(c,d) Left panels show brain sections stained for
tau phosphorylated at
Ser262/Ser356 (12E8) (c) and Ser396/Ser404 (PHF1) (d). The area of
delineated by dotted lines is enlarged and shown in the right corner of the
image. Right panels shows the same slice stained with 4',6-diamidino-2-phenylindole. Scale
bar, 50 μm. DG, dentate gyrus; Hi, dentate hilar
area.
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f2

Brain cortical tissue obtained from Nrf2 (−/−) (6 months old,
one male and two female) or wild-type (11 months old, two male and one
female) mice were separated into sarkosyl-soluble (SS) and insoluble (SI) fractions as
described in the Methods. The level of total tau was analysed by immunoblotting
using a monoclonal antibody to total tau (Tau5). The relative molecular masses (kDa) are indicated
to the left of each blot. Bar graph represents the relative optical density
of sarkosyl-insoluble
tau normalized with
that of sarkosyl-soluble
tau. n=3. Data
shown are mean±s.e. and were analysed using Student’s
t-test. (*P0.05).
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f3

(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM,
SFN) for 12 or
24 h. (b) Stably transfected CN1.4 cortical neurons were
maintained in the presence of doxycycline
(1 μg ml−1) to
induce the expression of tau for 24 h, and subsequently treated with
SFN for 12 or
24 h. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404
were analysed by immunoblotting using the 12E8 and PHF1 antibodies, respectively.
Tau was detected with
a polyclonal phospho-independent tau antibody (Tau). The relative molecular masses (kDa) are indicated
to the left of each blot. (c,d) Bar graphs represent the
relative optical density of phosphorylated tau normalized with that of total
tau. Data shown are
mean±s.e. of three independent experiments and were analysed using
Student’s t-test. (*P0.05;
**P0.01).
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f4

Mouse brains were obtained from wild-type (11 months old, one male; 5 months
old, one female; 12 months old, two female) and Nrf2
(−/−) (10 months old, two male and two female) mice.
(a)Mouse brain tissues were homogenized in the lysis buffer, and
10 μg of lysates was incubated with
2 μg of GST-tau protein at 37 °C for
30 min in the presence or absence of 1 mM
ATP. The levels of
tau phosphorylated at
Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using a 12E8
and PHF1 antibodies,
respectively. GST-tau was
detected with a monoclonal GST antibody (GST-tau; Supplementary Fig. 4). Bar graph of the
relative optical density of phosphorylated tau normalized to actin. Data shown
are mean±s.e. of duplicated independent experiments and were
analysed using Student’s t-test. (b) Mouse brain
tissues were homogenized in the phosphatase storage buffer, and phosphatase
activity in the lysates was quantified using the serine/threonine
phosphatase assay system (Promega) by measuring the dephosphorylation of a
phosphopeptide, RRA(pT)VA in the presence or absence of okadaic acid (OA, 20 nM). PP2A
activity was defined as the activity inhibited by the addition of
OA to the phosphatase
reaction mixture. n=4. Data shown are mean±s.e. of three
independent experiments and were analysed using Student’s
t-test.
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f5

CN1.4 mouse cortical cells stably expressing inducible tau were treated with doxycycline (Dox,
1 μg ml−1) for
24 h. On the next day (a), the cells were washed twice
with PBS and the media were replaced with Dox-deficient media with either
vehicle, epoxomicin
(12.5 nM, Epox), MG132 (7 μM) or 3-methyladenine (10 mM,
3-MA) for
24 h. (b) Cells were treated with either chloroquine
(50 μM, CQ) or bafilomycin
A1 (10 nM, Baf A1) for 24 h. Samples in first lane were
from cells maintained in the presence of Dox without medium exchange. (c) HEK293-TN cells
were co-transfected with the plasmid expressing tau together either with siRNA of
beclin-1 or scramble
RNA as a control, which was kept for 48 h. (d) Stably
transfected CN1.4 cortical cells were maintained in the presence of
Dox
(1 μg ml−1) to
induce the expression of tau for 24 h, and subsequently treated with
SFN
(10 μM) for 12 h. The culture medium was
exchanged for fresh media containing Dox
(1 μg ml−1)
but no SFN after washing
with PBS. The cells were then incubated for an additional 18 h in
the absence or presence of CQ (50 μM). The levels of
tau phosphorylated at
Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using the
12E8 and PHF1 antibodies,
respectively. Tau,
beclin-1 and LC3 were
detected with the appropriate polyclonal antibodies. The relative molecular
masses (kDa) are indicated to the left of each blot. (e) Bar graphs
represent the relative optical density of phosphorylated tau, tau or LC3-II normalized with that
of actin. n=4. Data shown are mean±s.e. and were analysed
using Student’s t-test. (*P0.05;
**P0.01).
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f6

