Acridine Orange

A chemical library of 658-natural compounds was kindly provided by Dr. Sang Jeon Chung of Sungkyunkwan University (Suwon, Korea). Kaempferide (69545), dimethylsulfoxide (D2650), bafilomycin A1 (B1793), rapamycin (553210), tiliroside (79257), chloroquine (C6628), orlistat (O4139), palmitic acid (P5585), oleic acid (O1383), acridine orange (A6014), oil-red-O (O0625), dexamethasone (D8893), insulin (I0516), and 3-isobutyl-1-methylxanthine (I5879) were purchased from Sigma-Aldrich. BODIPY 493/503 (D3922), Hoechst33342 (H3570), lipofectamine LTX (94756), lipofectamine 2000 (52887), Plus reagent (10964), protease and phosphatase inhibitor solution (78441), M-PER kit (89842Y), DMEM, fetal bovine serum (FBS), bovine serum, and antibiotics were purchased from Invitrogen ThermoFisher Scientific. For in vivo experiments, Kaempferide (K0057) was purchased from TCI Chemicals. siRNA targeting TUFM was purchased from Dharmacon. mRFP-GFP-LC3B plasmids were kindly provided by Dr. Jaewhan Song of Yonsei University (Seoul, Korea).

For acridine orange (AO) stain in the screen, HeLa cells were grown in 96-well plates. Cells were treated with DMSO or indatraline, bafilomycin A1, and 658-natural chemicals for 24 h, and stained with 5 μg/mL AO. Fluorescence intensity was measured by victor plate reader, where fluorescence intensity of each well in the plate is promptly displayed as numerical readout. For confocal microscopy, 3T3-L1 or HeLa cells were grown on 15 mm coverslips at a density of 1.0 × 10⁵ cells/well in 6-well plates The cells were then treated with drugs for the time periods indicated, followed by treatment with 5 μg/mL AO (Sigma-Aldrich). Nuclei were stained with Hoechst. Following incubation for 20 min, the cells were fixed with 4% PFA and washed three times with PBS. Images were obtained using an LSM880 confocal microscope at ×400 magnification. Red fluorescence intensity was quantified using Image J2 software. For BODIPY FL-Pepstatin A stain, HeLa cells were grown on 15 mm coverslips at a density of 1.0 × 10⁵ cells/well in 6-well plates. The cells were then treated with drugs for the time periods indicated, followed by treatment with 1 μM BODIPY FL-Pepstatin A (Invitrogen) for 30 min. Nuclei were stained with Hoechst. Following incubation for 20 min, the cells were fixed with 4% PFA and washed three times with PBS. Images were obtained using an LSM880 confocal microscope at 400× magnification. For DQ-BSA analysis, HeLa cells were grown on 15 mm coverslips at a density of 1.0 × 10⁵ cells/well in 6-well plates. The cells were then treated with 10 μg/mL DQ-BSA for 2 h. After change medium, cells were treated with drugs for the time periods indicated. Nuclei were stained with Hoechst with incubation for 20 min, the cells were fixed with 4% PFA and washed three times with PBS. Images were obtained using an LSM880 confocal microscope at ×400 magnification.

