Hop depletion reduces HSF1 levels and activity and coincides with
reduced stress resilience
Abantika Chakraborty, Adrienne Lesley Edkins*
Biomedical Biotechnology Research Unit (BioBRU), Department of Biochemistry and Microbiology, Rhodes University, Grahamstown 6140, South Africa
article info
Article history:
Received 25 March 2020
Accepted 15 April 2020
Available online xxx
Keywords:
Hop
STIP1
HSF1
Heat-shock
KRIBB11
abstract
Heat-shock factor 1 (HSF1) regulates the transcriptional response to stress and controls expression of
molecular chaperones required for cell survival. Here we report that HSF1 is regulated by the abundance
of the Hsp70-Hsp90 organizing protein (Hop/STIP1). HSF1 levels were significantly reduced in Hop￾depleted HEK293T cells. HSF1 transcriptional activity at the Hsp70 promoter, and binding of a bio￾tinylated HSE oligonucleotide under both basal and heat-shock conditions were significantly reduced.
Hop-depleted HEK293T cells were more sensitive to the HSF1 inhibitor KRIBB11 and showed reduced
short-term proliferation, and reduced long-term survival under basal and heat-shock conditions. HSF1
nuclear localization was reduced in response to heat-shock and the nuclear staining pattern in Hop￾depleted cells was punctate. Taken together, these data suggest that Hop regulates HSF1 function un￾der both basal and stress conditions through a mechanism involving changes in levels, activity and
subcellular localization, and coincides with reduced cellular fitness.
© 2020 Elsevier Inc. All rights reserved.
1. Introduction
Heat-shock factor I (HSF1) is a transcription factor that regulates
the expression of chaperone genes in response to cellular stress [1].
HSF1 also mediates a distinct transcriptional program in onco￾genesis [2]. Inactive HSF1 held in the cytoplasm in complex with
chaperones undergoes activation when stress-induced misfolding
competes for chaperone binding [3]. HSF1 undergoes nuclear
translocation, trimerization, and posttranslational modifications to
bind DNA and become transcriptionally active. HSF1 is subse￾quently deactivated through posttranslational modifications,
chaperone binding, and/or degradation [3e5]. HSF1 binds motifs
known as heat-shock elements (HSE), comprised of inverted re￾peats of the sequence GAAn [1,6,7]. HSF null mice and fibroblasts
failed to express elevated levels of chaperones when exposed to
thermal shock coupled with reduced survival [8]. HSF1 controls the
expression of a restricted number of predominantly chaperone
genes critical for restoring protein homeostasis and cell survival
after stress [9,10]. HSF1 has a strong nuclear localization signal
(NLS) [11], and nuclear localization of HSF1 is critical for prevention
global protein aggregation and stress survival [9].
While the role of Hsp70 and Hsp90 in HSF1 activation and sta￾bilization has been studied [12e14], there have been limited re￾ports on the Hsp70/Hsp90 organizing protein (Hop/STIP1) [15].
Hop is a co-chaperone that binds to and interacts with the mo￾lecular chaperones, Hsp70 and Hsp90 [16e19]. Hop is essential in
the mouse where it is required for proper embryonic development
[20]. Hop functions as an adaptor protein facilitating the folding
and transfer of client proteins between the molecular chaperones
Hsp70 and Hsp90 [21], although Hop is not required for this
function [22]. Hop was originally identified in yeast in a screen for
proteins regulating yeast Hsp70 SSA4 (HSPA1A in humans)
expression [23]. Yeast lacking STI1 showed normal growth under
basal conditions but reduced growth at elevated or lowered tem￾peratures suggesting a role in temperature stress adaption. STI1
overexpression transactivated the SSA4 promoter, suggesting a role
of STI1 in the stress response [23]. Here, we show in a mammalian
cell line model that Hop levels change HSF1 levels and activity.
2. Materials and methods
2.1. Cell culture and RNA interference
The pGL3-Hsp70pro-Luc plasmid, containing the Hsp70 stress￾inducible promoter regulating luciferase expression was a gift
from Stuart Calderwood [24]. The pLV-eGFP plasmid encoding GFP
* Corresponding author.
E-mail address: [email protected] (A.L. Edkins).
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
0006-291X/© 2020 Elsevier Inc. All rights reserved.
