pH-Assisted multichannel heat shock monitoring in the endoplasmic reticulum with a pyridinium fluorophore

Heat shock is a global health concern as it causes permanent damage to living cells and has a relatively high mortality rate. Therefore, diagnostic tools that facilitate a better understanding of heat shock damage and the defense mechanism at the sub-cellular level are of great importance. In this report, we have demonstrated the use of a pyridinium-based fluorescent molecule, PM-ER-OH, as a ‘multichannel’ imaging probe to monitor the pH change associated with a heat shock in the endoplasmic reticulum. Among the three pyridinium derivatives synthesized, PM-ER-OH was chosen for study due to its excellent biocompatibility, good localization in the endoplasmic reticulum, and intracellular pH response signaled by a yellow fluorescence (λmax = 556 nm) at acidic pH and a far red fluorescence (λmax = 660 nm) at basic pH. By changing the excitation wavelength, we could modulate the fluorescence signal in ‘turn-ON’, single excitation ratiometric and ‘turn-OFF’ modes, making the fluorophore a ‘multichannel’ probe for both ex vitro and in vitro pH monitoring in the endoplasmic reticulum. The probe could efficiently monitor the pH change when heat shock was applied to cells either directly or in a pre-heated manner, which gives insight on cellular acidification caused by heat stress.


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All reagents and dry solvents were purchased from commercial sources and were used as such.
Column chromatography was done using silica gel 100-200 mesh. 1 H and 13 C NMR were recorded on a Bruker Advance II spectrometer at 500 MHz and 125 MHz, respectively with solvents mentioned with the NMR data.Data are reported as follows: chemical shift in ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, coupling constant (Hz) and integration.HRMS analysis was recorded on a Thermo Scientific Exactive LCMS instrument by electrospray ionization method with ions given in m/z using Orbitrap analyzer.
Electronic absorption spectra were recorded in a Shimadzu UV-2600 spectrophotometer.All emission spectra were taken in Horiba Fluorolog ─ 3 Jovin Yoon.The cell imaging experiments were carried out by a Zeiss LSM710 Airyscan CLSM (confocal laser scanning microscope).
Optical density (OD) for the cellular experiments was recorded using the microplate reader (infinite M200, TECAN).

Optical Measurements
Electronic absorption spectra were recorded in a Shimadzu UV-2600 UV-Vis spectrophotometer and emission studies were performed in Horiba Fluorolog ─3 Jovin Yoon.The stock solution of all the molecules were prepared in spectroscopy grade DMSO and diluted with phosphate buffer as per need.The pH solutions were made by using phosphate buffer and adjusted by using dilute HCl/ NaOH solution.and PM-ER-OH (10 µM for all) in chloroform.

PM-C 3 and PM-ER-OMe pH response:
To understand whether the fluorescence change of PM-ER-OH with pH variation is due to the two -OH groups present in it, we studied the effect of pH on our control molecules, i.e., PM-C 3 and PM-ER-OMe.In the case of PM-C 3, we could not find any significant change in the peak maximum and intensity for both absorbance and emission with change in pH from 4 to 10.However, in the case of PM-ER-OMe, though there was no shift in peak maximum with increase in pH, the intensity decreased for both absorbance and emission.As the fluorophore core is same for both molecules, the decrease in intensity of the peak for PM-ER-OMe may be due to the weak photo induced electron transfer (PET) effect coming from the sulphonamide group at the basic pH value.
But for both molecules, there was no shift in the peak maximum in the emission spectra.

Fluorescence Quantum Yield measurement:
Fluorescence quantum yields of the molecules were determined using Coumarin 153 as the Φ  reference according to the literature method.
Quantum yields were determined following this equation: Where 's' and 'r' represent the synthesized and reference samples, respectively.A, Φ  , and F were the absorbance (≤ 0.07) at λ ex , refractive index of the used spectroscopy grade solvent, and the integrated area under the corrected emission spectrum, respectively.

General Strategies for cellular studies:
5.1 Cell culture: The HeLa cells, that were obtained from American Type Culture Collection (ATCC, Manassas, VA), were grown in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin (PS) antibiotics at 37 ℃ in humidified environment of 5% CO 2 .Cells were plated on 6-well plates, and allowed to adhere for 12 h.
Thereafter, MTT solution (5 mg/mL, 10 µL per well) was added mixed with DMEM.Following 4 h incubation, the MTT solution was replaced with 100 µL of DMSO and absorbance of the formed formazan crystals was measured at 570 nm.The percentage of cellular viability was calculated according to the following equation:

Co-localization:
For the colocalization experiment, the HeLa cells (1 × 10 4 cells) were seeded in µ-Dish 35 mm microscopy dishes.24 h after adherence, media were replaced with 10 µM PM-ER-OH and 10 µM PM-C 3 , and allowed to incubate for 20 min in PBS at 37 ℃.Thereafter, the cells were washed three times with PBS, and replaced with PBS containing 1 µM ER tracker solution.20 minutes later, the cells were washed with PBS three times and imaged under Zeiss LSM710 CLSM.The samples were excited with 405 nm and 488 nm lasers.Image analysis was performed using Image j.

Fig S1 :
Fig S1: Structures of the pentacyclic pyridinium molecules used in this work.
Fig S2: a) Normalized absorbance and b) Normalized emission spectra of PM-C 3 , PM-ER-OMe

Fig
Fig S3: a) absorbance and b) emission spectra of PM-C 3 and PM-ER-OMe in PBS buffer of pH 4, 7.4 and 10.
Fig S4: a) Emission Spectra of PM-ER-OH from pH 4 -10 (λ ex = 405 nm).b) Fluorescence response of the probe at pH 4 -10 when excitation wavelength is shifted to 458 nm.c) Emission spectra of PM-ER-OH when the excitation wavelength is shifted to 488 nm at pH 4 -10.

Fig S6 :
Fig S6: Colocalization analysis.a) CLSM images of ER tracker and PM-ER-OH incubated live HeLa cells in blue, green and merged channels.b) Correlation of PM-ER-OH and ER tracker intensities.c) CLSM images of ER tracker and PM-C 3 stained live HeLa cells in blue, green and merged channels.d) Correlation of PM-C 3 and ER tracker intensities.Pearson's coefficients calculated using ImageJ are mentioned in the inset.Scale bar 10 µm.

Fig S7 :
Fig S7: Comparison of photostability of PM-ER-OH with commercially available ER tracker.(a) HeLa cells were treated with 1 μM of ER-Tracker Blue-White DPX dye and 10 μM of PM-ER-OH separately.Then cells were kept under light irradiation for 10 mins with intervals of 10 s.Images were acquired every 60 s.Scale bar is 20 μm.(b) The fluorescence intensity was measured from the cells and a comparison graph for the intensity ratio at each time after photobleaching was plotted.Scale bar 20 µm.