(a) Primary cortical neurons (DIV 5) were treated with SFN (10 μM)
for 12 h, and the relative mRNA level of each gene was compared
with that of cells not treated with SFN using qRT–PCR. (b) In hippocampal
tissues from Nrf2
(−/−) (5 months old, one male; 11 months old, one
female; 5 months old, one female; 12 months old, three female) or wild-type
(10 months old, four male and two female) mice, the relative mRNA level of
each gene compared with that of wild-type mice. n=6. (c) The
levels of p62/SQSTM1, Keap1, mouse NDP52 (mNDP52) and Hsp70 in Nrf2
(−/−) or wild-type mice were examined by
immunoblotting. The asterisk on the Nrf2 panel indicates a nonspecific band.
(d,e: upper panel) Primary cortical neurons (DIV 5)
(d) and SH-SY5Y cells (e) were treated with SFN for 24 h, and the
expression levels of rat and human NDP52 (rNDP52 and hNDP52) examined by immunoblotting using
anti-NDP52 antibody.
The relative molecular masses (kDa) are indicated to the left of each blot.
(d,e: low panel) Primary cortical neurons (DIV 5)
(d) and SH-SY5Y cells (e) transiently transfected with the
pGL4.14/human NDP52
promoter (2,173 bp) luciferase reporter plasmid were treated with
SFN for
24 h, and assayed for the luciferase activity. (f) SH-SY5Y
cells were treated with SFN for 12 h, and cell lysates were used for
the ChIP assay using anti-Nrf2 antibody as described in the Methods. Data shown
are mean±s.e. and were analysed using Student’s
t-test. (*P0.05; **P0.01;
***P0.001).
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f7

(a) Diagram shows five putative ARE cis-acting elements in
human NDP52 promoter
region (2,137 bp). Red ellipsoids indicate the putative ARE
cis-acting elements. (b–d) The mutant
forms of pGL4.14-Luc reporter plasmid with the human NDP52 promoter gene were prepared
by digestion with specific restriction enzymes (for example, HindIII, SmaI
or EcoRI) by cloning of specific regions following PCR amplification or with
the QuikChange site-directed mutagenesis
kit (Agilent Technologies).
(b) CN1.4 cortical cells and SH-SY5Y cells were transiently
transfected with the reporter plasmids with the human NDP52 promoter (2,317 bp
or deleted mutants), and luciferase activity was assayed 24 h
after transfection. (c) SH-SY5Y cells were co-transfected with the
reporter plasmids with human NDP52 promoter (wild or deleted mutants) along with
either the mock plasmid (pcDNA3.1) or the plasmid expressing Nrf2, and the luciferase activity
was assayed 24 h after transfection. (d) SH-SY5Y cells
were transiently co-transfected with the reporter plasmids with human
NDP52 promoter or its
mutant plasmid in which the putative ARE sequence was site-directly mutated
(X), together with the mock plasmid (pcDNA3.1) or the plasmid expressing
human Nrf2. The
luciferase activity was assayed after 24 h. The induced
luciferase activity with co-expression with Nrf2 was not observed in a mutant
NDP52 promoter in
which all three ARE sites (−2,057, −1,023 and
−94) were site-directly mutated, indicating these three sites of
five ARE cis-acting elements are functional. Data shown are
mean±s.d. of three independent experiments and were analysed using
Student’s t-test. (*P0.05;
**P0.01; ***P0.001).
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f8