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Please cite this article as: A. Chakraborty, A.L. Edkins, Hop depletion reduces HSF1 levels and activity and coincides with reduced stress
resilience, Biochemical and Biophysical Research Communications, for mammalian expression was a gift from Pantelis Tsoulfas
(Addgene#36083) [25]. HEK293T cells were stably transfected with
TRIPZ plasmids encoding doxycycline-inducible shRNA against Hop
(referred to henceforth as HEK-shHOP or KD) or a control, non￾targeting shRNA (referred to as HEK-shNT or NT) [26]. HEK-shNT
and HEK-shHOP cells were grown in DMEM with 10% (v/v) fetal
bovine serum (FBS), 100 U/ml penicillin, streptomycin and
amphotericin (PSA), 1 mM sodium pyruvate, 0.1 mM non-essential
amino acids (NEAA), 500 mg/ml G418, and 2 mg/ml puromycin under
9% CO2 at 37
C. Hop-specific and control shRNA expression was
induced for 72e96 h by daily addition of 1 mg/ml doxycycline. As￾says were conducted with and without doxycycline in both cell
lines. Heat-shock was performed at 42
C for 1 h.
2.2. SDS-PAGE and western blot analysis
Equal amounts of protein were analyzed using the standard
modifications of the Laemmli SDS-PAGE protocol [27] and the
expression levels of various proteins were determined by western
blot analysis [28]. Details on antibodies used can be found in the
supplementary file.
2.3. Clonogenic assay
HEK-shNT and HEK-shHOP cells were seeded at 1000 cells/ml,
left untreated or heat-shocked, and allowed to grow for 8e12 days
with medium supplementation every third day. Colonies were fixed
with 3:1 methanol: acetic acid for 2 min, washed in PBS, stained
using 0.5% (w/v) crystal violet in methanol, washed in distilled
water, and air-dried. Images were captured, and crystal violet dye
solubilized in 1% (v/v) acetic acid and absorbance read at 595 nm.
2.4. HSF1 reporter assay
HEK-shNT and HEK-shHOP cells were seeded at 1 105 cells/ml
and transfected for 48 h with pGL3-Hsp70pro-Luc plasmid and
pLV-eGFP plasmid (transfection efficiency control) with X-trem￾eGENE HP transfection reagent (Roche) according to the manufac￾turer’s protocol. Cells were untreated or heat-shocked for 1 h,
followed by recovery for 8 h and lysed with 10% (v/v) Triton-X 100.
Luciferase activity was quantified with FLAR buffer (20 mM Tricine
pH 7.4, 100 mM EDTA, 2.67 mM MgSO₄, 17 mM DTT, 250 mM ATP,
250 mM D-luciferin) [29]. The reporter activity was determined as
the ratio of the luminescence to the EGFP signal (ex 485 nm, em
525 nm).
2.5. Nuclear-cytoplasmic fractionation
Nuclear-cytoplasmic fractionation of HEK-shNT and HEK-shHOP
cell lysates was done by NE-PER kit as per the manufacturer’s in￾structions (Thermo Fischer Scientific) and fractions analyzed by
SDS-PAGE and western blot analysis.
2.6. Immunofluorescence and confocal microscopy
HEK-shNT and HEK-shHOP (on poly-L-lysine-coated glass cov￾erslips) were permeabilized with 0.1% (v/v) PBS-T for 15 min at RT
and blocked with 1% (w/v) BSA-PBS-T for 1 h at RT. Primary anti￾bodies were used at 1:100 at 4
C overnight, and coverslips washed
in 0.1% (w/v) BSA/TBS-T. Fluorescently-conjugated species-specific
secondary antibodies were diluted 1:500 in 0.1% (w/v) BSA/TBS-T
and incubated at RT for 1 h. Coverslips were washed in 0.1% (w/v)
BSA/TBS-T. Nuclei were stained with 1 mg/ml Hoechst 33342 and
slides analyzed using the Zeiss LSM780 Confocal Microscope.
2.7. Resazurin cytotoxicity and cell proliferation assay
HEK-shNT and HEK-shHOP cells were seeded at a density of
1 105 cells/ml and treated with a series of KRIBB11 concentrations
for 72 h. For proliferation assay, cells were incubated at 37
C for
72 h. In both cases, 0.54 mM resazurin solution was added, incu￾bated for 4 h and fluorescence (ex 560 nm, em 590 nm) determined.
The half-maximal inhibitory concentration (IC50) was calculated by
non-linear regression using GraphPad Prism.
