Propidium iodide

Forskolin was purchased from Sigma (USA). PCI-34051 and Tubastatin were procured from Cayman chemicals. DMEM, FBS, 1% Penicillin & Streptomycin Antibiotics were obtained from Himedia. Monoclonal antibody of HDAC8 (A-4008) was obtained from Epigenetik, Beta actin ab8227 (Abcam), GAPDH (MA5–15738), Alpha tubulin (B-5-1-2) and anti-acetylated alpha tubulin (Cat no: 322700) were purchased from Thermo scientific Life technologies respectively. Protein A Agarose beads were obtained from Santa Cruz. Poly-L-Lysine (Sigma P8920), 4′, 6-Diamindino-2-phenylindole dichloride DAPI as a nuclear stain (Sigma), Alexa Fluor® 555 Dye (Thermo Fischer Scientific). HDAC8 FLUOR DE LYS fluorometric assay kit (BML-AK518–0001) was purchased from Enzo life sciences. Propidium iodide was purchased from Himedia. GST Column (GE Healthcare 17–5132-01), Custom synthesized peptides of alpha tubulin 33–46 amino acids, Acetylated alpha tubulin peptide, at Lys40 DGQMPSDKTIGGGD and Unacetylated Alpha tubulin peptide DGQMPSDKTIGGGD from SIGMA (USA) (resuspended in MilliQ water).

Ruthenium (Ru-1) with molecular weight 722 Dalton was dissolved in DMSO, and a 10 mM stock solution was prepared and synthesised as in our previous study [21 (link)]. Regorafenib (BAY 73-4506) was ordered from selleckchem.com in the United States in a concentration of 10 mM. To maintain the DMSO concentration below 0.5% during experimental studies, further working dilutions were prepared, which are not toxic for cells. Fetal bovine serum (FBS) and DMEM medium were obtained from Gibco (Thermo Fisher Scientific, Mumbai, India). Propidium iodide was purchased from HI Media (HI media labs, Mumbai, India), 3-(4,5-dimethylthiazol-2-yl) −2,5-diphenyl tetrazolium bromide (MTT), DCFH-DA, JC-1 staining kits were purchased from G-BIOSCIENCES (Noida, India). Molecular structures of Ru-1 and Regorafenib are provided as Supplementary Data.

Confocal laser scanning microscopy (CLSM) for salt and PGPR-treated roots was performed to check membrane damage and cell viability. Fresh roots were cut into small pieces, followed by dual staining with 30 μM propidium iodide (PI; HiMedia, India) and 10 μM acridine orange (AO; HiMedia, India) solution for 15 min. After being treated with a dye mixture, mustard roots were then rinsed with phosphate buffer (0.1 M, pH 7.0), and finally mounted on glass slides. Stained roots were inspected for both live and dead tissues under confocal laser scanning microscope Leica Microsystems TCS-SP5. Dead and live tissues showed PI and AO staining, respectively. Control roots were used for assessing the difference between treated and untreated roots.

Strophanthidin was procured from Sigma-Aldrich chemicals, while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and propidium iodide were obtained from HiMedia Chemicals. + + + + + All the antibodies were purchased from Cell signaling technologies and Elabscience. Alexa Fluor 488 and ProLong Gold Antifade Mountant were procured from Thermo Fisher Scientific, 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Roche Chemicals, and SYBR Green Master Mix was procured from Origin (India). Strophanthidin was stored as stock solution (10 mM) in DMSO in amber-colored glass containers at −20°C. Final working concentrations were prepared in the media before the experiment. The control contained the highest DMSO concentration (0.001%).

Spheroids were first pelleted in a sterile 15-mL tube. Cells were then fixed using 3.7% formaldehyde (24005, Thermo Fisher Scientific) at $4^{\circ }$ C for 30 min. After fixation, cells were washed and resuspended in PBS. Following this, 20 μL of spheroid suspension was dried in eight well chambered cover glass at ${37}^{\circ }$ C for 30 min. Permeabilization was achieved using 0.5% Triton X-100 in PBS (PBST) (MB031, HiMedia) for 2h at RT. ${\text{Alexa}\text{Fluor}}^{TM}$ 633-conjugated phalloidin (A22284, Thermo Fisher Scientific) was added to cells at 1:500 dilution in 0.1% PBST and incubated overnight at $4^{\circ }$ C. Cells were washed thrice with 1 $\times$ PBS for 5 min, counterstained with 1 μg/mL DAPI (D1306, Thermo Fisher Scientific).
To determine live and dead cells in the culture, cells were stained with 0.5 mg/mL Calcein AM (C1430, Invitrogen) for 15 min. Cells were washed with PBS, counterstained with Propidium Iodide (TC252, HiMedia) for 5 min, and imaged under the Olympus IX73 fluorescence microscope.

For the proliferation assay, non adherent cells enriched for lymphocytes after monocyte depletion for DCs culture were prepared from allogenic and autologous PBMCs. Matured DCs primed with rhSPAG9 (250, 500, 750 and 1000 ng/ml per million cells), were co-cultured with PBMCs, stained with carboxyfluorescein, succinimidyl ester [CFSE (Invitrogen, Carlsbad, CA, USA)]. Briefly, SPDCs were co-cultured at the ratio of 1:50 (DCs: allogeneic PBMCs) and 1:10 (DCs: autologous PBMCs) to check for proliferation as described in our previous study [27 (link)]. The cells were cultured for 8 days. The cells were centrifuged at 1580 rpm and subsequently pellet was resuspended and washed with PBS, blocked with 5% FBS. The cells were further probed with anti-CD4-PC5, anti-CD8-APC, anti-CD56-PE, anti-CD25-ECD, anti-FOXP3-PE antibodies and Propidium iodide (1 µg/ml; Himedia, India) to verify the response of the autologous or allogeneic PBMCs upon stimulation with SPDCs. Unstimulated PBMCs were used as negative controls in all experiments and the percentage proliferation of these cells has been subtracted from all DC stimulated wells to arrive at the representative data. Cells were acquired using a MoFlo XDP cell sorter/ Flow cytometer (Beckman Coulter, Carlsbad, CA, USA) and FCS express 7 was used for analyzing the proliferating population.