Preferential pathways of water movement through the surface of ‘Sonsation’ fruits were identified by selectively sealing different portions of the fruit surface with silicone rubber. Two experiments were carried out with the rates of osmotic water uptake and transpirational water loss being quantified gravimetrically.
The first experiment was conducted in two phases (‘control’/’treatment’) using a repeated-measures design, comparing each treatment with its control (Fig. 4, Table 1). In phase I, fruit with calyx and peduncle (cut to 5 mm long) was incubated for 1.5 h. Rates of water movement were determined gravimetrically as described above. There was no sealing in phase I (‘control’). In phase II, selective sealing using silicone rubber or cutting was applied as follows (‘treatment’): (1) sepals (including episepals) and stamina were excised at their base and the cut surfaces, the calyx-receptacle junction, and the peduncle end were sealed (Fig. 3); (2) sepals and stamina were excised and the cut surfaces, the calyx-receptacle junction, and the peduncle end were left un-sealed, and (3) the peduncle end was sealed (Table 1). Each treatment had its own control. Fruit were held in a controlled temperature room for 1.5 h for the silicone rubber to cure. Fruits were then incubated for an additional 1.5 h.
In the second experiment, the portion of the fruit surface responsible for the increased water movement in the calyx region was identified by sealing a progressively increasing portion of the fruit surface—beginning at the proximal end of the fruit (Fig. 3, Table 2). The treatments were (a) ‘Control’: no sealing; (b) ‘Sepals sealed’: the sepals and the peduncle surface were sealed. The petal and stamina abscission zones, the calyx-receptacle junction, and the seedless zone of the receptacle were not sealed; (c) ‘Sepals, petal and stamina abscission zones and calyx-receptacle junction sealed’: the sepals, abscission zone, stamen, peduncle, and calyx-receptacle junction was cut and all surfaces sealed (the ‘seedless’ zone, strictly, the zone lacking the ‘pips’ or single-seeded achenes, was not sealed); and (d) ‘Seedless zone sealed’: the seedless zone of the receptacle was sealed, also the abscission zone and stamens, the calyx-receptacle junction, the peduncle, and sepals were removed and sealed. The cut end of the peduncle was sealed in all these treatments including the control.
Rates of transpiration and osmotic water uptake were calculated as described above. The total number of replications per treatment was 30. From these treatments, the amounts of water transpired or taken up through the sepals and through the peduncle surface or through the abscission zones of petals, stamina, the junction between calyx and receptacle, and through the seedless zone and through the receptacle bearing seeds were obtained by calculating the difference between the respective rates of water movement in phase I and phase II (Table 3).
To identify the sites of water uptake microscopically, ‘Sonsation’ fruits from the same batch were incubated in 0.1% (w/w) aqueous acridine orange (Carl Roth, Karlsruhe, Germany) for 1.5 h. The cuticle is impermeable to the fluorescent tracer acridine orange. The dye therefore penetrates only in regions where the cuticle barrier is bypassed. Fruits were rinsed with deionized water, blotted using soft tissue paper, and viewed under a fluorescence binocular microscope (MZ10F; Leica Microsystems, Wetzlar, Germany) (Figs. 5, 6).
To establish whether the same preferential pathways for water movement occurred in other strawberry cultivars, the relative contributions to water movement of the various pathways—through the petal and staminal abscission zones, and around the junction between calyx and receptacle, were determined on a whole fruit basis in ‘Clery’, ‘Sonsation’, ‘Florentina’, ‘Dream’, ‘Joly’, and ‘Elsanta’ (Table 4). To do this the abscission zone and the junction were sealed using silicone rubber. Unsealed fruit served as controls. Pathways of water movement were also viewed by fluorescence microscopy. Fruits from the same batch were incubated in a solution of the fluorescence tracer acridine orange at 0.1% (w/w) (Carl Roth, Karlsruhe, Germany) for 1.5 h. Thereafter, fruits were observed under a fluorescence binocular microscope (MZ10F; Leica Microsystems, Wetzlar, Germany).

Thawing was performed in a 37°C water bath and was completed under 3 min. Acridine orange (AO; Thermo Fisher) and propidium iodide (PI; Thermo Fisher) were used to stain cells for recovery calculations on a hemocytometer (Millipore Sigma). A 1:1 ratio of AO/PI and cells were used for counting on the hemocytometer.

A fluorescence microscope was used to visualize cell proliferation efficiency on the specimen. Both cancer and normal cells were seeded onto the sample in 12-well plates for 24 h at 310 K. Fresh solution of PBS (phosphate-buffer saline) was used twice to wash test samples for removing the dead or floating cells. The 4% paraformaldehyde solution fixed the adhered cells for 20 min. Then they were washed again with PBS and marked by AO (acridine orange) and EtBr (ethidium bromide) (100 μg mL⁻¹) fluorescent dye for 10 min. Again, these were rinsed with PBS for twice and consequently incubated for 5 min in dark. After that with a fluorescence microscope (Leica, Germany) images were collected.