2.8. Enzyme-linked immunosorbent assay (ELISA)
A total of 1 mg/well of cell lysate or known amounts of human
HSF1 (Abcam, ab204184) was coated on to wells of a high-binding
96-well plate overnight at 4
C. Wells were blocked with 200 ml of
3% (w/v) BSA in PBS for 1 h at room temperature. Anti-HSF1 anti￾body (Abcam, ab2923) was incubated at 1:2500 dilution in 1% (w/v)
BSA-PBS overnight, washed in PBS and species-specific HRP-con￾jugated secondary antibody in 1% (w/v) BSA-PBS incubated at
1:10 000 for 2 h at RT. HRP Buffer [25.7 mM citric acid, pH 5.0,
48.6 mM Na2HPO4, 1 mg/ml TMB in DMSO, 0.001% (v/v) H2O2] was
added, the reaction was stopped with 1 M H2SO4, and absorbance
read at 450 nm. The concentration of HSF1 per mg of lysate was
determined from the standard curve.
2.9. HSE binding assay
A total of 500 mg/well of cell lysate in PBS was incubated at 4
C
in a 96-well high binding plate overnight, followed by blocking in
3% (w/v) BSA-PBS for 1 h at RT. Double-stranded biotin-labelled
oligonucleotides containing three HSE repeats (50
-GATCTA￾GAACGTTCTAGAA CGTTCTAGAACGTTCTA-30 and 50
-CTA￾GATCTTGCAAGATCTTGCAAGATCTTGCAAGAT-30
) [30] (10 nM) with
or without unlabeled competitor (100 nM) were added and incu￾bated in Kingston buffer [24 mM HEPES, pH 7.9, 120 mM KCl, 4 mM
MgCl2, 0.24 mM EDTA, 0.6 mM PMSF, 0.6 mM DTT, 24% (v/v) glyc￾erol] [31] at RT for 2 h followed by washes in PBS. Streptavidin-HRP
was incubated at 1:1000 in PBS at RT for 2 h, followed by washes in
PBS. The HRP activity was developed using HRP buffer and read at
450 nm.
2.10. Statistical analysis and reproducibility
All data are representative of at least 3 independent biological
experiments unless otherwise stated. Statistical analysis was per￾formed by either t-test or ANOVA in GraphPad Prism 4.0, and values
below 0.05 were taken as significant.
3. Results and discussion
3.1. Hop depletion reduces HSF1 levels and activity
We previously conducted a global proteomics analysis to iden￾tify changes in the cellular proteome upon Hop depletion. HSF1 was
significantly reduced in the Hop-depleted cells (data not shown). To
validate these results, we analyzed HSF1 protein levels by western
blot analysis (Fig. 1A). Reduced HSF1 levels were observed upon
Hop depletion in the HEK-shHOP cells. In contrast, the lysates from
HEK-shNT cells (irrespective of doxycycline treatment) and the
HEK-shHOP cells (without doxycycline), showed higher and
equivalent levels of HSF1 protein.
Next, we quantified HSF1 levels in lysates (Fig. 1B). HSF1 levels
were not significantly different in the HEK-shNT lysates (irre￾spective of doxycycline treatment) (86.1 ± 11.5 ng HSF1/mg vs
63.6 ± 12.6 ng HSF1/mg, respectively) or in the HEK-shHOP lysates
2 A. Chakraborty, A.L. Edkins / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
Please cite this article as: A. Chakraborty, A.L. Edkins, Hop depletion reduces HSF1 levels and activity and coincides with reduced stress
resilience, Biochemical and Biophysical Research Communications, (without doxycycline) (67.2 ± 16.4 ng HSF1/mg). In contrast, a
significant reduction of HSF1 to ~50% of the untreated HEK-shHOP
lysates was detected the Hop-depleted lysates (33.3 ± 10.3 ng HSF1/
mg, p < 0.05). Upon heat-shock, HSF1 levels were significantly
increased compared to basal levels for all lysates, with no signifi-
cant difference between HEK-shHOP lysates with and without
doxycycline (189.5 ± 16.8 ng HSF1/mg compared to 166.1 ± 37.2 ng
HSF1/mg, respectively). These data suggest that Hop depletion
reduced HSF1 levels under basal conditions but did not prevent
heat-shock-induced increases in HSF1 levels. Ruckova and col￾leagues showed that siRNA against Hop reduced HSF1 protein
levels, while Hop overexpression had no significant effect on HSF1
levels [15]. The increase in HSF1 levels in Hop-depleted cells to
equivalent to the controls upon heat-shock suggested that either
Hop does not regulate stress-induced expression of HSF1, (and as￾sumes stability of HSF1 under normal and stress conditions is
different), or that HSF1 is somehow stabilized by changes associ￾ated with heat-shock.