(a) Primary cortical neurons were transduced with lentivirus
expressing sh-RNA for Nrf2 or rat NDP52 (rNDP52), or a scrambled sh-RNA at DIV 1, and were
maintained until DIV 6. The levels of tau phosphorylated at Ser262/Ser356 and Ser396/Ser404
were analysed by immunoblotting using 12E8- and PHF1-specific antibodies,
respectively. Total tau
was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses
(kDa) are indicated to the left of each blot. (b) Bar graph of the
relative optical density of phosphorylated tau normalized to actin. Data shown
are mean±s.e. of three independent experiments and were analysed
using Student’s t-test. (*P0.05;
***P0.001) (c,d) Primary cortical neurons
were transduced with a control lentivirus (FIGB) or with one expressing
human NDP52
(hNDP52) at DIV 1. To
induce autophagy, trehalose (150 mM) was added at DIV 5 and the
neurons incubated for 24 h (DIV 6). Primary cortical neurons were
fixed with 4% paraformaldehyde, and stained with the 12E8 or PHF1 antibodies. Scale bar,
20 μm. The optical density of tau phosphorylated at Ser262/Ser356
(12E8). (e) and Ser396/Ser404 (PHF1) (f) in the soma of ~30 neurons
randomly chosen was analysed with the ImageJ program. Data were analysed
using Student’s t-test (***P0.001).
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f9

(a) Schematic diagram of human NDP52, the domain-deleted constructs and mouse
NDP52. (b)
Naive cortical cells were co-transfected with the plasmids expressing
HA-hNDP52 or
HA-tagged domain-deleted mutants of human NDP52 together with
GFP-tau. Twenty-four
hours later, cell lysates were prepared and transfected tau was immunoprecipitated and
blots probed for both the HA-hNDP52 constructs and GFP-tau. Homogenates (input) used for
co-immunoprecipitation were analysed by immunoblotting using anti-HA or a
polyclonal tau-specific
antibody (Tau).
(c) Stably transfected CN1.4 cortical cells were maintained in the
presence of doxycycline
(1 μg ml−1) to
induce the expression of tau for 24 h. Homogenates from CN1.4 cortical
cells were used for the co-immunoprecipitation of mouse NDP52 (mNDP52) using a Tau5 antibody. Co-precipitation of
mNDP52 was examined
by immunoblotting using anti-NDP52 antibody. Input of the homogenates used for
co-immunoprecipitation was analysed by immunoblotting using
anti-NDP52 or
Tau antibody.
(d,e) CN1.4 cortical cells were transfected with the mock
or HA-mNDP52 plasmid
(d) or with mNDP52 siRNA or scramble RNA as a control (e).
The cells transfected with siRNA or scramble RNA were treated with
SFN
(10 μM) for 24 h. The levels of
tau phosphorylated at
Ser262/Ser356 and Ser396/Ser404 were analysed by immunoblotting using 12E8
and PHF1 antibodies,
respectively. Total tau
was detected with a polyclonal tau-specific antibody (Tau). The relative molecular masses
(kDa) are indicated to the left of each blot.
Nrf2 reduces levels of phosphorylated
                tau protein by inducing autophagy
            adaptor protein NDP52 (2014)
f10

