Paper
Journal of Materials Chemistry B
in DMEM media supplemented with 10% FBS and 1% penicillin/ References
streptomycin. Cells were allowed to attach overnight at 37 1C and
1
2
3
C. Xu, B. Bailly-Maitre and J. C. Reed, Endoplasmic reticulum
stress: cell life and death decisions, J. Clin. Invest., 2005, 115,
in 5% CO . After cells attached, ER-Trackert Red (Sigma E34250)
2
was added to the cell media at a final concentration of 1 mM
along with a final concentration at 500 nM of the flavonoid dye
to be tested. Cells were left to incubate for 2 hours, and then
washed 5 times with PBS. Live Cell Imaging Solution (Thermo-
Fisher A14291DJ) was then added, along with 10 mM of DRAQ5
2656–2664.
P. Walter and D. Ron, The unfolded protein response: from
stress pathway to homeostatic regulation, Science, 2011, 334,
1081–1086.
I. Kim, W. Xu and J. C. Reed, Cell death and endoplasmic
reticulum stress: disease relevance and therapeutic opport-
unities, Nat. Rev. Drug Discovery, 2008, 7, 1013–1030.
H. Yoshida, ER stress and diseases, FEBS J., 2007, 274,
630–658.
D. J. Todd, A. H. Lee and L. H. Glimcher, The endoplasmic
reticulum stress response in immunity and autoimmunity,
Nat. Rev. Immunol., 2008, 8, 663–674.
S. Fu, S. M. Watkins and G. Hotamisligil, The Role of
Endoplasmic Reticulum in Hepatic Lipid Homeostasis
and Stress Signaling, Cell Metab., 2012, 15, 623–634.
L. Ozcan and I. Tabas, Role of Endoplasmic Reticulum
Stress in Metabolic Disease and Other Disorders, Annu.
Rev. Med., 2012, 63, 317–328.
P. van Bergeijk, C. C. Hoogenraad and L. C. Kapitein, Right
Time, Right Place: Probing the Functions of Organelle
Positioning, Trends Cell Biol., 2016, 26, 121–134.
N. Baumann and D. Pham-Dinh, Biology of Oligodendrocyte
and Myelin in the Mammalian Central Nervous System,
Physiol. Rev., 2001, 81, 871–927.
(Sigma 62251) fluorescent probe, to stain cell nuclei. Cells were
then left to incubate at room temperature for 30 minutes before
imaging.
4
5
Zebrafish breeding and staining
Zebrafish were maintained as described in the Zebrafish Book
by the University of Oregon. They were kept in 28.5 1C water for
mating, male and female zebrafish were maintained in one
tank on a 12 h light/12 h dark cycle. Spawning was triggered by
light stimulation in the morning and eggs were immediately
fertilized. All embryos were maintained in E3 medium (15 mM
6
7
8
9
NaCl, 0.5 mM KCl, 1 mM MgSO
4
, 1 mM CaCl
2
, 0.15 mM
À5
KH PO , 0.05 mM Na HPO , 0.7 mM NaHCO
2
4
2
4
3
, and 10
%
methylene blue at 7.5 pH). All animal related procedures were
approved by the Care and Use of Animals in Research Committee
at The University of Akron.
A stock solution of the dye was prepared in DMSO at 1 mM.
All stains were done at 10 mM with a 12 h incubation time and
the fish were washed 3 times with E3 medium to limit potential
background noise from aggregates. All fished were anesthetized
with 0.5 mL of MS-2,2,2 20–30 minutes prior to imaging to limit
movement. Once imaging was completed all the fish were
euthanized using excess MS-2,2,2 before disposal.
10 S. E. Pfeiffer, A. E. Warrington and R. Bansal, The oligo-
dendrocyte and its many cellular processes, Trends Cell Biol.,
1993, 3, 191–197.
1
1 J. Praet, C. Guglielmetti, Z. Berneman, A. Van der Linden
and P. Ponsaerts, Cellular and molecular neuropathology of
the cuprizone mouse model: clinical relevance for multiple
sclerosis, Neurosci. Biobehav. Rev., 2014, 47, 485–505.
Conclusion
In summary, we report the first example of flavonoid-based
probes for ER sensing in living eukaryotic cells. The D–A type
flavonoid 2 exhibits many attractive features, which include
fluorescence turn-on, protein interaction, low toxicity, good
biocompatibility, efficient cellular uptake, and ER-targeting.
The peculiar ER-targeting has been independently observed
in all the cell lines examined. The low molecular mass of 2
1
2 A.-S. Dhaunchak and K.-A. Nave, A common mechanism of
PLP/DM20 misfolding causes cysteine-mediated endoplasmic
reticulum retention in oligodendrocytes and Pelizaeus–
Merzbacher disease, Proc. Natl. Acad. Sci. U. S. A., 2007,
104, 17813–17818.
1
3 J. Wu, W. Liu, J. Ge, H. Zhang and P. Wang, New sensing
mechanisms for design of fluorescent chemosensors emerging
in recent years, Chem. Soc. Rev., 2011, 40, 3483–3495.
4 J. M. Meinig, L. Fu and B. R. Peterson, Synthesis of Fluoro-
phores that Target Small Molecules to the Endoplasmic
Reticulum of Living Mammalian Cells, Angew. Chem., Int.
Ed., 2015, 54, 9696–9699.
(o500 Dalton), in addition to its low toxicity, is anticipated to
cause less perturbation to cellular function during imaging. It
appears that the amide group plays an important role in the
observed ER selectivity, as the control compounds without the
amide side chain do not exhibit good selectivity to ER (ESI,†
Fig. S3). The observed ER selectivity from 2 could be attributed
to flavonoid’s binding to specific proteins. Further study will be
carried out to fine tune the ER selectivity.
1
1
5 F. M. Ashcroft and F. M. Gribble, Correlating structure and
function in ATP-sensitive K+ channels, Trends Neurosci.,
1998, 21, 288–294.
1
6 A. Hambrock, C. L o¨ ffler-Walz and U. Quast, Glibenclamide
binding to sulphonylurea receptor subtypes: dependence on
adenine nucleotides, Br. J. Pharmacol., 2002, 136, 995–1004.
Acknowledgements
This work was supported by NIH (Grant No. 1R15EB014546-01A1). 17 A. J. Smith, T. K. Taneja, J. Mankouri and A. Sivaprasadarao,
We also thank the Coleman endowment from the University of
Akron for partial support.
Molecular cell biology of KATP channels: implications for
neonatal diabetes, Expert Rev. Mol. Med., 2007, 9, 1–17.
J. Mater. Chem. B
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