We next analyzed HSF1 activity. First, we assessed the ability of
HSF1 in cell lysates to bind a biotinylated canonical HSE￾oligonucleotide (Fig. 1C). The binding of the HSE was inferred
from the absorbance after detection with a streptavidin-HRP con￾jugate. The data shown are from equivalent amounts of lysate, have
had background binding from an unlabeled competitor oligonu￾cleotide subtracted, and have been normalized to the HEK-shNT
cells without doxycycline under basal conditions (Fig. 1C). There
was equivalent binding of the HSE-containing oligonucleotide
above background under basal conditions in the HEK-shNT cell
lysates (with or without doxycycline), and the HEK-shHOP cell ly￾sates without doxycycline. However, Hop-depleted HEK-shHOP
cells had a reduction in the amount of HSE-containing oligonucle￾otide bound. Upon heat-shock, there was a significant increase
relative to basal conditions in HSE-oligonucleotide binding in the
HEK-shNT (irrespective of doxycycline treatment) and in the HEK￾shHOP lysates lacking doxycycline. However, while there was a
minor increase in HSE-oligonucleotide binding in the heat-shocked
HEK-shHOP lysates with doxycycline treatment, this was not
significantly different from the basal conditions. The HSE￾oligonucleotide binding in the Hop-depleted HEK-shHOP in
response to heat-shock was, however, significantly lower than the
heat-shock induced binding detected in other lysates.
We next analyzed HSF transcriptional activity from an Hsp70
promoter reporter (Fig. 1D) [24]. Equivalent levels of transcriptional
activity were detected in the untreated and doxycycline-treated
HEK-shNT cells, as well as the HEK-shHOP without doxycycline,
under basal conditions. However, transcriptional activity was
reduced in Hop-depleted HEK-shHOP cells (with doxycycline).
Heat-shock induced a significant increase in transcriptional activity
in the HEK-shNT cells irrespective of doxycycline treatment, and in
the HEK-shHOP cells without doxycycline. In contrast, transcrip￾tional activity was significantly reduced in the heat-shocked HEK￾shHOP cells with doxycycline (Fig. 1D). These data suggest that both
HSE binding and HSF1 transcriptional activity are reduced in Hop￾depleted cells. Total concentration of HSF1 is a determining factor
for the stress response. A 25% decrease in HSF1 from basal levels at
37
C translates into an equivalent decrease in transcriptional
Fig. 1. Hop depletion reduced HSF1 levels and activity. (A) Western blot of Hop and HSF1 levels in lysates of HEK-shNT (NT) and HEK-shHOP (KD) cells. GAPDH served as loading
control. (B). Quantification of HSF1 per mg of lysate (±SD, n ¼ 3) in HEK-shNT and HEK-shHOP cells under basal and heat-shock (HS) conditions. Statistical analysis by unpaired t-test
comparing doxycycline-treated HEK-shHOP cells with other treatments (*p˂0.05, ns not significant). (C) Binding of biotinylated HSE-containing oligonucleotide and (D) tran￾scriptional activity from an HSF1 reporter plasmid. In (C), the data have undergone subtraction of background binding of an unlabeled competitor HSE probe and are normalized to
the basal HEK-shNT sample without doxycycline (Dox). Error bars represent ±SD (n ¼ 3). Statistical analysis by two-way ANOVA with Bonferroni post-test (***p˂0.0001). Cells were
treated with (þ) and without () doxycycline.
A. Chakraborty, A.L. Edkins / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 3
Please cite this article as: A. Chakraborty, A.L. Edkins, Hop depletion reduces HSF1 levels and activity and coincides with reduced stress
resilience, Biochemical and Biophysical Research Communications, response and vice versa [32]. This suggested that the reduced HSF1
activity and HSE binding under basal conditions upon Hop deple￾tion was due to the reduction in HSF1 protein. However, HSF1 levels
increased in Hop-depleted cells with stress, suggesting that
reduced binding and transcriptional activation in response to heat￾shock were not solely due to HSF1 levels.