(a) Homogenates from cerebral cortical tissue of a diagnosed AD case
were used for the co-immunoprecipitation of human NDP52 (hNDP52) using a Tau5, 12E8 or PHF1 antibody. (b)
Homogenates used for co-immunoprecipitation in a were analysed by
immunoblotting using anti-NDP52, Tau5, 12E8 or PHF1 antibody. (c) Cerebral cortical tissues from
diagnosed AD cases were separated into sarkosyl-soluble and insoluble fractions. The levels of
total tau and
tau phosphorylated at
Ser262/Ser356 and Ser396/Ser404 in the sarkosyl-insoluble fractions were analysed by
immunoblotting using 12E8 and PHF1 antibodies, respectively. Samples were also
immunoblotted with a polyclonal to total tau (Tau) antibody. The levels of hNDP52, p62/SQSTM1 and LC3 in the sarkosyl insoluble fractions also
were examined by immunoblotting. The relative molecular masses (kDa) are
indicated to the left of each blot. (d–f) The
regression graphs show the relative amount of tau phosphorylated at Ser262/Ser356
(12E8) (d), at Ser396/Ser404 (PHF1) (e) or total tau (f) compared to
hNDP52 present in the
sarkosyl-insoluble
fractions.
A systems-level study reveals host-targeted repurposable drugs against SARS-CoV-2 infection (2021)
Figure 5

Figure 5. Suppression of SARS-CoV-2 infection by identified compounds.

Vero-E6 cells were pretreated with compounds (salmeterol, rottlerin (R077), temsirolimus, torin-1 or ezetimibe) for 1 hour prior to SARS-CoV-2 inoculation. 48 hours post infection cells were fixed and fluorescently labeled for SARS-CoV-2 S protein.

(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm.

(B) Violin plots of Vero-E6 cells labeled for Spike protein. The Multi-wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell. The plots show the population distribution of the integrated signal for all of the treatments. The Boxes in the plot show the interquartile range (IQR) with the top and bottom edges marking the 75th and 25th percentiles, respectively. The horizontal line in the box is the median value, and the whiskers are defined to be 1.5 IQR. The ordinate is a log scale. The effect of the treatment is assessed quantitatively by changes in the median signal level, and qualitatively by observing changes in the modes. The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut-off to quantify the number of cells that are positive or negative for the Spike signal. The statistics table below the plots shows the number of cells counted in each treatment group, and the median of the population.

(C) Pie charts showing the effect of treatment on the preventing infection of Vero-E6 cells. The number of cells above and below the cut-offs for being positive for Spike were counted and the percent cells in each category were determined. All analyses were done in Tibco Spotfire.
A systems-level study reveals host-targeted repurposable drugs against SARS-CoV-2 infection (2021)
Figure 6

Figure 6. Dose-response curves for selected compounds in the syncytia assay.

HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates, pretreated with 7-point gradients of test compounds for 1-2 h, and co-cultured for 4 hours with HEK293 donor cells expressing SARS-CoV-2 spike and GFP, or donor cells expressing GFP only (no spike). Images of GFP-positive objects were acquired on a confocal high-content imager and analyzed for syncytia formation and integrated GFP area (total GFP) as a measure of cytotoxicity, using a CNT algorithm as described in the Materials and Methods. Data are the aggregate of 8 independent biological repeats; where errors are shown they represent SD from matching concentrations in at least three experiments.

TABLES and their legends

Table 1. Antiviral and anti-inflammatory signature-genes derived from SARS-CoV-2-infected cells

aProtein: gene product from UniProt (UniProt, 2019) ;

bgenes observed to be upregulated in the transcriptome of A549 cells (Dataset 1); cgenes observed to be downregulated in the transcriptome of A549 cells (Dataset 1); dgenes observed to be upregulated in the transcriptome of ACE2-overexpressing A549 cells (Dataset 2); All genes are ordered by log2fold change in descending order. See Tables S1A-B and S2A-B for the log2foldchange values and associated GO biological processes or cellular components. See also Figure 2C and D for the respective log2fold change profiles observed in SARS-CoV-2-infected-A549 and -A549-ACE2 cells.

Table 2. Four modules mediating host cell response during SARS-CoV-2 infection, corresponding pathways, and proteins(*)

Module

(# of proteins)

Gene names of the host cell proteins involved in

the module

Viral Entry

(27 proteins)

Endocytosis,

lysosome pathway

Viral replication & translation

(45 proteins)

Regulation and signaling

Immune response

(32 proteins)

(*) see Appendix Table S4 for the full names of the proteins whose gene codes are listed in column 3. Genes corresponding to some key proteins targeted by the proposed compounds/drug and/or mentioned in the text are written in bold face, including: ARF6 (ADP ribosylation factor 6); ATP6AV1A (ATPase H+ transporting V1 subunit A); TBK1 (TANK-binding kinase 1); PRKACA (protein kinase CAMP-activated catalytic subunit α, or the catalytic subunit α of protein kinase A (PKA); RAB7A (Ras-related protein Rab-7A; RHOA (recombinant human RhoA); CTSL and CTSB (cathepsin L and B).

Table 3. High priority compounds with potential antiviral effects (*)

Drug/

Compound(a)

(a) Those tested in experiments are indicated by asterisks in the first column

(b) Rank refers to the proximity to the module in the third column, the lower the better.

Table 4. Compounds proposed to help attenuate hyperinflammation based on Dataset 2

Bacterial 50S ribosomal subunit inhibitor

autophagy inhibitor

TGFβ receptor inhibitor, furin inhibitor, anti-fibrotic,

autophagy enhancer

Expanded View Figure Legends
Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes (2015)
Figure 1

Alternative MPC complexes are found dependent on the carbon source

Mpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions. MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose (YPD) or glycerol (YPG). Mitochondrial matrix protein aconitase (Aco1), 23‐kDa translocase of the inner membrane subunit (Tim23), and 70‐kDa translocase of the outer membrane subunit (Tom70) were used as loading controls.
Kinetics of the switch from Mpc2 to Mpc3. Yeast cells were grown in YPD until mid‐log phase and then shifted to YPG medium. Whole cell extracts were taken at the indicated time points after the shift, and HA‐tagged Mpc2 and Mpc3 detected by Western blotting. Mpc3 is induced very rapidly by switch from YPD to YPG, while Mpc2 levels decrease only gradually.
Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN‐PAGE. Isolated mitochondria expressing HA‐tagged MPC proteins were run on blue native gels, and MPC complexes detected with an HA‐specific antibody. MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions, and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions. In addition, a complex of ˜300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3. Non‐specific reactivity of the anti‐HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk (*).

Source data are available online for this figure.
Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes (2015)
Figure 2

Constitutive expression of alternative MPC complexes

A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1∆mpc2∆mpc3∆ triple deletion mutant. Growth tests were performed with cells transformed with MPC1, MPC2, or MPC3 expression plasmids or with empty vectors in indicated combinations. A serial dilution of yeast cells was spotted on agar plates with glucose‐containing synthetic minimal medium with (SD+AA) or without (SD−AA) amino acids.
B Plasmid‐encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co‐expressed with either Mpc2 or Mpc3. Cells were grown in selective medium containing glycerol as a carbon source. MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1∆mpc2∆mpc3∆ background and detected by Western blot using anti‐HA or anti‐Flag antibodies. Mitochondrial enzymes aconitase (Aco1) and malate dehydrogenase (Mdh1) were used as loading controls.
C BN‐PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid‐based protein expression. Non‐specific reactivity of the antibody is denoted by an asterisk (*). It has to be noted that all complexes migrate slightly faster than in Fig C, most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA‐tag in Fig C.
D Mpc1 is co‐immunoprecipitated with Mpc2 or Mpc3. Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol‐containing medium were lysed in 1% digitonin, and either Mpc2‐Flag or Mpc3‐Flag was immunoprecipitated. Mpc1‐HA was detected by Western blot with an antibody directed against the epitope tag. The matrix protein aconitase (Aco1) and 40‐kDa translocase of the outer membrane subunit (Tom40) were not co‐immunoprecipitated.
E, F Stoichiometry of MPC complexes. Chemical cross‐linking indicates that Mpc1/Mpc2 and Mpc1/Mpc3 form heterodimers of the predicted molecular weight. DSG, disuccimidyl glutarate. Cross‐linking products with unknown partners are labeled with a question mark (?).

Source data are available online for this figure.