3.2. HSF1 subcellular distribution is altered in Hop-depleted cells
HSF1 nuclear localization is required for transcriptional activity.
We conducted biochemical fractionation of the cytoplasmic and
nuclear fractions from HEK-shNT and HEK-shHOP cells (Fig. 2).
Tubulin and histone H3 served as loading controls and to confirm
successful isolation of the cytoplasmic and nuclear fractions,
respectively (Fig. 2B). HSF1 levels were reduced in the whole cell
lysates from Hop-depleted HEK-shHOP cells (with doxycycline),
compared to the controls of HEK-shHOP without doxycycline and
HEK-shNT (with and without doxycycline treatment) (Fig. 2A).
HSF1 levels were increased in both HEK-shNT and HEK-shHOP cells
(irrespective of doxycycline treatment) upon heat-shock (Fig. 2A),
which was consistent with the ELISA data (Fig. 1C). In the cyto￾plasmic fractions, similar levels of HSF1 were detected in all lysates
under basal conditions. However, an accumulation of HSF1 levels in
the cytoplasmic fraction was seen upon heat-shock only in the
Hop-depleted HEK-shHOP cells (with doxycycline) (Fig. 2B). In the
nuclear fractions, reduced HSF1 levels were detected for the Hop￾depleted HEK-shHOP cells (with doxycycline) compared to the
controls under both basal and heat-shock conditions (Fig. 2C).
Nuclear HSF1 accumulation under stress is due to reduced export
and is essential for HSF1 activity [11]. Reduced HSF1 nuclear
translocation under heat stress, and the concomitant cytoplasmic
accumulation, would explain the loss in HSF1 DNA binding and
transcriptional activity in Hop-depleted cells upon heat-shock.
To support the fractionation data, we conducted confocal mi￾croscopy on HEK-shNT and HEK-shHOP (Fig. 3 and Fig. S2). We
analyzed the localization and distribution of total HSF1 (green) and
phosphorylated HSF1 on residue Ser326 (red; HSF1-pSer326), which
is a classical site of HSF1 phosphorylation in response to heat-shock
[33,34]. Under basal conditions, the HEK-shNT with (Fig. 3A) and
without doxycycline (Fig. S1) showed a diffuse staining pattern
across the cytoplasm and nucleus for both the HSF1 and HSF1-
pSer326. Heat-shock treatment increased the proportion of HSF1
and HSF1-pSer326 in the nucleus in HEK-shNT irrespective of
doxycycline treatment (Fig. 3B and Fig. S1). In contrast, in the HEK￾shHOP cells with doxycycline treatment, the staining pattern of
HSF1 and HSF1-pSer326 was punctate in the nucleus (Fig. 3A). Upon
heat-shock, the HEK-shHOP cells without doxycycline showed an
increase in HSF1 and HSF1-pSer326 in the nucleus, like the HEK￾shNT cells (Fig. S1). In heat-shocked Hop-depleted HEK-shHOP
cells (with doxycycline), punctate nuclear staining for HSF1 and
HSF1-pSer326 was observed, as was an accumulation of total HSF1
but not HSF1-pSer326 in the cytoplasm (Fig. 3B). The HSF1 nuclear
morphology in Hop-depleted cells resembled nuclear stress bodies
(nSB) [35], which represent transcriptionally active HSF1 com￾plexes [36]. However, this is not consistent with reduced HSF1
activity in Hop-depleted cells, which suggests that the punctae
likely do not represent functional nSB. However, these data do
suggest a change in HSF1 DNA interactions in Hop-depleted cells
under both basal and stress conditions.
Fig. 2. Hop depletion reduced nuclear HSF1 during heat-shock. Western blot of (A) whole cell lysate (WCL), (B) cytoplasmic (CYT) and (C) nuclear (NUC) fractions from HEK-shNT
(NT) and HEK-shHOP (KD) lysates treated with (þ) and without () doxycycline (Dox) under basal and heat-shock (HS) conditions. GAPDH, tubulin and histone H3 served as loading
controls and markers for fractionation of cytoplasm and nucleus.
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Please cite this article as: A. Chakraborty, A.L. Edkins, Hop depletion reduces HSF1 levels and activity and coincides with reduced stress
resilience, Biochemical and Biophysical Research Communications, 3.3. Hop-depleted cells are more sensitive to HSF1 inhibition
We next tested Hop depletion and the sensitivity of cells to the
HSF1 inhibitor, KRIBB11 (Fig. 4A). KRIBB11 inhibits HSF1 tran￾scription by blocking p-TEFb recruitment to transcriptional com￾plexes [30]. Hop-depleted HEK-shHOP (with doxycycline) had an
IC50 value for KRIBB11 (2.8 ± 1.2 mM) approximately ten-fold lower
than the respective control cell lines (HEK-shNT with and without
doxycycline) (36.3 ± 1.1 and 37.1 ± 1.0 mM, respectively) and HEK￾shHOP without doxycycline (43.0 ± 1.2 mM) (Fig. 4A). This sug￾gested that Hop-depleted cells are more sensitive to HSF1 inhibi￾tion which correlates with reduced basal levels of HSF1 upon Hop
depletion. KRIBB11 treatment and HSF1 depletion by RNAi have
been shown to produce the same phenotype [37], and hence it is
likely that Hop depletion and KRIBB11 combine in inhibiting HSF1.
3.4. Hop-depletion reduces viability and stress resilience
We next analyzed the survival of HEK-shNT and HEK-shHOP
cells (Fig. 4B). Cell viability after 72 h was normalized to the HEK￾shNT cells without doxycycline, which was taken as 100%. There
was a significant reduction in average viability of the Hop-depleted
HEK-shHOP cells (with doxycycline) (18.9 ± 3.6%) compared to the
HEK-shNT with or without doxycycline and the HEK-shHOP
without doxycycline (96.7 ± 5.0%, 100 ± 3.6% and 94.4 ± 12.1%,
respectively) (Fig. 4B). We next used clonogenic assays to measure
the long-term survival of cells (Fig. 4C and D). Images were
captured (Fig. 4C) and average cell survival was quantified by sol￾ubilization of crystal violet dye (and normalized to the HEK-shNT
without doxycycline under basal conditions) (Fig. 4D). HEK-shNT
cells (irrespective of doxycycline treatment) and HEK-shHOP cells
without doxycycline showed significantly higher cell survival
compared to Hop-depleted HEK-shHOP cells (with doxycycline)
under both basal and heat-shock conditions. The Hop-depleted
HEK-shHOP cells (with doxycycline) showed significantly reduced
long-term survival under basal conditions, which was further
reduced upon heat-shock. Taken together, these data suggest that
Hop depletion reduces cell proliferation and survival under stress
which is consistent with reduced growth at both 30
C and 37
C in
sti1 null yeast [23].
4. Conclusion
Our data demonstrate a role for Hop in the regulation of HSF1
levels and activity. Depletion of Hop impaired HSF1 function. The
most plausible explanation for this based on our data and those of
others in the field is that, in addition to reducing basal HSF1 levels,
Hop depletion restricts the nuclear localization of HSF1, which re￾duces its ability to bind HSE and become transcriptionally active,
culminating in reduced cell survival.
Fig. 3. Hop depletion alters HSF1 subcellular localization. Confocal microscopy of total HSF1 (green) or HSF1-pSer326 (red) in HEK-shNT and HEK-shHOP with doxycycline (Dox)
treatment under (A) basal and (B) heat-shock conditions. Nucleus stained with Hoechst 33324 (blue). Upper panels show cells captured at 63x magnification. Panels (i-iv) show
magnified images of the areas in white boxes. Arrows indicate nuclear punctae and cytoplasmic accumulation. Cell images without doxycycline are in Fig. S2. (For interpretation of
the references to colour in this figure legend, the reader is referred to the Web version of this article.)
A. Chakraborty, A.L. Edkins / Biochemical and Biophysical Research Communications xxx (xxxx) xxx 5
Please cite this article as: A. Chakraborty, A.L. Edkins, Hop depletion reduces HSF1 levels and activity and coincides with reduced stress
resilience, Biochemical and Biophysical Research Communications, Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
This research was supported by funding from the South African
Research Chairs Initiative of the Department of Science and Inno￾vation (DSI) and National Research Foundation of South Africa
(NRF) (Grant No 98566), National Research Foundation CPRR (Grant
No105829), and Rhodes University. A.C was supported by an NRF
Innovations postgraduate bursary. The views expressed are those of
the authors and should not be attributed to the DST, NRF, or Rhodes
University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
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